Analysis of circulating transcription factors

Abstract

The present invention relates to a method for detecting disease in a subject, using a minimally invasive body fluid test to detect circulating chromatin fragments comprising a transcription factor and an associated DNA sequence as an indication of the presence of disease, such as cancer, in the subject, the method comprising sequencing the associated DNA sequence and / or removing cell-free nucleosomes from the body fluid.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method for detecting disease in a subject by minimally invasive testing of body fluids.The present invention also relates to the measurement or detection of circulating chromatin fragments containing transcription factors as an indicator of the presence of disease in a subject. [Background technology]

[0002] BACKGROUND OF THEINVENTION Cancer is a common disease with a high mortality rate. It is understood that the biology of the disease involves progression from precancerous stages to stage I, II, III, and finally to stage IV cancer. Mortality rates vary widely for most cancer diseases depending on whether the disease is detected at an early, localized stage, when effective treatment options are available, or, if at a later stage, when the disease spreads into affected organs or becomes more difficult to treat. Symptoms of late-stage cancer are varied and include visible blood in the stool, blood in the urine, blood in the sputum, irregular vaginal bleeding, unexplained weight loss, persistent unexplained lumps (e.g., in the breast), indigestion, difficulty swallowing, transformation into a wart or mole, and many other possible symptoms depending on the type of cancer. However, most cancers diagnosed because of such symptoms will already be late-stage and difficult to treat. Most cancers are asymptomatic in the early stages or present with non-specific symptoms that do not aid in diagnosis. For these reasons, cancer should ideally be detected early using cancer screening.

[0003] To address the need for simple routine cancer blood tests, many blood-borne proteins have been investigated as potential cancer biomarkers, including carcinoembryonic antigen (CEA) for CRC, alpha-fetoprotein (AFP) for liver cancer, CA125 for ovarian cancer, CA19-9 for pancreatic cancer, CA15-3 for breast cancer, and PSA for prostate cancer. However, they are considered better used for patient monitoring because their clinical accuracy is too low for routine diagnostic use.

[0004] More recently, researchers in the field have been investigating circulating tumor DNA (ctDNA) as a blood-based biomarker for cancer detection. Cell-free DNA (cfDNA) circulates in the blood as chromatin fragments that are believed to result from cell death, mainly by apoptosis, of a vast number of cells every day. During the apoptotic process, chromatin is fragmented into mono- and oligonucleosomes, some of which are released from the cell and circulate as cell-free nucleosomes. Each circulating cell-free nucleosome is associated with a small DNA fragment less than 200 base pairs (bp) in length. Similarly, circulating cell-free chromatin fragments consisting of DNA-binding transcription factors or other non-histone chromatin proteins have also been inferred from fragmentomics analysis. Circulating chromatin fragments in healthy subjects are believed to be of hematopoietic origin and their levels are low. High levels of circulating nucleosomes, and therefore cfDNA fragments, are found in subjects with a variety of conditions, including many cancers, autoimmune diseases, inflammatory conditions, stroke, and myocardial infarction (Holdenrieder and Stieber, 2009).

[0005] At least a portion of the cfDNA in cancer patient blood is believed to originate from nucleosomes and other chromatin fragments released into the circulation from dying or dead cancer cells (i.e., cfDNA contains some ctDNA). Studies of matched blood and tissue samples from cancer patients have shown that cancer-associated mutations present in the patient's tumor (but not in the patient's healthy cells) are also present in the cfDNA of blood samples taken from the same patient (Newman et al., 2014). Similarly, differentially methylated (epigenetically altered by methylation of cytosine residues) DNA sequences in cancer cells can also be detected as methylated sequences in cfDNA in the circulation. Furthermore, the proportion of circulating cfDNA containing ctDNA is associated with tumor burden, so that disease progression can be monitored both quantitatively by the proportion of ctDNA present, and qualitatively by its genetic and / or epigenetic composition. Analysis of ctDNA can provide highly useful and clinically accurate data on DNA originating from all or many different clones in the tumor, thus spatially integrating tumor clones. Moreover, repeated blood sampling over a long period of time is a much more practical and economical option than, for example, repeated tissue biopsies. Analysis of ctDNA has the potential to revolutionize tumor detection and monitoring, as well as recurrence detection and acquired drug resistance detection for early tumor treatment selection through tumor DNA investigation without invasive tissue biopsy procedures. Such ctDNA testing may be used to investigate all kinds of cancer-related DNA abnormalities (e.g., point mutations, nucleotide modification status, translocations, gene copy number, microsatellite abnormalities, and DNA strand integrity) and may be applicable to routine cancer screening, regular and more frequent monitoring, and regular checks of optimal treatment regimens (Zhou et al., 2017).

[0006] Typically, plasma is used as the substrate for ctDNA assays. cfDNA fragments (including any ctDNA) are extracted from the plasma (and thus removed from association with nucleosomes, transcription factors, or other proteins) and analyzed for nucleotide sequence. Any DNA analysis method can be used, but typically analysis is performed by deep sequencing using next-generation sequencer equipment.

[0007] ctDNA testing is applicable to all cancer diseases because DNA abnormalities are characteristic of all cancer diseases and ctDNA has been observed in all cancer diseases that have been investigated, including but not limited to bladder cancer, breast cancer, colorectal cancer, melanoma, ovarian cancer, prostate cancer, hepatic lung cancer, endometrial cancer, ovarian cancer, lymphoma, oral cancer, leukemia, head and neck cancer, and osteosarcoma (Crowley et al., 2013; Zhou et al., 2017; Jung et al., 2010).

[0008] An example of cfDNA analysis method includes identifying the tissue or cell of origin of cfDNA fragments of a subject.The basis of this approach is that all cfDNA fragments present in the circulatory system avoid digestion by nuclease during cell death or in the circulatory system, because they are protected from nuclease action by protein binding in nucleosome.This approach includes determining the nucleosome fragmentation pattern of cfDNA in a blood sample taken from a subject, and identifying the genomic location of said cfDNA fragments in a reference genome.The fragmentation pattern is different for different cell types and can be used to identify the cell of origin of cfDNA of a subject.

[0009] This approach involves extracting cfDNA (including any ctDNA) from the plasma sample and whole genome sequencing the DNA to detect nucleosome-bound DNA patterns exhibited by the cfDNA fragments. The end sequences of the cfDNA fragments are identified using computational bioinformatics to identify their genomic location within one or more reference genomes. The genomic locations of the cfDNA ends within the reference genomes provide a nucleosome-protected cfDNA coverage map for that genome.

[0010] The percent contribution of different cell types or tissues to cfDNA in a subject may also be determined by comparison of the subject's nucleosome fragmentation pattern to a calibration sample containing known relative abundances of cfDNA from different cellular sources using computational bioinformatics as described in International Publication No. WO 2017 / 012592.

[0011] cfDNA fragments associated with nucleosome-containing chromatin fragments are typically 120-200 bp in length. However, protein binding and protection of cfDNA is not limited to histone binding of cfDNA within nucleosomes. Other cfDNA fragments, including active gene promoter sequences, are bound by transcription factors, cofactors, or other non-histone chromatin proteins, either in addition to nucleosomes or in the absence of nucleosomes. In the absence of nucleosomes, these proteins often bind and protect short cfDNA fragments in the 35-80 bp range. However, these short cfDNA fragments are observed in experiments only when the DNA fragment library preparation method used is suitable for the isolation, amplification, and sequencing of short DNA fragments less than 100 base pairs in length (Snyder et al., 2016).

[0012] Since different DNA sequences, including different promoter and gene sequences, are active in different cells, the protein binding patterns of DNA throughout the genome in living cells vary from cell type to cell type. The protein binding patterns of DNA in any cell type can be determined by nuclease accessible site mapping, which involves digesting chromatin extracted from cells with nuclease enzymes and sequencing the undigested DNA in the resulting protein-protected chromatin fragments. Thus, if cfDNA fragments in blood are considered to be the product of in vivo nuclease digestion, the cfDNA sequences found should match the DNA sequences bound to proteins in the cells from which the cfDNA originates. Thus, in principle, the pattern of cfDNA fragment sequences in blood should resemble the sequence pattern of chromatin fragments created by nuclease accessible site mapping of its cell of origin. Thus, the fragmentation patterns of cfDNA sequences determined from blood samples can be compared to known DNA fragmentation patterns created by nuclease accessible site analysis of cells of known tissues or cancer types using bioinformatics methods to determine the tissue of origin of the cfDNA. Results from samples from healthy subjects indicate that the cell of origin of cfDNA is hematopoietic. Results from this approach in samples from cancer patients indicate that cfDNA and ctDNA originate from a cell mixture that includes hematopoietic and other cells. In many cases, the non-hematopoietic cell types represented have been shown to correlate with the histology of the patient's cancer disease (Snyder et al., 2016).

[0013] Other researchers have used a similar cfDNA fragment end analysis approach that involves whole genome cfDNA sequencing (including any ctDNA), but with bioinformatic computational analysis targeting transcription factor binding site (TFBS) sequences. The goal of this approach is to determine TFBS accessibility in plasma samples taken from cancer patients and to identify TFBS DNA sequences with altered accessibility (Ulz et al., 2019). In this approach, plasma samples are taken from subjects, and cfDNA is extracted and amplified using a DNA library preparation method suitable for small DNA fragments less than 100 bp in length. This DNA library is sequenced using next generation sequencing methods. This sequencing data is used to identify cfDNA fragmentation patterns within genomic regions proximal to the TFBSs using bioinformatics methods. This analysis involves determining the nucleosome positioning profile of cfDNA fragments across the TFBSs and their flanking sequences within gene promoter sequences to determine whether the TFBSs were bound or not by transcription factors within the chromatin fragments that contain the cfDNA. The method is complex but can be summarized as follows:

[0014] If the cfDNA fragmentation pattern observed within the DNA sequence spanning the TFBS and flanking sequences in the genome shows a periodicity of approximately 200 bp, which is associated with alternating stronger protein-bound protection (at the central site of the nucleosome-bound position) from degradation with weaker protein-bound protection (at sites between nucleosomes where the DNA is unbound and unprotected), then it is presumed that the TFBS and flanking sequences were covered by nucleosomes within the chromatin fragments containing the cfDNA in the plasma sample.

[0015] If the presence of a cfDNA fragmentation pattern further indicates protein binding protection of the TFBS and its flanking sequences, but the nucleosome-associated periodicity is absent (or attenuated), this is associated with transcriptional regulatory protein binding at the TFBS and its flanking sequences, inferring that the TFBS are bound to one or more transcription factors and / or other regulatory proteins in the chromatin fragments that comprise the cfDNA in the plasma sample.

[0016] The cfDNA fragmentation patterns seen in healthy subjects generally correlate with those obtained in nuclease accessible site experiments in hematopoietic cells. Thus, transcription factor bound or nucleosome covered TFBS sequences in cfDNA correlate with transcription factors expressed or not expressed in hematopoietic cells. The patterns in cancer patients refer to a mixture of cell types in which TFBS may be transcription factor bound in cancer cell types and nucleosome bound in hematopoietic cell types. As most of the cfDNA is from hematopoietic cells and only a small amount is from cancer cells, the fragmentomics signal from cancer is small compared to the hematopoietic signal. However, fragmentomics bioinformatics methods have been developed to separate the small signal of transcription factor protected TFBS fragments present in ctDNA from the much larger periodic signal of superimposed nucleosomes present in hematopoietic-derived cfDNA components. This fragmentomics analysis showed that the mixed pattern included cfDNA TFBS sequences bound to transcription factors that are not expressed in hematopoietic cells but are expressed by cancer tissues.

[0017] Chromatin immunoprecipitation followed by sequencing of chromatin-associated DNA (ChIP-Seq) is an analytical technique used to map the genomic location of cellular chromatin proteins. A typical method involves extraction of chromatin from cells, followed by digestion of the chromatin into mononucleosomes or other chromatin fragments by physical disruption (e.g., sonication) or with nuclease enzymes that cleave DNA (e.g., DNase or micrococcal nuclease). The fragmented chromatin is then exposed to a solid support coated with an antibody that binds to a specific chromatin protein of interest, such as a specific modified histone. Chromatin fragments containing specific structures are adsorbed (immunoprecipitated) onto the solid phase. DNA associated with the adsorbed chromatin is then extracted from the solid phase and amplified by polymerase chain reaction (PCR). The amplified DNA fragment library is sequenced to determine the genomic location of the chromatin protein of interest. ChIP using antibodies against transcription factors has also been used to identify the genomic location of transcription factor binding sites (TFBS) of specific transcription factors, or to identify whether specific TFBSs are occupied by specific transcription factors in different cell types.

[0018] We have previously reported immunoassay tests for circulating cell-free nucleosomes containing specific epigenetic signals, including specific post-translational modifications, histone isoforms, modified nucleotides, and non-histone chromatin proteins, for the detection of cancer and other diseases (referenced in International Publication Nos. WO2005 / 019826, WO2013 / 030577, WO2013 / 030579, and WO2013 / 084002), as well as immunoassay tests for chromatin fragments containing transcription factor-bound DNA for the detection of cancer (referenced in International Publication No. WO2017 / 162755).

[0019] We report here a method with excellent analytical and clinical specificity and sensitivity for the isolation and direct analysis and measurement of circulating cell-free chromatin fragments containing one or more transcription factors along with associated DNA fragments. Isolation of transcription factor-DNA complexes from the vast majority of nucleosomal fragments simplifies the analysis and obviates the need to separate the signals of transcription factor-covered TFBS from the predominant periodic signal of nucleosomes. This method can be used in blood samples as a non-invasive or minimally invasive blood test for diseases including cancer, autoimmune diseases, and inflammatory diseases. Summary of the Invention

[0020] (Summary of the invention) In a first aspect of the present invention, there is provided a method for detecting cell-free chromatin fragments, comprising transcription factors and DNA fragments, in a body fluid sample obtained from a human or animal subject, comprising: (i) contacting the bodily fluid sample with a binding agent that binds the transcription factor; (ii) detecting or measuring the DNA fragment associated with the transcription factor; and (iii) using the presence or amount of the DNA fragment as a measure of the amount of cell-free chromatin fragment containing the transcription factor in the sample.

[0021] In a further aspect of the invention there is provided a method for detecting a disease in a human or animal subject comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds a transcription factor; (ii) detecting or measuring DNA associated with the transcription factor; and (iii) using the presence or amount of said DNA as an indicator of the presence of disease in said subject.

[0022] In a further aspect of the invention there is provided a method for detecting diseased tissue in a human or animal subject comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds a transcription factor; (ii) sequencing the DNA associated with the transcription factor; and (iii) using the presence of said transcription factor and the sequence of said associated DNA as a combined biomarker for determining tissue affected by said disease in said subject.

[0023] In a further aspect of the invention there is provided a method for assessing the suitability of an animal or human subject for a medical treatment comprising: (i) detecting, measuring, or sequencing DNA associated with cell-free chromatin fragments containing the transcription factor in a bodily fluid sample obtained from the subject; and (ii) using the level and / or sequence of the associated DNA detected in step (i) as a parameter for selecting an appropriate treatment for the subject.

[0024] In a further aspect of the invention there is provided a method for monitoring treatment of an animal or human subject comprising: (i) detecting, measuring, or sequencing DNA associated with cell-free chromatin fragments that contain the transcription factor in a bodily fluid sample obtained from the subject; (ii) repeating the step of detecting, measuring or sequencing DNA associated with cell-free chromatin fragments containing the transcription factor in a bodily fluid sample obtained from the subject one or more times; and (iii) using a change in the level and / or DNA sequence of the associated DNA detected in step (i) compared to step (ii) as a parameter for a change in the condition of the subject.

[0025] In a further aspect of the invention there is provided a kit for detecting cell-free chromatin fragments comprising a transcription factor and a DNA fragment as a combined biomarker, said kit comprising a ligand or binder for said transcription factor, optionally together with reagents for amplifying and / or sequencing DNA associated with said transcription factor, and / or a ligand or binder for nucleosomes, and / or instructions for using the kit according to the methods defined herein.

[0026] In a further aspect of the invention, there is provided a method of treating cancer in a subject in need thereof, comprising: (a) contacting a bodily fluid sample obtained from a human or animal subject with a binding agent that binds a transcription factor; (b) detecting, measuring, or sequencing DNA fragments associated with the transcription factor; and (c) using the presence or amount of the DNA fragment as an indication of the presence of cancer in the subject; and (d) administering a treatment if the subject is determined to have cancer in step (c).

[0027] 1. A method for detecting a disease in a human or animal fetus, comprising: (i) obtaining a body fluid sample from a pregnant human or animal subject; (ii) contacting the bodily fluid sample with a binding agent that binds a transcription factor; (iii) detecting, measuring, or sequencing DNA associated with the transcription factor; and (iv) using the presence, sequence, or amount of said DNA as an indicator of the presence of disease in the fetus. [Brief description of the drawings]

[0028] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]Figure 1: Illustration of the co-binding of various transcription factors at the promoter sites of surfactant protein B, thyroglobulin, thyroperoxidase, and thyrotropin receptor (TSH receptor) genes. CRE: cyclic adenosine monophosphate response element, GABP: GA binding protein, HNF-3: hepatocyte nuclear factor 3, NF-1: nuclear factor 1, PAX-8: paired box gene 8, Runx2: Runt-related transcription factor 2, TRα / RXR dimer: thyroid hormone receptor α / retinoid X receptor dimer, TTF-1: thyroid transcription factor 1 (also known as NK2 homeobox 1, NKX2-1), TTF-2: thyroid transcription factor 2.

[0029] [Diagram 2] Figure 2: An illustration of an example of a DNA loop structure of a transcription complex to illustrate the co-binding of some of the various regulatory proteins involved in the transcription complex, including but not limited to general transcription factors (GTFs), gene-specific transcription factors (TFs), cofactors, activators, repressors, mediators, DNA bending proteins, and RNA polymerases. Regulatory proteins bind to regulatory DNA sequences located proximal to the gene as well as to regulatory sequences distal to the gene, including promoter sequences, TATA box sequences, enhancer sequences, and repressor sequences. Other regulatory proteins (e.g., chromatin remodeling proteins) and other regulatory sequences are also possible.

[0030] [Diagram 3] Figure 3: Western blot analysis of recombinant mononucleosomes adsorbed onto magnetic beads coated with an antibody that binds to histone H3. The results demonstrate dose-dependent adsorption of mononucleosomes.

[0031] [Figure 4]Figure 4: Nucleosome ELISA results of human plasma samples and recombinant mononucleosome solutions after immunoprecipitation of nucleosomes using uncoated magnetic beads or magnetic beads coated with an antibody that binds to histone H3. The results demonstrate that in solution, both native human circulating nucleosomes and recombinant nucleosomes were unaffected by uncoated magnetic beads, but were quantitatively removed by immunoprecipitation using magnetic beads coated with an antibody that binds to histone H3.

[0032] [Diagram 5] FIG. 5: Levels of ERα measured in women diagnosed with ER-negative breast cancer (ER-BC), ovarian cancer, or ER-positive breast cancer (ER+BC) with an ER score of 7 or 8.

[0033] [Figure 6] Figure 6: Effect of washing magnetic polystyrene particles exposed to plasma samples obtained from cancer patients with regular wash buffer alone containing 0.1% Tween (0.1%) or strong wash buffer containing a detergent mixture totaling 1.2% detergent (1.2%). Non-specific IgG-coated particles showed a large reduction in background binding by using a strong detergent wash (lanes 4 and 5), without disrupting the specific antibody-bound proteins (a mixture of poly ADP-ribosylated (parylated) proteins) (lanes 6 and 7).

[0034] [Figure 7]Figure 7: Western blot analysis of chromatin fragments immunoprecipitated from four pooled cross-linked EDTA plasma samples from patients diagnosed with CRC by ChIP using mouse anti-CTCF antibodies immobilized on magnetic polystyrene beads and washed using a strong 1.2% detergent mix wash buffer. All four plasma samples showed a band at approximately 140 kD corresponding to CTCF protein (anti-CTCF; lanes 3, 5, 7, and 9). No band corresponding to CTCF was seen in a negative control experiment using a nonspecific mouse IgG (NS-IgG; lanes 2, 4, 6, and 8). This experiment demonstrated that CTCF protein could be isolated from the plasma samples and that the use of a strong wash buffer leads to a relatively pure CTCF extract from plasma.

[0035] [Figure 8] Figure 8: Electropherograms showing amplified adaptor-ligated cfDNA fragment library analysis obtained by ChIP of CTCF chromatin fragments in cross-linked EDTA plasma samples from patients. The sharp peak at approximately 140 bp represents adaptor dimers, so adaptor-ligated fragments of 175-220 bp represent cfDNA fragments of 35-80 bp (visible in the electropherogram). (a) The specific CTCF ChIP library contained small cfDNA fragments in the 35-80 bp range with a fluorescence peak of approximately 1000 FU. (b) The nonspecific control IgG library also contained small cfDNA fragments with a fluorescence peak of approximately 80 FU.

[0036] [Figure 9]Figure 9: Normalized coverage of 9780 published CTCF TFBS loci by transcription factor-bound cfDNA fragments (35-80 bp) or nucleosome-bound cfDNA fragments (135-155 bp or 156-180 bp). (a) Specific CTCF coverage by cfDNA sequence libraries obtained for CRC patients. (b) Non-specific coverage by cfDNA sequence libraries obtained from chromatin fragments non-specifically bound to mouse IgG-coated particles. The results show that peaks of specific cfDNA coverage resulting from plasma circulating CTCF-DNA complexes correlate with published CTCF TFBS loci. The periodic oscillatory coverage pattern predicted by nucleosome binding is minimal in the 5 kb span investigated. No cfDNA coverage peaks were observed at CTCF-bound loci in control samples.

[0037] [Figure 10] Figure 10: Normalized coverage of 1041 published CTCF TFBS loci occupied by CTCF in cancer cells but not in normal cells. Coverage is shown for transcription factor bound cfDNA fragments (35-80 bp) or nucleosome bound cfDNA fragments (135-155 bp or 156-180 bp). (a) CTCF occupancy of cancer-associated loci by cfDNA sequence libraries obtained for CRC patients. The results show coverage within the 35-80 bp size range, confirming CTCF occupancy of some or all of these 1041 sites, and thus indicating cancer in the subject from whom the sample was taken. (b) No CTCF occupancy peaks were observed in non-specific control experiments.

[0038] [Figure 11]Figure 11: Western blot analysis of chromatin fragments immunoprecipitated from eight cross-linked EDTA plasma samples by ChIP with mouse anti-AR antibody immobilized on magnetic polystyrene beads and washed with a strong 1.2% detergent mix wash buffer. All eight plasma samples showed a band of approximately 140 kD corresponding to the AR protein (S1-S8; lanes 2-9). The densest bands were observed in samples S1 and S2. Lane 10 represents a positive control using sheared chromatin from LnCAP prostate cancer cells.

[0039] [Figure 12] Figure 12: Electropherograms (S1-S8) showing amplified adaptor-ligated cfDNA fragment library analysis derived from ChIP of AR chromatin fragments in cross-linked EDTA plasma samples from eight prostate cancer patients. The sharp peak at approximately 140 bp represents adaptor dimers, so the adaptor-ligated fragments of 175-220 bp represent cfDNA fragments of 35-80 bp. Electropherograms of the negative control (ctrl) are also shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Detailed Description of the Invention Transcription factors are involved in cancer and account for approximately 20% of all known oncogenes (Lambert et al., 2018). We previously described the use of tissue-specific transcription factor-containing chromatin fragments as biomarkers in serum for the detection or diagnosis of cancer in subjects. The tissue specificity of the transcription factor can be used to indicate the tissue of origin of the cancer. For example, the transcription factor TTF-1 has been reported to be expressed in thyroid and lung tissues, but not in other tissues. Thus, the presence of circulating chromatin fragments containing TTF-1 indicates that the tissue of origin is lung or thyroid. We also described an immunoassay method for the measurement of circulating cell-free chromatin fragments containing transcription factors. The immunoassay includes a double antibody (or other binder) method, where one antibody binds to the transcription factor and another antibody binds to DNA associated with the transcription factor or to nucleosomal components contained in the chromatin fragment. In one embodiment, a binding agent that targets and binds a transcription factor is immobilized on a solid phase, and chromatin fragments containing the transcription factor are isolated (i.e., the chromatin fragments are immunoprecipitated). The isolated chromatin fragments are then detected using a second binding agent that binds to DNA. This immunoassay method is simple, low-cost, and non-invasive.

[0041] ChIP-Seq is a method typically applied to cellular chromatin extracts after fragmentation by enzymatic digestion with nucleases or by sonication. There are few reports of the application of the ChIP-Seq method in EDTA plasma. As the chromatin in plasma is already fragmented, nuclease digestion or sonication of the sample is not necessary. Reports of ChIP-Seq in plasma involve the isolation of histone proteins from EDTA plasma using anti-histone antibodies, followed by extraction, amplification, and sequencing of DNA fragments associated with histones (Deligezer et al., 2008; Mansson et al., 2021; Sadeh et al., 2021; Vad-Nielsen et al., 2020).

[0042] To the best of the authors' knowledge, no ChIP-Seq methods have been described in the literature for the direct isolation, analysis, or mapping of intact circulating transcription factor DNA chromatin fragments and associated TFBS DNA sequences. Instead, researchers in the field have developed indirect methods based on the analysis of DNA fragments.

[0043] Fragmentomics is one such indirect method, where deep sequencing of cfDNA extracted from EDTA plasma is analyzed by bioinformatics methods to identify DNA fragmentation patterns indicative of transcription factor-DNA binding in the original sample (Snyder et al., 2016; Ulz et al., 2019). It is an indirect method because the first step of fragmentomics is the extraction of the total DNA in the investigated sample, which necessarily involves the disruption of all transcription factor-DNA complexes present. This destroys all information directly linking all DNA fragments or sequences in the sample to all transcription factors or other chromatin proteins. TFBS occupancy is inferred from the presence of short cfDNA fragments (35-80 bp) of appropriate sequence in the extracted DNA library. However, the characteristic nature of the chromatin proteins bound to the DNA fragments (before DNA extraction) cannot be known, especially when many proteins are bound in close proximity to the site of interest as shown in Figures 1 and 2. Therefore, one drawback of the fragmentomenomics approach is that the binding of specific transcription factors to specific TFBSs can only be inferred and not determined.

[0044] Another recent indirect method is nucleosome ChIP-Seq in EDTA plasma to directly map cell-free nucleosome positions and use the nucleosome position data to indirectly infer the locations of transcription factors ( Sadeh et al., 2021 ).

[0045] Direct ChIP of transcription factor-DNA complexes has not been reported because of significant technical difficulties or obstacles that have not been addressed. These include (i) the recognition that some transcription factor-DNA complexes are stably associated in plasma, while others will dynamically associate in vivo and dissociate in blood or other body fluids, (ii) the recognition that the most common class of transcription factor-DNA complexes dissociate in EDTA plasma, but this can be prevented, (iii) cell nuclear extracts from cell or tissue material are relatively pure chromatin preparations that can be obtained in μg or mg quantities. In contrast, blood, serum, or plasma contain low levels of highly impure chromatin that is "contaminated" with high levels of other circulating proteins, and (iv) there are at least several hundred transcription factors, and a particular transcription factor-DNA complex will be only one of thousands of different transcription factor-DNA complexes present in the plasma. Second, the total fraction of transcription factor-DNA complexes in cfDNA is a small fraction of the total cfDNA (most of which contains nucleosomal fragments), and the fraction of cfDNA attributable to cancer cells is a small fraction of the total cfDNA. Thus, transcription factor-DNA complexes containing a particular transcription factor are a small fraction of a small fraction that is highly contaminated with other proteins and other substances. One consequence of this is that the specific signal generated in plasma by the transcription factor-DNA ChIP-Seq method is small (smaller than the background signal), making effective data analysis problematic.

[0046] We report here a method for the detection of circulating cell-free chromatin fragments containing transcription factor-DNA complexes with excellent analytical sensitivity and tissue specificity, which also broadens the scope of applicable transcription factors to include most or all transcription factors.

[0047] We also report the use of combination biomarkers consisting of chromatin fragments containing transcription factors combined with sequences of DNA fragments associated with said transcription factors for the detection of disease, which are furthermore highly tissue specific and can be used as biomarkers for cancer.

[0048] Analytical sensitivity is important for circulating cell-free chromatin fragments containing transcription factors, which occur at low levels close to or below the detection limit by immunoassays. The analytical detection limit of immunoassays will be in the picomolar range, depending on the design of the assay and the affinity of the binding agent (usually an antibody) used. However, the analytical sensitivity of polymerase chain reaction (PCR) detection of DNA is several orders of magnitude lower. Digital PCR can detect concentrations as low as a few molecules per sample. Thus, using PCR amplification methods to detect DNA associated with transcription factors, rather than using antibodies that bind to DNA (or nucleosomal epitopes), allows the detection of circulating chromatin fragments containing transcription factors at extremely low levels.

[0049] Similar to the increased sensitivity achieved by using PCR for detection, analysis of chromatin fragments containing transcription factors based on their associated DNA content also provides high analytical sensitivity by addressing a large pool of transcription factors that do not contain associated nucleosomes.

[0050] Thus, in a first aspect of the present invention, there is provided a method for detecting cell-free chromatin fragments, comprising transcription factors and DNA fragments, in a body fluid sample obtained from a human or animal subject, comprising: (i) contacting the bodily fluid sample with a binding agent that binds the transcription factor; (ii) detecting or measuring the DNA fragment associated with the transcription factor; and (iii) using the presence or amount of the DNA fragment as a measure of the amount of cell-free chromatin fragment containing the transcription factor in the sample.

[0051] In one embodiment, the antibody or other binding agent of the transcription factor used in step (i) is immobilized on a solid phase in order to isolate the transcription factor from the sample.

[0052] In one embodiment, the method comprises a step of isolating the transcription factor bound in step (i), i.e. isolating it from the remaining body fluid sample, prior to detecting the associated DNA fragment. For example, a wash buffer may be applied to the transcription factor in the sample bound to the (solid phase) binding agent in step (i) to remove the remaining sample that is not bound to the binding agent.

[0053] In one embodiment, in step (ii) a DNA fragment associated with the transcription factor is extracted from the transcription factor and the DNA fragment is detected, measured or sequenced.

[0054] In one embodiment, DNA is detected or measured using common DNA binding agents such as anti-DNA antibodies or DNA chelators or DNA intercalators, e.g., ethidium bromide and cyanine dyes such as SYBR Green and SYBR Gold.

[0055] In one embodiment, step (ii) comprises sequencing the DNA fragment associated with the transcription factor. Sequencing methods are well known in the art.

[0056] In some embodiments, the detection or measurement of the DNA fragments in step (ii) is performed by amplification of the DNA fragments, e.g. using quantitative PCR techniques, to determine the presence and / or amount of the DNA fragments. Thus, a further aspect of the invention provides a method for detecting cell-free chromatin fragments comprising a transcription factor and a DNA fragment in a human or animal subject, comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds a transcription factor; (ii) isolating DNA associated with the transcription factor; (iii) amplifying the DNA; and (iv) using the presence or amount of the DNA fragment as a measure of the presence or amount of cell-free chromatin fragments comprising the transcription factor in the sample.

[0057] In one embodiment, the amplified DNA is detected or measured using DNA hybridization techniques.

[0058] In a further embodiment, amplification of transcription factor-bound DNA fragments is performed following ligation of an adaptor oligonucleotide to the DNA fragment. The adaptor oligonucleotide may contain a primer sequence that facilitates amplification of the DNA fragment by PCR, and then the primer sequence may be added. Methods using adaptor oligonucleotides are well known in the art and are routinely used for library creation for next generation sequencing. Thus, in one embodiment of the present invention, a method for detecting cell-free chromatin fragments containing transcription factors and DNA fragments in a human or animal subject is provided, comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds a transcription factor; (ii) isolating DNA associated with the transcription factor; (iii) ligating an adaptor oligonucleotide to the isolated DNA; (iv) amplifying the DNA; and (v) using the presence or amount of the DNA fragment as a measure of the amount of cell-free chromatin fragment containing the transcription factor in the sample.

[0059] In one embodiment, the amplification of transcription factor-bound DNA fragments is performed using PCR primer oligonucleotides of specific sequence(s) designed for the amplification of DNA fragments containing the specific sequence(s). This embodiment facilitates the amplification of selected DNA fragments containing TFBS sequence(s) and / or flanking sequence(s). This embodiment is also rapid, low-cost, easily automated for high throughput, and can be performed in any PCR laboratory, and further enhances the tissue-of-origin specificity of healthy or diseased cfDNA by combining the binding tissue specificity of transcription factor expression with the specificity of identifying its binding location in the genome through the analysis of the TFBS sequence and / or flanking sequence of the associated DNA in the chromatin fragments. Thus, in one embodiment of the present invention, a method for detecting cell-free chromatin fragments containing transcription factors and DNA fragments in a human or animal subject, comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds a transcription factor; (ii) isolating DNA associated with the transcription factor; (iii) amplifying the DNA using sequence-specific PCR primer oligonucleotides; and (iv) using the presence or amount of the DNA fragment as a measure of the amount of cell-free chromatin fragment containing the transcription factor in the sample.

[0060] In one embodiment, the method comprises the step of extracting the DNA fragments associated with the transcription factor. In a further embodiment, the method comprises the step of amplifying the extracted DNA fragments. Thus, a further aspect of the invention provides a method for detecting cell-free chromatin fragments comprising a transcription factor and a DNA fragment in a body fluid sample obtained from a human or animal subject, comprising: (i) contacting the sample with a binding agent that binds the transcription factor; (ii) isolating the bound transcription factor; (iii) extracting DNA associated with the transcription factor; (iv) amplifying the extracted DNA; (v) detecting the amplified and extracted DNA; and (vi) using the presence or amount of said DNA as a measure of the amount of cell-free chromatin fragments containing said transcription factor in said sample.

[0061] In a preferred embodiment, the amplification of the DNA associated with the transcription factor is carried out by PCR.There are many PCR methods known in the art, including but not limited to quantitative PCR, real-time PCR, reverse transcriptase PCR, nested PCR, digital PCR, multiplex PCR, arbitrarily primed PCR, cold PCR (co-amplification PCR at low denaturation temperature).In some embodiments, the amplification method comprises DNA quantification.

[0062] In a further aspect of the invention there is provided a method for detecting a disease in a human or animal subject comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds a transcription factor; (ii) detecting or measuring the DNA associated with the transcription factor; and (iii) using the presence or amount of DNA as an indicator of the presence of disease in the subject.

[0063] In another aspect of the invention, there is provided a method for detecting a disease in a human or animal subject, comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds a transcription factor; (ii) isolating DNA associated with the transcription factor; (iii) contacting the DNA with a DNA binding agent; (iv) detecting the DNA binding agent; and (v) using the presence or amount of the DNA binding agent as an indicator of the presence and / or characteristic of disease in the subject.

[0064] Any DNA binding agent, including antibodies, may be suitable for use in the present invention. The DNA binding agent may be directly or indirectly (e.g., via a linker system such as biotin / avidin or glutathione) labeled with a detectable moiety, such as a fluorescent, enzymatic, or radioactive moiety.

[0065] In another aspect of the invention there is provided a method for determining the genomic TFBS positions occupied by a particular transcription factor (and therefore which genes are regulated) by detecting cell-free chromatin fragments containing the transcription factor and the bound DNA fragment, wherein the DNA fragment associated with the transcription factor is sequenced to determine the genomic position where the transcription factor is bound. Thus, in another aspect of the invention there is provided a method for determining the genomic position where a transcription factor is bound, comprising: (i) contacting the sample with a binding agent that binds the transcription factor; (ii) isolating the bound transcription factor; (iii) extracting DNA associated with the transcription factor; (iv) amplifying the extracted DNA; (v) sequencing the amplified extracted DNA; and (vi) using the sequence of the extracted DNA to determine the location of the TFBS on the genome.

[0066] The present invention finds particular use for analyzing small DNA fragments bound by transcription factors, usually in the size range of 35-80 bp. Thus, in one embodiment, the extracted DNA sequenced relates to DNA fragments comprising small DNA fragments, such as those less than about 100 bp, less than about 80 bp, in particular less than about 60 bp. It should be noted that these DNA fragment sizes relate to DNA fragments without or before adaptor ligation. In one embodiment, the extracted DNA sequenced comprises DNA fragments in the size range of less than 100 bp, such as 35-80 bp (without / before adaptor ligation). In one embodiment, the extracted DNA sequenced comprises multiple DNA size ranges, which are then compared, for example as shown in Figures 10 and 11.

[0067] In a preferred embodiment, the sample is a body fluid sample. In a further embodiment, the body fluid sample is a blood, serum, or plasma sample.

[0068] In a preferred embodiment, the binding agent used is an antibody that binds to a specific transcription factor. Thus, in one embodiment, the binding agent that binds to the transcription factor is an antibody or a fragment thereof (i.e., a binding fragment).

[0069] In a preferred embodiment, the antibody is immobilized on a solid phase, facilitating isolation of transcription factor-DNA complexes or chromatin fragments that are bound to the antibody.

[0070] The presence of both a transcription factor and a DNA fragment associated with a sequence known to match the transcription factor in a circulating chromatin fragment in vivo further confirms the distinctive identity of both the transcription factor and the DNA fragment. The combination of a transcription factor and the sequence of the DNA fragment associated with it is a powerful combination biomarker for the diagnosis or evaluation of a wide variety of disease conditions. Furthermore, since many transcription factors present in a healthy subject bind to different sets of TFBS in different tissues, identifying the TFBS location to which a transcription factor is bound via the DNA present in association with the transcription factor will identify the tissue of origin of the chromatin fragment. Furthermore, the same approach is also applied to disease conditions. Thus, the presence of a disease condition can be identified from the set of TFBSs bound to a commonly expressed transcription factor (even if the transcription factor itself is expressed in many or all tissues). For example, the commonly expressed transcription factor CTCF binds to over a thousand specific locations on the genome in immortalized cancer cells but not in other non-cancer cells (Wang et al., 2012; Liu et al., 2017). Thus, upon identification of the presence of circulating CTCF-DNA complexes, the associated DNA fragments are sequenced and, if found to have a sequence matching one of the cancer-specific TFBS locations to which CTCF binds, this identification is indicative of cancer disease in the subject from which the sample was taken. Thus, in a highly preferred embodiment of the present invention, there is provided a method for detecting the stage of disease in a subject by detecting, in a body fluid sample obtained from a human or animal subject, cell-free chromatin fragments comprising transcription factors and DNA fragments which together form a combined biomarker identifying transcription factors occupying genomic TFBS locations which correspond to a disease condition or a particular tissue: (i) contacting the sample with a binding agent that binds a transcription factor; (ii) isolating the bound transcription factor; (iii) extracting DNA associated with the transcription factor; (iv) amplifying the extracted DNA; (v) sequencing the amplified extracted DNA; and (vi) using the sequence of the associated DNA fragments as an indicator of the tissue of origin of the chromatin fragments or the stage of disease in the subject.

[0071] Determining the stage of disease in a subject can include, for example, detection, diagnosis, treatment selection, monitoring, or prognosis of the disease.

[0072] In one embodiment, the method includes using the transcription factor and the sequence of the associated DNA as a combined biomarker that indicates the presence of a disease in the subject. The term "biomarker" refers to a characteristic biological or organism-derived indicator of a process, event, or disease state. Biomarkers can be used in diagnostic methods, such as, for example, clinical screening and prognostic evaluation, as well as in monitoring treatment outcomes, identifying subjects most likely to respond to a particular therapeutic treatment, drug screening and drug development. Such biomarkers include, for example, the presence (e.g., sequence), level, concentration, or amount of DNA associated with a transcription factor. The reference herein to a "combined biomarker" refers to a biomarker that includes more than one biological or organism-derived indicator, such as a transcription factor and associated DNA, in particular the level, concentration, or amount of a transcription factor associated with one or more specific sequences of DNA.

[0073] Tissue specificity is important because most transcription factors do not have perfect (single cell type) expression specificity. The tissue specificity of immunoassays for circulating chromatin fragments containing transcription factors is limited by both the analytical specificity of the antibody used and the tissue specificity of the transcription factor or panel of transcription factors used. This tissue specificity can then be improved by combining a particular transcription factor component with the sequence of the cfDNA fragment to which it is bound.

[0074] The reason for the above is that transcription factors bind to different DNA sequences in the genome of different cells. Gene expression is controlled by transcription factors specifically binding to short TFBS DNA sequences, also called response elements or binding motifs. TFBSs are usually, but not always, located in gene promoter regions proximal to the transcription start site of the regulated gene. Transcription factors bind to TFBSs in a sequence-specific manner via DNA binding domains (DBDs). TFBS sequences are usually 5-15 bp long in the promoters of their target genes, and transcription factor proteins can usually bind to a similar set of DNA sequences with different binding affinities. The length of the DNA fragment associated with the circulating chromatin fragment containing the transcription factor varies depending on whether the fragment is also bound by transcription factors, cofactors, nucleosomes, or other chromatin proteins and contains DNA-protected sequences. Many such chromatin fragments have been reported to occur in the range of 35-80 bp (Snyder et al., 2016). Furthermore, we note that this is consistent with the size range of chromatin fragments generated by nuclease digestion of chromatin extracted from cancer patient cells, and that this range of small fragments, approximately 35–80 bp, represents a larger proportion of the total chromatin fragments than the nucleosome-associated fragments (Corces et al., 2018). We conclude that these associated DNA fragments are longer than typical DNA response elements, and therefore contain flanking DNA sequences. However, the size of the DNA fragments associated with nucleosomes is usually greater than 100 bp DNA. We therefore conclude that the range of 35–80 bp DNA fragments does not include intact nucleosome-associated DNA fragments.

[0075] Transcription factor response elements or TFBS sequences can occur repeatedly at many positions in the genome, and some transcription factors occur at thousands of positions. Thus, the same transcription factor may be bound at numerous positions in the chromatin of a single cell. This means that, in principle, the death of a single cell could generate many circulating chromatin fragments containing the same transcription factor.

[0076] Furthermore, transcription factors do not operate alone, but tend to work in concert with other transcription factors or cofactors, or other components necessary for the regulation of a particular gene. Thus, one transcription factor can bind to one response element in the promoters of many different genes, each in concert with a different transcription factor. Thus, the same TFBS sequence for the same transcription factor or the DNA flanking sequence around the response element will be different in the promoters of different genes, because it contains binding motifs for different combinations of transcription factors. This is true for all or most transcription factors.

[0077] Furthermore, the binding sequence of the response element itself may be degenerate, allowing the transcription factor to bind to a variety of different motif sequences. For example, the transcription factor TTF-1 is expressed in a tissue-specific manner in healthy lung tissue and healthy thyroid tissue. In the lung, two protein TTF-1 factors bind to the promoter region of the lung-specific surfactant protein B (SPB) gene. The DNA binding sequence or binding motif of TTF-1 in the promoter of SPB is: [ka] where A, C, G, and T are designations for the DNA bases adenine, cytosine, guanine, and thymine, respectively, and N is a designation for any of these bases. The broad consensus promoter DNA sequence surrounding TTF-1 binding is: [ka] (where (-64) is the designation for bp distance from the SPB transcription start site.) In the SPB promoter in lung tissue, TTF-1 binds cooperatively with the transcription factor hepatocyte nuclear factor 3 (HNF3) as shown in Figure 1 (Matys et al., 2006 and Bohinski et al., 1994).

[0078] In the thyroid gland, TTF-1 regulates a number of genes, including thyroglobulin, thyrotropin receptor, and thyroperoxidase. The consensus binding sequence for TTF-1 in the promoter region of the thyroglobulin gene is distinct from that in the lung. [ka] It has been reported that in the promoter of the thyroglobulin gene, TTF-1 binds in a cooperative manner with the TTF-2, PAX8, and Runx2 transcription factors, and that a wide sequence including the 50 bp flanking sequences at the 5' and 3' ends is [ka] Similarly, TTF-1 also binds to the promoter regions of the thyroid stimulating hormone receptor and thyroperoxidase genes, in each case in cooperation with different co-acting transcription factors. Thus, as shown in FIG. 1, not only are the DNA sequences surrounding the TTF-1 binding sites in the promoter sequences of genes regulated in thyroid tissue or in lung tissue different, but the cofactors associated with TTF-1, and therefore the surrounding DNA sequences, are also different for binding to different genes in the same tissue (Matys et al., 2006; Maenhaut et al., 2015). This demonstrates that the combination of knowledge of the circulating chromatin fragments containing TTF-1 together with the sequence of the DNA associated with the chromatin fragments is sufficient to identify the origin of the chromatin fragments, either lung or thyroid.

[0079] There are about 1000-3000 human transcription factors, each of which binds to a specific location in the genome, resulting in dynamic transcriptional changes that drive a vast array of cellular process pathways. The principles of the present invention have been described with respect to TTF-1 as an example. However, in principle, any transcription factor can be used in the method of the present invention. Even transcription factors that are ubiquitously expressed in many cell types and bind to discrete DNA sequences, such as Hox protein transcription factors, can co-operably bind to cofactors and specifically bind to different sequences to control different genes in different tissues (Merabet and Mann, 2016; Mann et al., 2009). This means that all or most transcription factors, together with their TFBS sequences (optionally including flanking sequences), can be used as combination biomarkers in the method of the present invention. For example, the estrogen receptor alpha (ERα) transcription factor binds to more than a thousand binding sites or estrogen response elements (EREs) in the human genome in cooperation with a combination of at least 60 other transcription factors at different genomic locations (Lin et al., 2007). Similarly, the androgen receptor (AR) binds to androgen response elements (AREs) associated with thousands of genes in cooperation with other co-acting transcription factors at thousands of distinct sequence loci. Thus, the method of the present invention can identify the tissue of origin of chromatin fragments containing ERα or AR through the sequence of the associated DNA, even if these transcription factors are expressed in multiple tissues.

[0080] Furthermore, when the genome-wide binding of transcription factors to DNA loci is reprogrammed in cancer and transcription factors are expressed, the TFBSs that they bind in cancer cells are different from those that they bind in healthy cells of the same tissue, so that the identification of chromatin fragments containing transcription factors in the circulation, combined with sequence data of the associated DNA fragments, allows both the identification of subjects with cancer and the identification of the cancer type, e.g., prostate or lung cancer (Pomerantz et al., 2015). This is possible because chromatin is remodeled during tumorigenesis, which includes the upregulation of tumor-associated proteins via remodeled transcription factor binding patterns in cancer cells. Thus, the expression of many transcription factors is upregulated in cancer cells. This is a widespread phenomenon, but can be illustrated here with some non-limiting examples. For example, the well-known cancer-associated transcription factors c-Myc and p53 are upregulated in most cancers. The binding site sequences bound by AR are highly altered in prostate cancer (Pomerantz et al., 2015). Similarly, epithelial-mesenchymal transition (EMT) in cancer cells is associated with metastasis and therapeutic resistance and involves upregulation of Jun / Fos family transcription factors, including Fosll, Fosb, Fos, and Junb. ETS (E26 transformation specific) family transcription factors and Runxl, Tead, and Nfkb transcription factors have also been found to be highly enriched in open chromatin of tumor cells. In addition, p63, Klf, Grhl, and Cepba have been reported to be upregulated in tumor cells and their binding sites are enriched in open chromatin regions. Klf5 and p63 transcription factors are associated with carcinomas and act as drivers in lung and head and neck carcinomas. EMT-related transcription factors further include bHLH, Runx, Nfat, Tbx1, Tcf7I1, and Smad2 (Latil et al., 2017).

[0081] Transcriptional control of eukaryotic genes involves multiple regulatory proteins bound to multiple regulatory DNA sequences, located both proximal to the transcription start site (TSS) of the gene and distal to the TSS in the genome of the transcription complex, as shown, for example, in FIG. 2. Distal regulatory sequences in the DNA may be located hundreds to more than a million bases away from the TSS, or even further away. Transcription complexes usually contain DNA loops, which may contain DNA bending proteins, in which case the more distal regulatory sequences, as well as the regulatory proteins bound to them, come into contact with proteins bound to the regulatory sequences proximal to the TSS, as also shown, for example, in FIG. 2. TATA boxes are so named because they contain a repeat sequence of thymine / adenine nucleotides that binds the general transcription factors required for transcription. In addition, gene-specific transcription factors (e.g., those required for expression of surfactant protein B, thyroglobulin, thyroperoxidase, and TSH receptor genes, as shown in FIG. 1) are also required for expression of certain genes. In addition, numerous other proteins are also required, including, but not limited to, cofactors, mediators, activators, coactivators, repressors, co-repressors, chromatin remodeling proteins, DNA bending proteins, insulators, RNA polymerase components, elongation factors, chromatin remodeling factors, STAT components or cytokine or cytokine-associated factors bound to STAT components, upstream binding factors (UBFs), or other components associated with such gene regulatory or transcription complexes. Such complexes may also contain multiple lengths of nucleosome-protected DNA. Transcription complexes can promote stable and abundant transcription. Thus, circulating chromatin fragments from healthy and / or diseased sources may contain large protein / DNA complexes containing multiple proteins that may be resistant to nuclease activity. As shown in FIG. 2, some large transcription complexes containing proximal and distal regulatory sequences have been named super-enhancers. Super-enhancers are large clusters with high levels of transcription factor binding that mediate the expression of genes involved in controlling characteristic aspects of the cell.Super-enhancers are also central to stimulating oncogene transcription in cancer. Cancer cells acquire super-enhancers, and cancer phenotypes depend on aberrant transcription driven by super-enhancers. Thus, the methods described herein provide a method to identify the cellular origin of chromatin fragments, including cancer-initiating cells, by detecting the presence of chromatin fragments that contain all or part of a super-enhancer complex and / or a combination of cfDNA fragment sequences that correspond to proximal and distal regulatory sequences of a super-enhancer. We also hypothesize that super-enhancer complexes, based on their properties, are more likely to contain stably bound, rather than transiently bound, transcription factors.

[0082] The DNA loops in such chromatin fragments from the transcription complex may in principle be intact or may be digested at one or more positions to generate either (i) two circular chromatin fragments corresponding to the proximal and distal regulatory sequences, or (ii) a large chromatin fragment containing the two DNA fragments. Thus, cfDNA may contain small DNA fragments corresponding to both the proximal and distal regulatory sequences of a gene.

[0083] In a further aspect of the invention there is provided a method for detecting a disease in a human or animal subject comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds a transcription factor; (ii) determining the sequence of one or more DNA fragments associated with the transcription factor; and (iii) using the presence of said transcription factor and the sequence of said associated DNA as a combined biomarker for determining the presence and / or characteristic of said disease in said subject.

[0084] It is understood that any non-histone chromatin protein that binds to DNA and whose cfDNA binding patterns differ between healthy and diseased subjects is suitable for use in the methods of the invention, including not only transcription factors, but other non-histone chromatin proteins including chromatin modifying proteins, genetic and epigenetic read, write, and delete proteins, proteins involved in RNA transcription (e.g., RNA polymerase molecules), and architectural or structural chromatin proteins (e.g., DNA bending proteins).

[0085] Thus, in a further aspect of the invention there is provided a method for detecting a disease in a human or animal subject, comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds non-histone chromatin proteins; (ii) determining the sequence of one or more DNA fragments associated with the non-histone chromatin protein; and (iii) using the presence of the non-histone chromatin protein and the sequence of the associated DNA as a combined biomarker for determining the presence and / or characteristic of the disease in the subject.

[0086] In one preferred embodiment, the non-histone chromatin protein is an RNA polymerase, in particular RNA polymerase II. RNA polymerase II is a DNA-binding enzyme responsible for transcribing the DNA sequence of a gene to generate an RNA copy. The RNA copy may be a messenger RNA (mRNA) molecule that directs protein production by the ribosome, but it may also be a non-coding RNA (ncRNA) molecule that is not translated into a protein. Thus, the presence of RNA polymerase II in a circulating chromatin fragment indicates that the fragment originates from a gene that was active in its developing cell. Thus, a library of DNA fragment sequences derived from chromatin fragments associated with RNA polymerase II provides a library of active and dynamic genes present in the sample. In healthy individuals, this library represents the majority of active genes present in hematopoietic tissues. do In diseased patients, the library further includes genes active in the tissue(s) affected by the disease. This is true for any tissue affected by the disease. For example, genes active in liver cells or kidney cells may be represented in an RNA polymerase II library made from samples taken from patients with liver disease or kidney disease, but such genes are not present in libraries from healthy individuals. Similarly, genes upregulated in cancer may be represented in an RNA polymerase II library made from samples taken from patients with cancer disease, but such genes are not present in libraries from healthy individuals. The use of RNA polymerase II in this aspect of the invention allows the identification of active dynamic genes represented in the sample. This allows the detection of cancer disease and the determination of the tissue(s) affected by cancer.

[0087] Accordingly, a further aspect of the present invention relates to a method for detecting a disease in a human or animal subject, comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds RNA polymerase; (ii) determining the sequence of one or more DNA fragments associated with the RNA polymerase; and (iii) using the sequence of the DNA fragment associated with the RNA polymerase as a biomarker for determining the presence and / or characteristics of the disease in the subject.

[0088] In one embodiment, the disease is selected from cancer, an autoimmune disease, or an inflammatory disease. In a further embodiment, the disease is cancer. In a further embodiment, the autoimmune disease is selected from systemic lupus erythematosus (SLE) and rheumatoid arthritis. In a further embodiment, the inflammatory disease is selected from Crohn's disease, colitis, endometriosis, and chronic obstructive pulmonary disorder (COPD).

[0089] In a preferred embodiment, the disease is cancer. In a further embodiment, the cancer is selected from breast cancer, bladder cancer, colorectal cancer, skin cancer (such as melanoma), ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, intestinal cancer, liver cancer, endometrial cancer, lymphoma, oral cancer, head and neck cancer, leukemia, and osteosarcoma.

[0090] In further embodiments, the disease is a fetal disease or condition. For example, it is well known in the art that chromatin fragments of fetal origin, including Y chromosome DNA sequences resulting from (XY) male fetuses, circulate in the blood of pregnant animals and human mothers (XX). It has been reported that circulating cfDNA in pregnant subjects contains both cfDNA fragments of the expected length of nucleosome-protected DNA fragments (about 160 bp) and short cfDNA fragments in the range of more than 50 bp. Furthermore, it has been reported that maternal cfDNA fragments of less than 140 bp in length are enriched for cfDNA of fetal origin (Hu et al., 2019). Thus, the method of the present invention is applicable not only to the disease stage of the subject from which the sample was taken, but also to prenatal testing or testing of fetal conditions in maternal blood samples.

[0091] Accordingly, a further aspect of the present invention provides a method for detecting a disease in a human or animal fetus, comprising: (i) obtaining a body fluid sample from a pregnant human or animal subject; (ii) contacting the bodily fluid sample with a binding agent that binds a transcription factor; (iii) detecting, measuring, or sequencing DNA associated with the transcription factor; and (iv) using the presence, sequence, or amount of said DNA as an indicator of the presence of disease in said fetus.

[0092] In a further aspect of the invention there is provided a method for detecting diseased tissue in a human or animal subject comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds a transcription factor; (ii) determining the DNA sequence of the DNA associated with the transcription factor or chromatin fragment; and (iii) using said transcription factor / DNA sequence combination biomarker as an indicator of diseased tissue in said subject.

[0093] In one preferred embodiment, the disease is cancer. In another embodiment, the tissue affected by the disease is the organ where the disease originates, such as an organ where cancer originates.

[0094] In another aspect of the invention, there is provided a method for detecting a disease in a human or animal subject, comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds a transcription factor; (ii) isolating DNA associated with the transcription factor; (iii) amplifying the isolated DNA by PCR; (iv) determining the sequence of the amplified DNA; and (v) using the presence of said transcription factor and the sequence of said associated DNA as a combined biomarker for determining the presence and / or characteristic of disease in said subject.

[0095] It will also be apparent to one skilled in the art that multiple sequences corresponding to the various loci bound by a particular transcription factor can be obtained and data on the various sequences can be combined to determine the characteristics of the disease and / or tissues affected by the disease.

[0096] In another aspect of the invention, there is provided a method for detecting a disease in a human or animal subject, comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds a transcription factor; (ii) isolating DNA associated with the transcription factor; (iii) amplifying the isolated DNA by PCR, e.g., using sequence-specific primers; (iv) detecting the amplified DNA; and (v) using the presence, amount and / or sequence of said amplified DNA as an indicator of the presence and / or characteristic of disease in said subject.

[0097] In one embodiment, amplification of the isolated DNA fragment associated with the transcription factor is performed following ligation of an adaptor oligonucleotide to said DNA fragment. Thus, in one embodiment of the invention, there is provided a method for detecting a disease in a human or animal subject, comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds a transcription factor; (ii) isolating DNA associated with the transcription factor; (iii) ligating an adaptor oligonucleotide to the isolated DNA; (iv) amplifying the DNA; and (v) using the presence, amount and / or sequence of said DNA fragments as an indicator of the presence and / or characteristic of disease in said subject.

[0098] In another aspect of the invention, there is provided a method for detecting a disease in a human or animal subject, comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds a transcription factor; (ii) isolating DNA associated with the transcription factor; (iii) amplifying the isolated DNA using sequence-specific primer oligonucleotides; (iv) detecting the amplified DNA; and (v) using the presence, amount and / or sequence of the amplified DNA as an indicator of the presence and / or characteristic of disease in the subject.

[0099] This embodiment takes advantage of the tissue specificity of the transcription factor / DNA sequence combination biomarkers of the invention, while avoiding the creation of DNA fragment adapter libraries and next generation DNA sequencing by PCR amplification of selected DNA fragments containing the TFBS sequence(s) of interest and / or flanking sequence(s) of interest of the biomarkers. The method is rapid, low cost, easily automated for high throughput, and can be performed in any PCR laboratory.

[0100] The DNA sequences isolated in step (i) or (ii) can be amplified by any method known in the art. In some embodiments, the isolated DNA is amplified using PCR techniques with adapters ligated to the DNA fragments. In other embodiments, PCR primers are used to amplify the DNA. The primers may be designed to amplify all the DNA sequences isolated in step (i) or (ii) or may be designed to amplify specific DNA sequences associated with the sequences of the response elements of the transcription factors, optionally including flanking regions.

[0101] In another aspect of the invention, there is provided a method for detecting a disease in a human or animal subject, comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds a transcription factor; (ii) isolating (and optionally amplifying) DNA associated with the transcription factor; (iii) detecting the DNA by hybridization; and (iv) using the presence, amount and / or sequence of hybridized DNA as an indicator of the presence and / or characteristic of disease in the subject.

[0102] This embodiment takes advantage of the tissue specificity of the transcription factor / DNA sequence combination biomarkers of the present invention, while avoiding expensive next generation DNA sequencing by selective DNA hybridization of DNA fragments containing the TFBS sequence(s) and / or flanking sequence(s). The method is low cost and can be performed in any PCR laboratory.

[0103] In a preferred embodiment, the isolated DNA is amplified prior to hybridization. In a preferred embodiment, the hybridization method is a DNA microarray method (also known as a DNA chip method).

[0104] The methods of the invention can also be used to measure combination biomarkers of transcription factors and sequence-associated DNA.

[0105] (Selection of transcription factors) The control of gene transcription in eukaryotes is highly complex and can involve bending and looping of DNA to bring together multiple regulatory DNA sequences bound by multiple regulatory proteins in a regulatory transcription complex, for example as shown in Figure 2. Thus, the term "transcription factor" as used herein means a regulatory protein that binds directly or indirectly to a gene regulatory sequence in the genome to control the transcription of the gene, including but not limited to general transcription factors and specific transcription factors associated with the control of a particular gene(s), as well as enhancers, co-enhancers, repressors, co-repressors, mediators, activators, co-activators, repressors, co-repressors, chromatin remodeling proteins, DNA bending proteins, insulators, RNA polymerase components, elongation factors, STAT components, cytokine factors or cytokine-associated factors bound to STAT moieties, UBFs, or other components associated with such gene regulation or transcription complexes. Similarly, the term "transcription factor binding site" (TFBS) as used herein means DNA binding sites for regulatory proteins involved in the transcriptional control of genes, including but not limited to distal and proximal enhancer and repressor sequences, such as those shown in FIG. 2.

[0106] It is well known that the expression of transcription factors is altered in disease. Thus, the method of the present invention also relates to transcription factors whose expression is upregulated in disease and / or that are inappropriately expressed in diseased tissue, e.g., cancer tissue, when they are not normally highly expressed in their (healthy) tissue. Thus, the level of transcription factors present in a body fluid sample can be used as a biomarker of disease.

[0107] It is also known that TFBS occupancy profiles by transcription factors vary in different cell types and diseases (Wang et al., 2012). Therefore, TFBS occupancy profiles by transcription factors present in body fluid samples can be used as biomarkers for diseases.

[0108] Chromatin fragments present in the circulatory system of healthy subjects are predominantly of hematopoietic origin, and therefore the methods of the invention also relate to detecting the inappropriate presence of chromatin fragments that contain transcription factors together with associated DNA that are not normally expressed in hematopoietic tissues (but may be expressed in non-hematopoietic tissues).

[0109] For example, many cancer diseases originate from epithelial tissues. Epithelial GRHL2 transcription factor is expressed in many epithelial tissues and many cancer diseases originating from epithelial tissues, but not in hematopoietic tissues. The presence of GRHL2 in the circulation indicates the presence of cancer of epithelial origin, for example, colorectal cancer, prostate cancer, lung cancer, or breast cancer. Thus, the method of the present invention can be used not only to detect the presence of cancer itself, but also to identify the organ of origin of cancer using a combination of lineage-specific transcription factors and / or sequences of DNA associated with lineage-specific transcription factors. Thus, any transcription factor can be utilized in the method of the present invention. In a preferred embodiment, the level of chromatin fragments containing the selected transcription factor is elevated in the body fluids of diseased subjects (above the levels seen in other subjects), in partial or total tissues, and / or in a disease-specific manner, and / or has multiple response elements in its genome.

[0110] Thus, in one embodiment, the transcription factor is disease specific (i.e., levels of circulating chromatin fragments containing the transcription factor are upregulated in disease). In one embodiment, the transcription factor is tissue specific. In one embodiment, the transcription factor binds at more than one location in the genome, for example at more than 5, more than 10, more than 100, more than 1000, or more than 10,000 locations in the genome. Some transcription factor binding locations are occupied in some tissue types but not in other tissues. Some transcription factor binding locations are occupied in diseased cells but not in healthy cells of the same tissue.

[0111] Transcription factors may be classified by binding domain (see, e.g., Vaquerizas et al., 2009, incorporated herein by reference). In one embodiment, the transcription factor comprises a DNA binding domain selected from a homeodomain, HLH, bZip, NHR, forkhead, P53, HMG, ETS, IPT / TIG, POU, MAD, SAND, IRF, TDP, DM, heat shock, STAT, CP2, RFX, AP2, or zinc finger (e.g., zinc finger C2H2 or zinc finger GATA) binding domain. In one embodiment, the transcription factor comprises a non-zinc finger DNA binding domain.

[0112] Suitable transcription factors can be determined experimentally, for example, classical nuclease accessible site mapping methods may be used to identify transcription factors of interest in tissue(s) of interest. In a typical experiment, chromatin is extracted from cells of interest (e.g., cancer cells, healthy cells of the same tissue, and hematopoietic cells) and digested with appropriate nucleases. The chromatin fragments generated by digestion are exposed to an antibody that binds the transcription factor, and the DNA fragments bound by the antibody are isolated and sequenced to identify the TFBS sequence(s) (optionally including flanking sequences) bound by the transcription factor. The results can be used to select transcription factors for use in the present invention. For example, transcription factors and transcription factor / TFBS ​​(optionally including flanking sequences) combinations that are elevated in diseased cells but low or absent in hematopoietic cells can be used in the methods of the present invention. Classical nuclease accessibility methods have been recently improved and the art now includes, for example, the CUT & RUN method and other methods that are easier to perform and provide improved results (Skene and Henikoff, 2017). Any such method would be suitable for use in identifying transcription factors suitable for use in the present invention.

[0113] Many such and similar experiments have been performed, and therefore suitable transcription factors are available in the art. There are many publications on transcription factors and cancer that list transcription factors for use in the methods of the present invention. For example, Lambert et al., 2018, lists 294 known oncogenic transcription factors and regulators. Gurel et al., 2010, lists the transcription factor NKX3.1 as a prostate cancer marker. Darnell et al., 2002, lists STAT3, 5, STAT-STAT, GR, IRF, TCF / LEF, β-catenin, NF- ΚB, NOTCH (NICD), GLI, c-JUN, bZip proteins (including c-JUN, JUNB, JUND, c-FOS, FRA, ATF, and CREB-CREM families), cEBP family, ETS proteins, and MAD-box family. Vaquerizas et al., 2009, describes a number of tissue-specific transcription factors that can be used in the methods of the invention. Ulz et al., 2019, describes transcription factors such as epithelial transcription factor GRHL2, which is present in many cancer types but not in hematological tissues, as well as AR (androgen receptor), NKX3-1, and HOXB13. Corces et al., 2018 described many cancer- and tissue-specific transcription factors, including NR5A1, TP63, GRHL1, FOXA1, GATA3, NFIC, CDX2, RFX2, ASCL1, PAX2, HNF1A, NKX2.A, PHOX2B, DRGX, HOXB13, AR, MITF, HNF4, and POU5F1. Using ChIP-Seq, Wang et al., 2012 identified 77,811 distinct binding sites for the transcription factor CTCF in 19 different cell types, including 7 immortal cancer cell lines and 12 normal cell types. Of these 77,811 CTCF TFBS, 1236 sites were found to be differentially occupied in cancer cells. Occupancy of 195 sites was found to occur in normal cell types but not in cancer cells. Occupancy of the 1041 site was observed to occur in cancer cells, but not in normal cell types (Liu et al., 2017). The discovery of cfDNA fragments associated with CTCF, corresponding to cancer-specific TFBS in body fluids by ChIP-Seq, indicates the presence of cancer disease in the subjects studied and in this manner can be used as a biomarker. These documents are incorporated herein by reference.

[0114] Transcription factors suitable for use in the methods of the invention may also be selected using various transcription factor, cancer, and genomic databases, including, for example, the ENSEMBL database, which provides annotated genomic sequences for many species, including humans, the Encyclopedia of DNA Elements or (ENCODE) database (https: / / www.encodeproject.org), The Transcription Factor (TRANSFAC) database (Matys et al., 2006), The Gene Transcription Regulation Database (GTRD) version 18.01 (http: / / gtrd.biouml.org), the Human Transcription Factors database version 1.01 (http: / / humantfs.ccbr.utoronto.ca), the NIH Genomics Data Commons database (https: / / gdc.cancer.gov), The Cancer Genome Atlas (http: / / cancer.gov), and the NIH Genomics Data Commons database (http: / / cancer.gov). These include the Human Protein Atlas (TCGA) (https: / / www.cancer.gov / about-nci / organization / ccg / research / structural-genomics / tcga), the UCSC Xena browser (https: / / atacseq.xenahubs.net), and The Human Protein Atlas database (https: / / www.proteinatlas.org), which provides data on healthy tissues in which transcription factors are expressed, including their expression in cancer diseases, as well as other databases.

[0115] The use of these databases to characterize transcription factors and associated TFBS and flanking sequences for use in the methods of the invention can be illustrated by reference to some of these databases by way of example. The TRANSFAC database provides data for thousands of human and other eukaryotic transcription factors. Details provided for each transcription factor include the number of TFBSs it binds in the genome, a list of genes whose transcription it controls, the sequences and genomic locations of the TFBSs associated with each regulated gene, details of other transcription factors that act in a co-operative manner with it to control transcription, consensus TFBS DNA sequences, details of the DBD, and cancer association. The use of this data in the context of the invention is illustrated below for the transcription factors CDX2 and c-JUN for illustrative purposes. The TRANSFAC database lists 48 human CDX2 TFBSs that control 26 specific genes. The CDX2 TFBS sequences are provided along with their genomic locations and the genes regulated by each. The flanking sequences for each CDX2 TFBS can be determined by reference to the ENSEMBL human genome database for the sequences at each genomic location. A consensus CDX2 TFBS sequence is also provided. Similarly, the TRANSFAC database lists 265 human c-JUN TFBSs that control 166 specific genes. The c-JUN TFBS sequences are provided as well as their genomic locations and the genes controlled by each. The flanking sequences for each c-JUN TFBS can be determined by reference to the ENSEMBL human genome database for the sequence at each genomic location. A consensus c-JUN TFBS sequence is also provided.

[0116] Thus, transcription factors and / or TFBSs may be selected experimentally or from the literature and / or databases, such as the Human Protein Atlas database, to be useful in the methods of the invention. Transcription factors can be characterized in terms of (i) the healthy and diseased tissues in which they are expressed, (ii) the genes they regulate in those cells or tissues, (iii) the TFBS sequences (optionally including flanking sequences) they bind in those tissues, and (iv) the other factors they co-act with by co-binding on the TFBSs to regulate transcription. This characterization can be used to identify healthy or diseased tissues or cells of origin of chromatin fragments and / or transcription factor-associated cfDNA fragments in a body fluid sample, by the methods described herein.

[0117] Similarly, experimental data regarding chromatin fragments and / or cfDNA sequences in bodily fluid samples can be interpreted using these databases to identify all or part of the TFBS sequences contained in the cfDNA fragments, optionally including flanking sequences, which can then be used to identify the tissue or cell of origin of the cfDNA fragments.

[0118] There are currently three major groups of transcription factors that are recognized to be of particular importance in relation to cancer. The first group is the nuclear hormone receptor group, which includes estrogen receptors, androgen receptors, progesterone receptors, glucocorticoid receptors, thyroid receptors, and retinoic acid receptors. The nuclear hormone receptor group of transcription factors are cell surface receptors that can be viewed as inactive or latent transcription factors that can be activated by ligand binding. For example, estrogen receptors are activated by binding with estrogen. As a result of ligand binding, nuclear hormone receptors translocate to the nucleus, where they bind to target DNA sequences (e.g., estrogen receptors bind to estrogen response elements), and up-regulate or down-regulate genes associated with the DNA target sequences (e.g., estrogen-regulated genes).

[0119] The second group of transcription factors are the signal transducers and activators of transcription (STATs), known to be important in the initiation and development of cancer. These are latent cytoplasmic transcription factors that can be activated by a wide variety of molecular triggers in the cytoplasm and / or at the cell surface. STAT activation is usually associated with a cascade of biochemical events in the cytoplasm, such as kinase reactions, proteolytic reactions, and protein-protein interactions that result in the entry of proteins or protein complexes into the nucleus, and control the transcription of target genes. In many cases, the biochemical cascade that leads to the activation of transcription is triggered by receptor binding of a ligand at the cell surface, including, for example, binding of a cytokine component by a cytokine receptor, or binding of a growth factor, such as epidermal growth factor or platelet-derived growth factor, by a growth factor receptor, or by binding of a peptide or protein to a G protein-coupled receptor.

[0120] A third group of transcription factors important in cancer are the resident nuclear proteins, whose transcriptional effects are usually activated by a cascade of biochemical events involving serine kinase reactions. There are hundreds of serine kinase components and hundreds of nuclear proteins that are targets of serine kinases.

[0121] It will be apparent to one of skill in the art that cell-free chromatin fragments comprising (i.e., including or containing) any transcription factor involved in the initiation, development or maintenance of cancer, such as the three groups of transcription factors listed above, will be useful in the methods of the present invention. Some transcription factors or families of transcription factors known to play a role in cancer or known to be elevated in cancer diseases include, for example, but are not limited to, STATs, particularly STAT3, STAT5 and STAT-STAT dimer components, NF-κB, β-catenin, γ-catenin, Notch and notch intracellular domain (NICD), GLI, c-JUN, JUNB, JUND, c-FOS, FRA, ATF, CREB-CREM, cEBP, ETS, MYC, N-MYC, MAX, E2F, interferon regulatory factors (IRFs), T cell factors (TCFs), lymphocyte enhancer factors (LEFs), EN2, GATA3, CDX2, PAX8, WT1, NKX3.1, P63 (TP63), or P40, and helix-loop-helix proteins (Darnell, 2002). All such transcription factors may be useful in the methods of the invention.

[0122] Many transcription factors have been found to be lineage specific and associated with particular tissues and may therefore be considered tissue-specific transcription factors, i.e., transcription factors that are always or normally expressed in certain tissues or cancers, while being little or never expressed in other tissues or cancers. Tissue-specific transcription factors may be used in the methods of the invention, where combinational detection of associated DNA provides enhanced specificity and / or sensitivity.

[0123] Thyroid transcription factor 1 (TTF-1) is selectively expressed during embryonic development in the thyroid, diencephalon, and respiratory epithelium. TTF-1 is expressed in tissue samples from neuroendocrine and non-neuroendocrine lung cancers, but its expression frequency varies significantly between histological subtypes. Thus, the method of the present invention can also be used to identify cancer types and subtypes through the measurement of chromatin fragments containing transcription factors and the sequences of their associated DNA.

[0124] PAX8 is a transcription factor involved in the embryonic development of the thyroid gland, kidney, and Mullerian system. PAX8 shows high levels of expression in tissue samples taken from mucinous non-producing ovarian carcinoma, serous carcinoma, endometrioid carcinoma, clear cell carcinoma, and transitional cell carcinoma. PAX8 is also expressed in endometrioid adenocarcinoma, uterine serous carcinoma, endometrial clear cell carcinoma, and ductal and lobular breast carcinoma tissue.

[0125] CDX2 is a lineage-specific transcription factor that plays a key role in controlling the proliferation and differentiation of intestinal epithelial cells and is expressed in almost all colorectal adenocarcinoma tissue samples.

[0126] NKX3.1 is essential for normal prostate development and is a known marker expressed in almost all prostate cancers.

[0127] GATA3 is transcriptionally active as early as the fourth week of human pregnancy. GATA3 is highly expressed in samples from breast cancer, particularly in estrogen receptor-positive breast cancer tissue samples, as well as in tissue samples from urothelial and transitional cell carcinomas.

[0128] WT1 plays an important role in embryonic development. WT1 is an excellent marker for ovarian cancer tissues and is expressed to a very limited extent in healthy adult tissues.

[0129] EN2 plays a role in embryonic development and is expressed in some cancers but is barely expressed in healthy adult tissues. Its presence in urine has been used as the basis for a urine test to detect prostate cancer.

[0130] Other transcription factors may also be used in the method of the present invention. For example, UBF is a transcription factor that binds to ribosomal RNA gene promoters and activates RNA polymerase I-mediated transcription. It is known that UBF expression is elevated in some cancer tissues. Many other such examples undoubtedly exist and are suitable transcription factors for use in the method of the present invention. Furthermore, RNA polymerase I and RNA polymerase III are also elevated in cancer. These components are responsible for the transcription of tRNA and ribosomal RNA genes, providing the cellular machinery required for increased and accelerated protein production, proliferation and cell replication characteristic of cancer cells and tissues. In a further embodiment of the present invention, a method is provided for the detection or measurement of cell-free chromatin fragments containing UBF, RNA polymerase I or RNA polymerase III.

[0131] In another embodiment, the transcription factor is not a tissue-specific transcription factor. The method of the present invention can also detect commonly expressed transcription factors, i.e., transcription factors expressed in more than 5, more than 10, more than 15, more than 20, or more than 30 tissue types. By combining detection with the sequence of associated DNA (i.e., combination biomarkers), the method of the present invention can detect commonly expressed transcription factors to provide clinically useful results. Nuclear hormone receptor transcription factors are examples. As explained above, CTCF is also a further example investigated herein.

[0132] Transcription factors bind to their DNA target sequences in a highly co-operative manner with many other factors, including other transcription factors, cofactors, coactivators, corepressors, RNA polymerase components, elongation factors, chromatin remodeling factors, mediators, STAT components, UBFs, and others. This means that the circulating transcription factors detected by the present invention may include other moieties as part of a larger gene regulatory complex, including any or all of the following: nucleosomes associated with DNA, nuclear hormone receptors, steroids or other hormones bound to nuclear hormone receptors, other transcription factors, cofactors, coactivators, corepressors, RNA polymerase components, elongation factors, chromatin remodeling factors, mediators, STAT components or cytokine factors or cytokine-associated factors bound to STAT components, upstream binding factors (UBFs), or any other components associated with such gene regulatory or transcription complexes occurring within cell-free chromatin fragments.

[0133] Cell-free chromatin fragments containing transcription factor components may also contain, but are not limited to, intact nucleosomes or any histone proteins present in a complex, and all such cell-free chromatin complexes may be useful and included in the present invention.

[0134] In one preferred embodiment, the transcription factor is selected from STAT, NF-κB, β-catenin, γ-catenin, Notch, notch intracellular domain (NICD), GLI, c-JUN, JUNB, JUND, c-FOS, FRA, ATF, CREB-CREM, cEBP, ETS, MYC, MAX, E2F, interferon regulatory factors (IRFs), T cell factors (TCFs), lymphocyte enhancer factors (LEFs), and helix-loop-helix proteins, HOX proteins, EN2, GATA3, CDX2, TTF-1, PAX8, WT1, NKX3.1, P63 (or TP63), P40, or CTCF. In a further embodiment, the transcription factor is selected from EN2, CDX2, or TTF-1. In another embodiment, the transcription factor is CTCF.

[0135] Most of these transcription factors are not 100% tissue specific, but can be expressed in several adult tissues as well as in several cancer types. Blood testing for chromatin fragments containing transcription factors is enhanced by using sensitive analytical methods to detect the associated DNA fragment(s). The disease and / or tissue specificity of this method is improved by combining the distinctive nature of the transcription factor with the specific sequence(s) of the DNA with which it is associated.

[0136] In one embodiment, a body fluid sample taken from a subject is contacted with one or more selected transcription factor binding agents to test for one or more disease symptoms in a multiplex assay. For example, multiple transcription factors, each specific to one or more cancer diseases, can be tested, optionally in addition to a transcription factor expressed in many cancers, allowing a test to detect many different cancer diseases in addition to identifying cancer tissue with one blood test. Multiplex testing methods are well known in the art, for example, but not limited to, the multiplex bead system of Luminex Corporation allows many multiplex assays to be performed with one sample (Dunbar, 2006).

[0137] In a further aspect of the invention there is provided a method for detecting a disease in a human or animal subject comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a plurality of binding agents that bind a plurality of transcription factors; (ii) analyzing the DNA associated with the different transcription factors; and (iii) using the presence and / or amount and / or pattern of DNA associated with a plurality of transcription factors to determine the presence and / or characteristic of the disease in the subject.

[0138] In a further aspect of the invention there is provided a method for detecting a disease in a human or animal subject comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with two or more (e.g., multiple) binding agents that bind to two or more (e.g., multiple) transcription factors; (ii) determining the sequence of DNA associated with the transcription factor bound in step (i); and (iii) using the presence and / or amount and / or pattern and / or sequence(s) of DNA bound to the transcription factor to determine the presence and / or characteristic of disease in the subject. In one embodiment, each of the multiple transcription factors is attached to a separate solid support so that each transcription factor can be isolated for analysis or sequencing of the DNA fragment with which it is associated. For example, the Luminex multiplex bead system is composed of multiple bead types, each coated with a different transcription factor binding agent, which after exposure to a sample can be isolated from one another for (separate) sequencing of the DNA associated with each transcription factor individually.

[0139] (transcription factor-DNA chromatin fragment) Chromatin fragments present in the circulation can come from a variety of sources. One source is through the release of chromatin into the circulation after the death of cells, which may include diseased cells such as cancer cells. In some cases, chromatin may be actively released into the circulation.

[0140] The main source of chromatin fragments in the circulation comes from neutrophils and is mediated by neutrophil extracellular traps (NETs) that are generated by a process known as NETosis, in which neutrophils release chromatin material into the extracellular matrix to trap and neutralize pathogens locally at the site of infection (NETs). NETs and their metabolic products contain oligo- and mononucleosomes with component DNA fragments, the majority of which are 150 bp or larger in size.

[0141] Size profiling of cfDNA extracted from blood revealed that the major component of cfDNA is mononucleosomes with a size distribution peak of approximately 160-170 bp, ranging from approximately 130-200 bp, corresponding to mononucleosomes with associated linker DNA of various lengths. There may also be additional peaks corresponding to oligonucleosomes of various sizes, including dinucleosomes (approximately 340 bp), trinucleosomes (510 bp), etc. In samples affected by NETosis, there may also be broad peaks associated with large chromatin fragments up to several thousand bp in length.

[0142] Transcription factors bind short DNA sequences, and transcription factor-DNA complexes contain shorter DNA fragments in the 35-80 bp range (Snyder et al., 2016). In the normal size profile of double-stranded plasma cfDNA libraries, there is little or no visible material corresponding to cfDNA fragments less than 100 bp in length. However, single-stranded library preparations contain more cfDNA fragments in the 35-80 bp range (Snyder et al., 2016). This protein-bound cfDNA component of 35-80 bp is a minor component of the total circulating chromatin fragments.

[0143] A further important aspect of transcription factor-DNA binding in the context of the present invention relates to the kinetic stability of transcription factor-DNA binding. Some transcription factors bind stably to DNA in vivo at TFBS. Other transcription factors bind transiently at TFBS in vivo where they associate, dissociate, and reassociate in a dynamic manner. This is not an issue for ChIP-Seq methods using cell and tissue-based substrates, as both can be detected using cross-linking techniques. Dynamically bound transcription factors naturally alternate between bound and dissociated forms, but when cross-linked, they are "trapped" in the bound form. Thus, a short cross-linking time increases the detection rate of stably bound transcription factors, but decreases the detection rate of dynamically bound transcription factors. In contrast, a long cross-linking time increases the detection rate of dynamically bound transcription factors, as more transcription factors are cross-linked and "trapped" in the associated form over time (Poorey et al., 2013).

[0144] However, based on kinetic considerations, it is inferred that dynamically bound transcription factors are unlikely to be present in the circulation in blood or other body fluids. In vivo, the relatively high nuclear concentrations of both chromatin and transcription factors allow for the association, dissociation, and reassociation of dynamically bound transcription factor-DNA complexes. However, the levels of transcription factor-DNA complexes in body fluids are highly diluted and present at very low concentrations, making it unlikely that once dissociated, transiently or dynamically bound transcription factor and DNA components will reassociate. It was therefore inferred that cross-linking in plasma involves only stably bound transcription factors and therefore would always be rapid (since slower cross-linked and transiently bound transcription factors would dissociate and could be ignored). Thus, in one embodiment of the present invention, a method for detecting a disease in a human or animal subject is provided, comprising: (i) contacting a bodily fluid sample obtained from a human or animal subject with a binding agent that binds to a kinetically stable transcription factor-DNA complex; (ii) determining the sequence of the one or more DNA fragments associated with the transcription factor in the kinetically stable transcription factor-DNA complex; and (iii) using the presence of said transcription factor and the sequence of said associated DNA in combination as a biomarker for determining the presence and / or characteristic of said disease in said subject.

[0145] (transcription factor DNA binding domain) Transcription factors may be classified by their DNA-binding domain (DBD). Vaquerizas et al. (2009) surveyed 1391 known transcription factors and identified over 24 different types of transcription factors based on their DBD. The most commonly occurring transcription factors identified are those with zinc finger DBDs, which account for almost half (48.5%) of all transcription factors.

[0146] The preferred sample type for cfDNA, ctDNA or nucleosome analysis is EDTA plasma. The function of EDTA or citrate in plasma collection tubes is to chelate and capture calcium ions in the blood to prevent clotting (the clotting cascade in blood requires the presence of calcium ions). The collection tubes are centrifuged to separate the cellular components in the blood from the plasma supernatant, which can be removed and used as a sample matrix for many clinical diagnostic purposes.

[0147] Binding of zinc finger transcription factors to their DNA TFBS is dependent on the presence of zinc ions. However, calcium chelators used in plasma collection tubes also chelate zinc ions as well. Chelating away zinc ions from zinc finger transcription factors can result in loss of DNA binding of the transcription factors (Ralston, 2008). The interaction of zinc chelators with zinc finger transcription factors means that this family of transcription factors behaves differently in EDTA plasma than transcription factors that bind DNA via other DBD types.

[0148] The presence of zinc finger transcription factor-DNA complexes in blood has not been directly demonstrated. We reasoned that such complexes exist but have not been isolated because they are a small fraction of the small circulating chromatin fragment component in blood (the majority of circulating chromatin fragments are nucleosomes) and because they are dissociated in the plasma samples used by researchers in the field. As described herein, we address these two issues and demonstrate plasma ChIP-Seq of the zinc finger transcription factor CTCF.

[0149] (Transcription factor binders) Preferred transcription factor binding agents include antibodies that bind to a transcription factor or an oligonucleotide, e.g., a DNA sequence of a TFBS (optionally including flanking sequences). Preferred binding agents have high affinity for the transcription factor so that binding occurs at low transcription factor concentrations, as well as high specificity for binding of the transcription factor so that non-specific binding of other proteins is minimized.

[0150] The binding agent may be coated on a solid support, such as, for example, Sepharose, Sephadex, plastic beads, or magnetic beads. In one embodiment, the solid support comprises a porous material. In another embodiment, the binding agent may be derivatized to include a tag or linker, and the tag or linker may be used to bind the binding agent to a suitable support that has been derivatized to bind to the tag. Many such tags and supports are known in the art (e.g., Sortag, click chemistry, biotin / streptavidin, his tag / nickel or cobalt, GST tag / GSH, antibody / epitope tag, and many others). Subsequent isolation of the binding agent may be performed before, simultaneously, or after reaction of the binding agent with the transcription factor. For ease of use, the coated support may be included in an apparatus, such as, for example, a microfluidic device. Multiple solid phase binding agents may be used in a multiplex assay format to simultaneously test for the presence of multiple chromatin fragments containing different transcription factors in one test in one body fluid sample.

[0151] In other embodiments, a binding agent is added in solution, cross-linked, and the bound nucleosomes are isolated by precipitating with a precipitating agent such as polyethylene glycol (PEG). The precipitated pellet can then be isolated as a separate phase, for example, by centrifugation or filtration. Many immunoprecipitation methods are known in the art, any of which may be used in the methods of the invention.

[0152] In some embodiments, the DNA associated with the transcription factor is bound by a DNA binding agent. The DNA binding agent may be attached to a solid phase (e.g., plastic particles, magnetic particles, agarose, or many others). The DNA binding agent may be directly or indirectly bound to the solid phase (e.g., via a linker system such as biotin / avidin or glutathione).

[0153] We used commercially available antibodies that bind to transcription factors. For ChIP-Seq, the antibodies were immobilized on commercially available magnetic polystyrene particles. Thus, in one preferred embodiment of the present invention, the transcription factor binding agent is a solid-phase anti-transcription factor antibody (or a portion thereof) immobilized on a magnetic polystyrene particle.

[0154] (DNA library preparation) Some embodiments of the present invention include preparing a library of cfDNA fragments that associate with transcription factors in chromatin fragments.The library may be amplified using PCR to facilitate detection and sequencing.In principle, any library preparation method may be suitable for use in the method of the present invention.

[0155] The method of DNA fragment library preparation is well known in the art and typically involves ligating adaptor oligonucleotides to DNA fragments. Amplification of adaptor-linked DNA fragment library is usually carried out by PCR. PCR primers can also be used for DNA amplification, and can be degenerate to amplify all sequences present in the library, or can be designed using software known in the art to amplify specific DNA sequences associated with the sequence of the response element of the transcription factor, optionally including flanking regions as well.

[0156] The library preparation method can include single-stranded or double-stranded adaptor ligation of cfDNA fragments.Preferred library preparation method includes single-stranded cfDNA adaptor ligation.Preferred library preparation method has high efficiency of amplifying and isolating small DNA fragments less than 100 bp in length. Many such library preparation methods are known in the art, including, for example, (i) the TruSeq DNA Sample Preparation Kit (Illumina) using 5-10 ng of input DNA for 20-25 PCR cycles according to the manufacturer's protocol (Ulz et al., 2019), (ii) the use of the MagMAX cfDNA Isolation Kit (Applied Biosystems) followed by library preparation using the NEBNext Ultra II DNA Library Preparation Kit (New England Biolabs) (Ulz et al., 2019), or (iii) the use of the blood and body fluids protocol of the Qiagen QIAamp DSP DNA Blood Mini Kit and PCR amplification using the Life Technologies Ion Plus Fragment Library Kit (Hu et al., 2019). Other methods include those described by Sanchez et al., 2018, Skene and Henikoff, 2017, Snyder et al., 2016, and Liu et al., 2019. In the examples provided herein, a commercially available single-stranded DNA library preparation kit (Claret Bio SRSLY NGS Library Preparation Kit) was used.

[0157] It will be apparent to one skilled in the art that in an embodiment of the present invention, PCR amplification of DNA associated with the transcription factor is performed (only) to increase the sensitivity of detection or quantification of the transcription factor, and that subsequent amplification of only the response element sequence without the flanking sequences is sufficient.

[0158] (Immunoprecipitation of transcription factor-DNA complexes) Immunoprecipitation is a simple process in principle. Typically, a solid support is coated with an antibody that specifically binds to a protein of interest and exposed to a biological sample containing that protein. The protein of interest is bound by the antibody and adsorbed to the solid surface, while other proteins and other substances remain in solution. The solid phase is isolated from the sample and washed, leaving a pure sample of the protein of interest attached to the solid support.

[0159] Cell- and tissue-based ChIP-Seq methods are well described in the art. Typically, 20-30 μg of chromatin extracted from tissue or cultured cells and digested or sonicated is used as substrate material. As chromatin is composed of about 40% DNA, this represents about 8-24 ug of substrate DNA. However, the concentration of circulating cfDNA is low, measured at 30±14 ng / ml in healthy human subjects and 71±55 ng / ml in gastric cancer patients (Park et al., 2012). Thus, the chromatin material in a 1 ml plasma sample would be about 200-500 times less than the amount typically used in ChIP-Seq.

[0160] Since the majority of circulating cell-free chromatin is occupied by nucleosomes, the available circulating cell-free transcription factor-DNA chromatin fragment material is very small. Furthermore, the available circulating cell-free transcription factor-DNA chromatin fragment material will contain thousands of transcription factors. Thus, the substrate material available for analysis by the method of the present invention represented by a single transcription factor will be only a small portion of the small amount of circulating cell-free transcription factor-DNA material present in the circulation.

[0161] Moreover, chromatin extracts from cells are relatively pure chromatin materials. In contrast, body fluids, such as blood, serum, or plasma, contain not only small amounts of chromatin, but also a large number of proteins and other compounds at high concentrations, any of which may interfere with the method of the present invention by non-specifically adhering to the solid-phase transcription factor antibody or other binding agent used. A further complication for immunoprecipitating circulating transcription factor-DNA complexes from blood, serum, or plasma is that background non-specific binding may be high for the small amount of target transcription factor bound to the specific binding agent on the solid-phase support, obscuring the detection of the target transcription factor.

[0162] Due to all these difficulties, there are few literature reports of ChIP-Seq in plasma or other blood sample matrices. Where ChIP-Seq in plasma has been described, it has been for nucleosomes and nucleosomal histones, because their levels (compared to those of single transcription factors) are high.

[0163] We have addressed these difficulties by using high avidity antibodies and by reducing non-specific binding of other proteins onto the solid support to extremely low levels through a combination of the use of appropriate solid supports and stringent washing of the solid phase with solutions containing high concentrations of strong detergents.

[0164] Thus, the antibody-bound transcription factor-DNA complex may be washed with a strong (e.g., at least 1%, e.g., 1.2% concentration) detergent or mixture of detergents before extraction of the DNA associated with the transcription factor. In one embodiment, the transcription factor bound by the binder in step (i) is washed with a buffer containing a detergent at a concentration of at least 1% before detecting the associated DNA fragment. Many detergents can be used for this purpose. Some common examples include, but are not limited to, Triton detergent (e.g., Triton X-100), Tween detergent (e.g., Tween 20 and Tween 80), sodium deoxycholate, sodium dodecyl sulfate, octylphenoxypolyethoxyethanol (IGEPAL CA-630), tricosaethylene glycol dodecyl ether (Brij), n-dodecyl-β-maltososide, octyl-β-glucoside, octylthioglucoside, 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonic acid (CHAPS), and many others.

[0165] Magnetic polystyrene microbeads were used and washed repeatedly (five times) with a washing solution containing a mixture of 1% octylphenoxypolyethoxyethanol, 0.1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate.

[0166] In one preferred embodiment of the invention, the solid support is a polystyrene particle, e.g. a magnetic polystyrene particle. The antibody (or other binding agent of the transcription factor) used may be directly or indirectly attached to the support.

[0167] In one preferred embodiment of the invention, the solid-phase-bound transcription factor-DNA complexes isolated on the solid support are washed with a solution containing at least 0.25%, or at least 0.5%, or at least 1% of a detergent or surfactant. The detergent used may consist of a detergent or a mixture of detergents as described herein.

[0168] In one embodiment of the invention, the solid phase transcription factor binder support used comprises a multiplex system, such as a multiplex bead system (such as that provided by Luminex Corporation), in which multiple fluorescently distinguishable beads, each coated with a different specific binder for a different transcription factor, can be used simultaneously to interrogate multiple transcription factor-DNA complexes in one sample (Dunbar, 2006).

[0169] (DNA sequencing) Many methods for analyzing, quantifying, or identifying DNA sequences are known in the art, and any DNA analysis method can be employed in the methods of the present invention, including, but not limited to, next generation sequencing, isothermal DNA amplification, cold PCR (co-amplification PCR at low denaturing temperature), MAP (MIDI-activated pyrophosphorolysis), PARE (personalized analysis of rearranged ends), DNA hybridization methods (including gene chip and in situ hybridization). In addition, gene sequences can also be analyzed for epigenetically altered DNA sequences (e.g., sequences containing 5-methylcytosine using bisulfite conversion of unmodified cytosine to uracil) by epigenetic DNA sequencing analysis. Thus, in one embodiment, the associated DNA is analyzed using DNA sequencing, e.g., a sequencing method selected from next generation sequencing (of the targeted genome or the whole genome) and methylated DNA sequencing analysis, BEAMing, PCR including digital PCR and cold PCR (co-amplification PCR at low denaturing temperature), isothermal amplification, hybridization, MIDI-activated pyrophosphorolysis (MAP) or personalized analysis of rearranged ends (PARE).

[0170] In the examples described herein, Illumina NovaSeq sequencing was used. Thus, in one preferred embodiment of the present invention, DNA extracted from isolated transcription factors is analyzed by next generation sequencing.

[0171] (Sample Preparation) The sample can be any body fluid in which chromatin fragments can be detected. Chromatin fragments are known to be found in blood, feces, urine, and cerebrospinal fluid. We have detected chromatin fragments in sputum (saliva) as well. In a preferred embodiment, the body fluid sample is a blood, serum, or plasma sample. These samples can be used to measure and analyze circulating cell-free chromatin fragments, including transcription factors and DNA fragments.

[0172] When a blood sample is used in the method of the invention, the sample may be a whole blood, serum or plasma sample. Whole blood or serum samples may be used as substrates for the analysis of any (stably bound) transcription factor-DNA chromatin fragments, including transcription factors with any DBD type.

[0173] Plasma samples, such as EDTA plasma samples, may also be used in the methods of the present invention. In a typical plasma sampling method, whole blood is collected in a citrate or EDTA-containing blood collection tube and centrifuged within 2 hours. The resulting supernatant plasma may be used fresh or frozen until analysis. However, calcium ion sequestrants, which are used as additives to blood collection tubes for plasma generation, cause dissociation of circulating zinc finger transcription factor-DNA complexes. As mentioned above, the most common class of transcription factors are zinc finger transcription factors.

[0174] There are a number of ways to overcome this difficulty, including, but not limited to, (i) avoiding the use of zinc finger transcription factors and using transcription factors with other DBD types, (ii) using serum samples, (iii) using heparinized plasma or other plasma sample types without calcium sequestration, or (iv) preventing dissociation of the transcription factor-DNA complex, for example, by cross-linking proteins and / or DNA in the chromatin fragments in the blood sample.

[0175] In one embodiment, the body fluid sample is a serum sample. Serum is believed to contain contaminating chromatin material (e.g., NETs) from white blood cells. This contamination interferes with the analysis of cfDNA, and therefore plasma is the most commonly used sample matrix for ctDNA methods. However, isolation of the chromatin fragments containing the transcription factors from other chromatin material present prior to DNA analysis eliminates such interference. Furthermore, contamination of serum with chromatin material is the result of the formation of neutrophil extracellular traps (NETs) (known inducers of NETosis) by neutrophil cells in the blood sample, which is activated by coagulation. If the serum sampling tube with whole blood is processed within a time period of, for example, 15 to 60 minutes after venipuncture, the contaminating NETs material will be large chromatin rather than small chromatin fragments and will not interfere with the analysis of small transcription factor-DNA complexes. Thus, the broadening of the types of samples that can be used is a further advantage of the method of the present invention.

[0176] The presence of contaminating NETs in serum can be further minimized or eliminated by adding an inhibitor of NETosis to the serum collection tube, which prevents NETosis and therefore minimizes the level of background chromatin present in the serum sample. Many inhibitors of NETosis are known in the art. Preferred inhibitors include anthracyclines, in particular doxorubicin. Thus, in one embodiment of the present invention, a method for detecting cell-free chromatin fragments, including transcription factors and DNA fragments, in a serum sample obtained from a human or animal subject is provided, comprising: (i) obtaining a whole blood sample from a subject into a serum collection tube; (ii) contacting the whole blood sample with an inhibitor of NETosis; (iii) isolating a serum sample from the whole blood sample; (iv) contacting the serum sample with a binding agent that binds to a transcription factor; (v) detecting or measuring the DNA fragment associated with the transcription factor; and (vi) using the presence or amount or sequence of said DNA fragment as a measure of the amount of cell-free chromatin fragment containing said transcription factor in said serum sample.

[0177] It will be appreciated that this embodiment of the invention may also be used to provide information indicative of the stage of the disease in a subject, as described herein above.

[0178] In one embodiment, the body fluid sample is any plasma sample, including plasma samples made with a calcium sequestering agent such as EDTA plasma or citrate plasma, which plasma sample is obtained by contacting a whole blood sample with a cross-linking agent. The cross-linking agent may be contacted with the whole blood in a first step of a process that includes: (1) contacting a whole blood sample with the cross-linking agent; (2) contacting the cross-linked sample with a calcium ion chelator; and (3) isolating plasma from the sample.

[0179] Cross-linking is a technique well known in the art. The most commonly used cross-linking agent is formaldehyde, which binds protein molecules to each other and to DNA. However, excessive cross-linking can result in alteration of the structure of antibody binding epitopes in the transcription factor (hence loss of antibody binding) and may even lead to cross-linking of the transcription factor to separate protein molecules or complexes. To prevent this, cross-linking is often quenched seconds or minutes after addition of formaldehyde, for example by adding excess glycine or tris(hydroxymethyl)aminomethane (TRIS) to stop further cross-linking. Thus, in one aspect of the present invention, a method for detecting, analyzing or measuring chromatin fragments containing transcription factors and associated DNA fragments in a blood sample taken from a human or animal subject is provided, comprising: (i) contacting a blood sample obtained from the subject with a cross-linking agent; (ii) optionally, adding a quenching agent to stop further cross-linking; (iii) contacting the sample with a calcium ion chelator; (iv) isolating plasma from the sample; (v) contacting the plasma sample with a binding agent that binds to a transcription factor; (vi) isolating the bound chromatin fragments containing the transcription factor; and (vii) analyzing the isolated chromatin fragments (e.g., by a method described herein).

[0180] In another aspect of the invention, there is provided a method for detecting a disease in a human or animal subject, comprising: (i) contacting a blood sample obtained from the subject with a cross-linking agent; (ii) optionally, adding a quenching agent to stop further cross-linking; (iii) contacting the sample with a calcium ion chelator; (iv) isolating plasma from the sample; (v) contacting the plasma sample with a binding agent that binds to a transcription factor; (vi) isolating DNA associated with the transcription factor; (vii) optionally amplifying the isolated DNA by PCR; (viii) determining the amount and / or sequence of the DNA; and (ix) using the presence of said transcription factor and / or the sequence of said associated DNA as a biomarker for detecting said disease state in said subject.

[0181] In one preferred embodiment, formaldehyde or a formaldehyde releasing agent is used as the cross-linking agent. In one embodiment, EDTA is used as the calcium ion chelating agent for anticoagulation. In a preferred embodiment, formaldehyde is added to the whole blood sample immediately after collection, for example by placing the whole blood sample in a blood collection tube that already contains formaldehyde. The blood collection tube is left for a sufficient time for the cross-linking reaction to occur, after which the reaction is stopped by the addition of a quencher to prevent excessive cross-linking of plasma components. The quencher is usually an amine compound, such as glycine or TRIS, that reacts with formaldehyde. The quencher may be added together with EDTA, for example by adding a solution of glycine and EDTA in TRIS buffer. The whole blood sample is then centrifuged and the plasma containing the cross-linked transcription factor-bound DNA complexes is isolated for analysis by the method of the invention.

[0182] In another aspect of the invention, there is provided a method for detecting a disease in a human or animal subject, comprising: (i) contacting a blood sample obtained from the human or animal subject with a cross-linking agent; (ii) contacting the whole blood sample with a quenching agent and a calcium ion chelator; (iii) isolating the plasma produced from the sample in step (ii); (iv) contacting the plasma sample with a binding agent that binds to a transcription factor; (v) isolating DNA associated with the transcription factor; (vi) optionally, amplifying the isolated DNA; (vii) determining the amount and / or sequence of the DNA; and (viii) using the presence of the transcription factor and / or the sequence of the associated DNA as a biomarker for detecting the presence and / or characteristic of the disease in the subject.

[0183] As mentioned above, transcription factors present in the circulation are likely to be stably bound to DNA rather than transiently associating and dissociating from DNA in a dynamic manner. For most stably bound DNA-(transcription factor-containing) circulating chromatin fragments, cross-linking with formaldehyde in whole cultured cells or tissue samples is rapid, taking less than 1-2 minutes. We reasoned that a minute or two may be required for diffusion and entry of formaldehyde into the cells, subsequent entry into the cell nucleus, and subsequent cross-linking of the chromatin, but this time may be shortened in the whole blood environment where the chromatin fragments are free in solution and can be cross-linked immediately. The cross-linking agent used may be formaldehyde, or a formaldehyde-releasing agent (also called a formaldehyde generator, formaldehyde donor, or formaldehyde-releasing preservative). A formaldehyde-releasing agent is a component that slowly releases formaldehyde. Many formaldehyde-releasing agents are known in the art and are commonly used as antimicrobial preservatives in the cosmetic industry, for example in skin care and hair care products, where high levels of formaldehyde are avoided due to toxicity, but low protective levels are maintained by release.Therefore, in one embodiment, the crosslinking agent is a formaldehyde-releasing agent.

[0184] We reasoned that cross-linking of circulating cell-free transcription factor-DNA complexes in whole blood (as opposed to cells or tissues) may be rapid, and may occur more rapidly than zinc depletion of zinc finger proteins. Thus, in one embodiment of the present invention, a cross-linking agent may be added simultaneously with a calcium ion chelator. Blood collection tubes (BCTs) containing both EDTA and a formaldehyde releaser are commercially available, such as cell-free DNA BCTs available from Streck Inc. Whole blood added to such a blood collection tube is exposed to EDTA and a cross-linking agent simultaneously.

[0185] We performed a number of experiments using different EDTA sample preparation methods. For example, the estrogen receptor (ER) is a zinc finger transcription factor. We measured the levels of ER present in (normal) EDTA plasma samples using an ELISA method. ER was detectable as shown in Figure 5. We immunoprecipitated ER from EDTA plasma samples, extracted the DNA bound to the solid phase, and amplified the DNA present in the extract. However, no DNA was observed in the amplified samples. We reasoned that this was because the ER-DNA complex was dissociated in EDTA plasma.

[0186] CTCF (also called CCCTC-binding factor) is an evolutionarily conserved zinc finger transcription factor that binds to multiple sites in the genome through a combination of 11 zinc fingers and plays an important role in genome function. A survey of CTCF binding sites in the human genome identified 77,811 distinct binding sites across 19 different cell types (Wang et al., 2012). Of the 77,811 binding sites, 27,662 were occupied in all 19 cell types surveyed. CTCF binding at the remaining 50,149 binding sites showed tissue specificity. The 19 cell types investigated included 12 normal cell types and seven cancer or EBV-immortalized cell lines, including colorectal cancer (Caco-2), cervical cancer (HeLa-S3), hepatocellular carcinoma (HepG2), neuroblastoma (SK-N-SH_RA), retinoblastoma (WERI-RB-1), and EBV-transformed lymphoblastoid (GM06990). CTCF binding at 1,236 binding sites was confirmed to be cancer cell line specific, and occupancy of these binding sites distinguished normal from immortal and cancer cell lines for epithelium, fibroblasts, and endothelium (Liu et al., 2017).

[0187] Mouse anti-CTCF antibodies were used to immunoprecipitate CTCF-DNA from four pools of cross-linked EDTA plasma samples (collected with Streck cfDNA BCT) from 18 subjects diagnosed with cancer. Western blot analysis of proteins isolated on solid support by ChIP was performed. The results in Figure 7 show that a protein band with a molecular weight of approximately 140 kD corresponding to CTCF was present in all four pooled samples (but was absent in a control experiment in which nonspecific mouse IgG was used instead of anti-CTCF antibodies). The approximately 50 kD band corresponds to the binding of the labeled anti-mouse IgG antibody used in the Western blot to the heavy chain of the mouse anti-CTCF antibody used in ChIP.

[0188] The ChIP procedure was then repeated to immunoprecipitate CTCF-DNA complexes using cross-linked EDTA plasma samples (collected in Streck cfDNA BCT) from subjects diagnosed with breast cancer. The cfDNA fragments were extracted from the solid support, the extracted DNA fragments were ligated to adapter oligonucleotides, and the cfDNA present was amplified. The amplified cfDNA library was analyzed by electrophoresis, and the resulting electropherogram (Figure 8) showed that the library contained small fragments in the range of 35-80 bp (corresponding to peaks between 175-220 bp on the x-axis, which are considered to be adapter-ligated fragments). The main peak of adapter-ligated cfDNA fragments was observed at a length of approximately 50 bp (corresponding to a peak at 190 bp on the x-axis, which is considered to be the adapter-ligated fragment length). Although the amplified cfDNA libraries contained small fragments ranging from 35 to 80 bp, not all of these fragments bound to CTCF in the samples, because amplification of extracts from solid supports coated with nonspecific mouse IgG also yielded small DNA fragments, but the specific peak (1000 fluorescent units (FU)) obtained by ChIP with specific anti-CTCF antibodies was higher than the nonspecific IgG peak (80 FU).

[0189] The amplified cfDNA library isolated using anti-CTCF immunoprecipitation was sequenced by next-generation sequencing. The results of the amplified library prepared from the cross-linked EDTA plasma sample (collected by Streck cfDNA BCT) collected from a patient diagnosed with CRC are shown in Figure 9. Enrichment of small cfDNA fragment binding of 9780 published CTCF TFBS sequences (Kelly et al., 2012) was observed. In contrast, the cfDNA library obtained by binding with non-specific mouse IgG showed no enrichment. The results of peak calling of cfDNA fragment sequences referring to the input non-specific control showed that CTCF is the transcription factor with the most TFBS sequence fragments. It was concluded that the method of the present invention was successful in ChIP-Seq of transcription factors in plasma.

[0190] The androgen receptor (AR) is a zinc finger transcription factor that has been of interest in prostate cancer. To demonstrate that the method of the present invention can be applied to transcription factors that are less abundant than CTCF, the same method was applied to AR. Mouse anti-AR antibodies were used to immunoprecipitate AR from cross-linked EDTA plasma samples (collected with Streck cfDNA BCT) from eight subjects diagnosed with prostate cancer. Western blot analysis of proteins isolated on a solid support using AR by ChIP was performed with LnCAP prostate cancer cell line cells as a positive control. The results in Figure 11 show that a protein band with a molecular weight of approximately 10 kD corresponding to AR was present in all eight samples, and was particularly strong in two samples (lanes 2 and 3 in Figure 11). The band at approximately 50 kD corresponds to the binding of a labeled anti-mouse IgG antibody to the heavy chain of the mouse anti-AR antibody used in ChIP. Next, DNA was extracted from the solid support, and the extracted DNA fragments were ligated to adapter oligonucleotides, and the DNA present was amplified. The results in Figure 12 showed that the amplified cfDNA library contained small fragments in the range of 35-80 bp (peaks shown at 175-220 bp for adapter-ligated fragments, as described above). Although the amplified cfDNA library contained small fragments in the range of 35-80 bp, not all of these fragments bound to AR in the samples, because amplification of extracts from solid supports coated with nonspecific mouse IgG also yielded small DNA fragments. The amplified cfDNA libraries obtained from the two samples with the highest levels of AR observed by Western blot were then sequenced by next-generation sequencing.

[0191] (Dissociated transcription factor-DNA complex) The above aspects of the invention are methods for detecting, measuring or characterizing chromatin fragments containing transcription factors directly or indirectly bound to DNA. In one embodiment of the invention, there is a method for detecting transcription factors not bound to DNA (i.e., free or unbound transcription factors) in a body fluid sample taken from a subject. Detection of free transcription factors can be performed by using an oligonucleotide containing the TFBS DNA sequence of the transcription factor, optionally containing flanking sequences, as a binding agent for the free transcription factor. The free transcription factor bound to the oligonucleotide can then be detected, for example, using a labeled anti-transcription factor antibody (see, for example, Active Motif, 2006). Transcription factors are initially produced in an inactive form, which is subsequently post-translationally activated, for example, by phosphorylation. Active transcription factor forms bind to oligonucleotides containing their TFBS sequences. Inactive transcription factor forms do not bind to oligonucleotides containing their TFBS sequences (Lee et al., 2007). Thus, active, free transcription factors can be detected in bodily fluid samples using an assay that involves binding the free transcription factor to an oligonucleotide that contains a DNA sequence to which the transcription factor binds, such as the TFBS sequence of the transcription factor, followed by the addition of a second transcription factor binding agent, such as an anti-transcription factor antibody that specifically binds to the transcription factor, and using the presence or degree of antibody binding as a measure of the presence or amount of active, free transcription factor present in the sample. Thus, in one embodiment of the invention, there is provided a method for detecting a free transcription factor in a human or animal subject, comprising: (i) contacting a body fluid sample obtained from a human or animal subject with an oligonucleotide that binds to a transcription factor; (ii) isolating the oligonucleotide-bound transcription factor; (iii) contacting the isolated transcription factor with a second binding agent that binds the transcription factor; and (iv) using the presence or extent of said second binding agent to said transcription factor as a measure of the amount of cell-free transcription factor in the sample.

[0192] In a preferred embodiment, the oligonucleotide used to bind to the free transcription factor comprises a TFBS sequence. In a preferred embodiment, the oligonucleotide used to bind to the free transcription factor is attached to a solid support. In a preferred embodiment, the second binding agent is an antibody. In a preferred embodiment, the second binding agent is labeled so that its binding to the solid-phase oligonucleotide-bound transcription factor can be easily detected and / or quantified.

[0193] In one embodiment, zinc ions are added to the sample to facilitate binding of the oligonucleotide to the zinc finger transcription factor, either simultaneously with the addition of the oligonucleotide in step (i) or prior to step (i).

[0194] In another aspect of the invention, there is provided a method for detecting a disease in a human or animal subject, comprising: (i) contacting a body fluid sample obtained from a human or animal subject with an oligonucleotide that binds to a transcription factor; (ii) isolating the transcription factor bound to the oligonucleotide; (iii) contacting the isolated transcription factor with a second binding agent that binds the transcription factor; and (iv) using the presence or degree of binding of said second binding agent to said transcription factor as an indication of the presence and / or characteristic of disease in said subject.

[0195] In one embodiment, a body fluid sample taken from a subject is contacted with one or more oligonucleotides (e.g., TFBS sequences specific for binding to one or more transcription factors) to identify the presence and / or characteristics of a disease. In a further embodiment, the method is performed using a multiplex assay (i.e., comprising more than one oligonucleotide, preferably each oligonucleotide specific for a different transcription factor) to test for one or more diseases. For example, testing for multiple transcription factors, each specific for one or more cancer diseases, optionally in addition to transcription factors expressed in many cancers, allows a single blood test to be tested for detecting many different cancer diseases in addition to identifying cancer tissue. Methods for multiplex testing are well known in the art, including, but not limited to, DNA microarray technology or the Luminex Corporation multiplex bead system, which can be used to perform many multiplex assays on a single sample (Dunbar, 2006).

[0196] In a preferred embodiment, the disease is cancer. In a further embodiment, the characteristic of the disease is tissue affected by cancer.

[0197] Estrogen receptor (ER) is a ligand-activated nuclear hormone receptor zinc finger transcription factor. We reasoned that circulating chromatin fragments containing zinc finger transcription factors and DNA fragments would be disrupted in EDTA plasma samples. We performed enzyme-linked immunosorbent assays (ELISAs) for free (i.e., not bound to DNA) estrogen receptor alpha (ERα) in plasma samples from patients with gynecological cancers with estrogen receptor overexpression and patients with ER-negative breast cancer. ER is involved in the transcriptional regulation of numerous genes and is highly expressed in female reproductive tissues and reproductive system cancer tissues. ER is expressed at low levels in hematopoietic cells but highly in ER-positive breast and ovarian cancer cells. ER-positive cancer cells have estrogen receptors, are estrogen-sensitive, and their proliferation is stimulated by estrogen. ER-negative cancer cells do not have estrogen receptors and are insensitive to estrogen. Approximately 80% of ovarian and breast cancers are ER-positive. ER-positive cancers are associated with a better prognosis than ER-negative cancers. Because ER-positive cancers grow in response to estrogen, hormone therapy including tamoxifen and aromatase inhibitors, which bind to estrogen and inhibit the activation of estrogen receptors, thereby suppressing cancer growth, is effective.

[0198] The ER positive or ER negative status of a cancer is determined by immunohistochemistry of surgically removed cancer tissue. Typically, a labeled antibody that binds to ER is incubated with the cancer cells / tissue, and the level of antibody staining observed determines the status. An ER score is assigned to ER positive cancers. The percentage of cancer cells that are hormone receptor positive and the intensity of staining are measured. These two parameters are combined to score the sample on a scale of 0-8. Samples with more receptors visible with higher intensity will receive a higher score.

[0199] Since nuclear hormone receptors are cellular proteins, it is expected that ER would not be present in the circulation. We hypothesized that any free ERα present in plasma must result from circulating chromatin fragments that contained ERα, but were dissociated when EDTA was added to make plasma, releasing the free ERα from DNA binding. We predicted that the levels of such chromatin fragments would be so low as to be close to zero, and therefore that the levels of free ERα in plasma would be found to be undetectable by ELISA and below the minimum sensitivity (0.8 pg / ml) of the ELISA used. Surprisingly, free ERα was found to be present in plasma at levels up to 20 pg / ml (Figure 5). In light of this, interleukin-6 and tumor necrosis factor are commonly measured blood biomarkers, with normal ranges of approximately 5-15 pg / ml, with a maximum of 8 pg / ml, respectively. Furthermore, measured levels of ERα were higher in ovarian and ER-positive breast cancers than in ER-negative breast cancers, indicating that this ERα is of tumor origin.

[0200] Thus, in another aspect of the invention there is provided a method for detecting the presence or measuring the level of a zinc finger transcription factor in a biological sample, comprising: (i) contacting the sample with a zinc ion chelator; and (ii) analyzing said sample for the presence or level of said substituted zinc finger transcription factor.

[0201] In one embodiment, the biological sample is a body fluid sample, such as blood, serum, or plasma. In a further embodiment, the zinc ion chelator is EDTA. EDTA may be added to the body fluid sample to disrupt zinc finger-DNA binding.

[0202] In one preferred embodiment, the biological sample is a whole blood sample and the zinc ion chelator is EDTA, which is added to the whole blood sample to disrupt zinc finger-DNA binding and prevent blood clotting, resulting in the production of a plasma sample containing free zinc finger transcription factors. Analysis of the sample for transcription factors may use any method. In one preferred embodiment, the analytical method employed is an immunoassay, in particular a two-site "sandwich" immunoassay. Thus, in one preferred embodiment of the invention, a method for detecting the presence or measuring the level of circulating chromatin fragments containing zinc finger transcription factors in a whole blood sample taken from a subject is provided, comprising: (i) contacting a whole blood sample with EDTA to produce a plasma sample; and (ii) analyzing the plasma sample for the presence or level of the zinc finger transcription factor using an immunoassay method.

[0203] The zinc finger transcription factor family is the most abundant transcription factor family. Therefore, this aspect of the invention can be used to detect the majority of transcription factors of interest. The term "zinc finger transcription factor" refers to a transcription factor that contains a zinc finger binding domain.

[0204] Circulating zinc finger transcription factors may be used as biomarkers for the detection of disease, for example for the detection, diagnosis, treatment selection, monitoring, or prognosis of gynecological cancer. Thus, in one embodiment of the invention, a method of determining a disease status in a subject, for example for the detection, diagnosis, treatment selection, monitoring, or prognosis of or for a disease in a subject, is provided, comprising: (i) contacting a blood sample obtained from the subject with a zinc chelator to produce a plasma sample; (ii) analyzing the plasma sample for the presence or level of the zinc finger transcription factor; and (iii) using the presence or level of said zinc finger transcription factor in said sample as an indication of said disease state in said subject.

[0205] This aspect of the invention is also suitable for use in cell culture methods. Chromatin immunoprecipitation (ChIP) methods of transcription factors are complex, difficult, time-consuming, and not robust. A typical ChIP method involves extraction of chromatin material from cells, fragmentation of the chromatin by DNA digestion or by physical methods such as sonication, isolation of the chromatin fragments using an antibody, extraction of DNA associated with the antibody, and determination of the DNA sequence of the extracted DNA. Using the methods of the invention, the presence or amount of zinc finger transcription factors may be established by extracting chromatin material from cells into a fluid containing EDTA (or other zinc chelator) and measuring free zinc finger transcription factors (e.g., by ELISA).

[0206] Any method may be used to analyze a sample for the presence or amount of a zinc finger transcription factor, including but not limited to mass spectrometry and any immunochemical method. In a preferred embodiment, the method used to analyze a sample for the presence or amount of a zinc finger transcription factor is an immunoassay.

[0207] Since it has been shown that addition of a zinc ion chelator to samples containing chromatin fragments containing zinc finger transcription factors disrupts those chromatin fragments to generate free zinc finger transcription factors, and since EDTA is a strong chelator of zinc (as well as calcium) ions, it would appear that DNA bound to zinc finger transcription factors cannot be investigated in EDTA plasma samples using methods that isolate the transcription factor with an antibody (or other transcription factor binding agent) and then analyze the DNA associated with that transcription factor, since the DNA would no longer be associated with the transcription factor.

[0208] It will be appreciated that disruption of a zinc finger transcription factor bound to DNA will lead to both a free zinc finger transcription factor and an equally free DNA fragment comprising the TFBS sequence and the flanking DNA sequence in its genome.Accordingly, in a further aspect of the invention, there is provided a method for identifying the presence in a subject of circulating chromatin fragments comprising sequence(s) of a zinc finger transcription factor or a DNA fragment bound to said zinc finger transcription factor, comprising: (i) contacting a blood sample obtained from the subject with a zinc chelator to produce a plasma sample; and (ii) analyzing the plasma sample for the presence or level of free DNA fragments comprising a DNA sequence that includes a transcription factor binding site sequence or a flanking sequence to a zinc finger transcription factor binding site.

[0209] The presence of chromatin fragments containing a transcription factor and associated TFB can be used for clinical purposes, including for detection, monitoring, prognosis, or treatment selection of or for a disease, as described herein. Thus, in one aspect of the invention, there is provided a method for determining a disease status in a subject, e.g., for detection, monitoring, prognosis, or treatment selection of or for a disease, comprising: (i) contacting a blood sample obtained from the subject with a zinc chelator to produce a plasma sample; (ii) analyzing the plasma sample for the presence or level of free DNA fragments that contain DNA sequences that contain transcription factor binding site sequences or flanking sequences to a zinc finger transcription factor binding site; and (iii) using the presence and / or level and / or sequence of said DNA fragments in said sample as an indication of said disease state in said subject.

[0210] The presence and / or sequence of free DNA fragments in plasma or other samples in DNA fragments bound to nucleosomes or other proteins may be determined by a number of means including the use of complementary DNA sequences to bind DNA fragments in the sample. This may be accomplished, for example, by the use of DNA chips, which facilitate the probing of samples for multiple sequences simultaneously. Another embodiment of the invention includes the use of exogenous zinc finger transcription factors as specific DNA binders. In this method, the zinc chelator is removed to facilitate binding of the zinc finger transcription factor to DNA. This can be done, for example, by buffer exchange by dialysis or by using size exclusion chromatography, for example, using a Sephadex size exclusion chromatography column. DNA fragments containing the TFBS of a zinc finger transcription factor may be isolated, for example, using solid phase bound transcription factor as a binder for free DNA containing TFBS. The isolated DNA may be analyzed for sequence and / or DNA fragment length. Recombinant transcription factor proteins may be used for the purposes of the invention. The recombinant zinc finger transcription factor protein may be bound to a solid support or may include a linker moiety, and the transcription factor may be used in liquid form or isolated as a bound system. Many such bound preparations are known in the art, for example, zinc finger transcription factors may be biotinylated and isolated using solid phase streptavidin. Thus, in one embodiment of the present invention, a method for identifying the presence of a zinc finger transcription factor and / or circulating chromatin fragments comprising a sequence(s) of a DNA fragment bound to said zinc finger transcription factor in a subject is provided, comprising: (i) contacting a blood sample obtained from the subject with a zinc chelator to produce a plasma sample; (ii) removing the zinc chelator from the sample; (iii) contacting the sample with an exogenous zinc finger transcription factor; and (iv) analyzing the DNA fragments bound by the exogenous transcription factor.

[0211] Alternatively or additionally, the zinc chelator may simply be inactivated in the sample. In one embodiment of the invention, the zinc chelator is inactivated by the addition of excess ions, preferably zinc ions, prior to contacting with the exogenous transcription factor. Thus, in one embodiment of the invention, there is provided a method for identifying the presence of circulating chromatin fragments comprising a zinc finger transcription factor and / or a sequence(s) of a DNA fragment bound to said zinc finger transcription factor in a subject, comprising: (i) contacting a blood sample obtained from the subject with a zinc chelator to produce a plasma sample; (ii) adding an excess of zinc ions or other ions to inactivate the zinc chelator in the sample; (iii) contacting the sample with an exogenous zinc finger transcription factor; and (iv) analyzing the DNA fragments bound by the exogenous transcription factor.

[0212] The presence of chromatin fragments containing a transcription factor and associated TFB can be used for clinical purposes, including for detection, monitoring, prognosis, or treatment selection of or for a disease, as described herein. Thus, in one aspect of the invention, there is provided a method for determining a disease status in a subject, e.g., for detection, monitoring, prognosis, or treatment selection of or for a disease, comprising: (i) contacting a blood sample obtained from the subject with a zinc chelator to produce a plasma sample; (ii) removing or inactivating the zinc chelator in the sample; (iii) contacting the sample with an exogenous zinc finger transcription factor; (iv) analyzing the DNA fragments bound by the exogenous transcription factor; and (v) using the presence and / or level and / or sequence of said DNA fragment in said sample as an indication of said disease state of said subject.

[0213] (Removal of cell-free nucleosomes) Sample preparation can also optionally include a pre-purification step to remove most of the nucleosomes and nucleosome-associated DNA from the sample prior to analysis. This step can reduce background signals and improve the efficiency of isolation and amplification of the transcription factor-binding DNA fragments of interest, improving the analytical and clinical sensitivity of the method of the invention. Thus, in one embodiment, the method further includes a step of removing cell-free nucleosomes from the body fluid sample. Chromatin fragments containing nucleosomes may be removed from the sample (optionally for separate analysis) prior to using the method of the invention described herein. The purpose of this preliminary step is to remove most of the DNA fragments from the sample and reduce the background signal they may generate in the analysis. This may be done, for example, but not limited to, by contacting the sample with a binding agent that binds to nucleosomes, such as a solid-phase anti-nucleosome binding agent, including antibodies or nucleosome binding proteins such as those described in WO2021 / 038010. The antibody may selectively bind to histone proteins, such as core histone proteins, such as H2A, H2B, H3, or H4, or linker histone proteins, such as H1. Reference to histone proteins includes histone post-translational modifications and histone variants or histone isoforms. The nucleosome binding protein may be selected from chromatin binding proteins that bind to linker DNA, or proteins that bind to linker DNA associated with nucleosomes. For example, the chromatin binding protein that binds to linker DNA may be selected from chromodomain helicase DNA binding (CHD) proteins; DNA (cytosine-5)-methyltransferase (DNMT) proteins; high mobility group box protein (HMGB) proteins; poly[ADP-ribose]polymerase (PARP) proteins; or methyl-CpG binding domain (MBD) proteins, such as MBD1, MBD2, MBD3, MBD4, or methyl-CpG binding protein 2 (MECP2).The protein that binds to the linker DNA associated with the nucleosome may be selected from histone H1, macroH2A (mH2A), or fragments or engineered analogs thereof.

[0214] All or most of the nucleosomal material present in the sample may be adsorbed (e.g., onto a solid phase) and thereby removed from the sample. Thus, in one embodiment, the method includes, prior to contacting the bodily fluid sample with the transcription factor binding agent, contacting the sample with a binding agent that binds nucleosomes or components thereof, and removing the sample bound to the binding agent.

[0215] It has been reported that the majority or most of the short cfDNA fragments in plasma less than 100 bp in length are not derived from chromatin fragments containing regulatory proteins, but from nucleosome-associated DNA, where one or both DNA strands are nicked or broken. In this case, the short cfDNA fragment may not be a single 150 bp cfDNA fragment, but rather, for example, a 150 bp nucleosome-associated DNA fragment that is nicked at one or more locations into two or more small cfDNA fragments (e.g., two 75 bp fragments) (Sanchez et al., 2018). Thus, removing nucleosomes from a sample prior to exposing the sample to a transcription factor binding agent has the added benefit of removing short cfDNA fragments less than 100 bp that result from nicked nucleosome-associated DNA. This further reduces the background of nucleosome-associated cfDNA in the sample, as compared to size separation of cfDNA fragments extracted, for example, by gel separation methods.

[0216] We demonstrated the quantitative depletion of nucleosome-containing chromatin fragments from human plasma samples using anti-H3 antibodies.

[0217] In one preferred embodiment, magnetic beads are used as solid support, but any suitable material may be used.Similarly, any method may be used as nucleosome binding method described in International Publications WO2016 / 067029, WO2017 / 068371 and WO2021 / 038010 as nucleosome removal method.Thus, in one embodiment, the sample used in the method of the present invention does not contain nucleosomes.In a further embodiment, the cell-free chromatin fragments detected by the method of the present invention consist of transcription factors and DNA fragments.

[0218] In one embodiment of the invention, there is provided a method for detecting a disease in a human or animal subject, comprising: (i) removing cell-free nucleosomes from a body fluid sample obtained from a human or animal subject; (ii) contacting the sample with a binding agent that binds the transcription factor; (iii) isolating DNA associated with the transcription factor; (iv) amplifying the isolated DNA by PCR; (v) determining the sequence of the amplified DNA; and (vi) using the presence of the transcription factor and the sequence of the associated DNA as a combined biomarker for determining the presence and / or characteristic of disease in the subject.

[0219] In some embodiments of the invention, the presence or sequence of DNA or chromatin fragments associated with cell-free transcription factors may be determined without DNA isolation, which can be accomplished by a variety of methods including, but not limited to, amplification methods that do not require DNA isolation.

[0220] The term "binding agent" as used herein refers to a ligand or binding agent, such as a naturally occurring compound or a chemically synthesized compound, capable of specifically binding to a biomarker (i.e. to a particular transcription factor). The ligand or binding agent of the present invention may comprise a peptide, an antibody or fragment thereof, or a synthetic ligand, such as a plastic antibody, or an aptamer or an oligonucleotide, or a molecularly imprinted surface or device, capable of specifically binding to a biomarker. The antibody may be a monoclonal antibody or a fragment thereof capable of specifically binding to a target. The ligand or binding agent of the present invention may be labeled with a detectable marker, such as a luminescent, fluorescent, enzymatic or radioactive marker, alternatively or additionally, the ligand of the present invention may be labeled with an affinity tag, such as a biotin, avidin, streptavidin or His (e.g. hexa-His) tag. In one embodiment, the binding agent is selected from an antibody, an antibody fragment or an aptamer. In a further embodiment, the binding agent used is an antibody. The terms "antibody", "binding agent" or "binding agent" are used interchangeably herein.

[0221] In one embodiment, the sample is a biological fluid (used interchangeably herein with the term "body fluid"). Any type of body fluid sample can be used in the present invention, including but not limited to blood, plasma, menstrual blood, endometrial fluid, feces, urine, saliva, mucus, semen, and exhaled air (e.g., as condensed exhaled air), or extracts or purifications therefrom, or dilutions thereof. Biological samples also include specimens from living subjects, or specimens taken after death. The sample can be prepared, for example, by dilution or concentration, if appropriate, and can be stored in the usual manner. In one preferred embodiment, the biological fluid sample is selected from blood or serum or plasma. It will be clear to the skilled person that the detection of chromatin fragments in a body fluid has the advantage of being a minimally invasive method that does not require a biopsy.

[0222] In one embodiment, the subject is a mammalian subject. In a further embodiment, the subject is selected from a human or an animal (such as a companion animal or a mouse) subject. In an even further embodiment, the subject is a human subject. In one embodiment, the human subject is a non-embryonic subject (i.e., a human at any stage of development that is not an embryo). In a further embodiment, the human subject is an adult subject, i.e., a subject over 16 years of age, such as over 18, 21, or 25 years of age. In another embodiment, the subject is an animal subject. In a further embodiment, the animal subject is selected from a rodent (e.g., a mouse, a rat, a hamster, a gerbil, or a chipmunk), a feline (i.e., a cat), a canine (i.e., a dog), an equine (i.e., a horse), a porcine (i.e., a pig), or a bovine (i.e., a cow) subject.

[0223] It will be appreciated that the uses and methods of the present invention can be practiced in vitro or ex vivo.

[0224] In a further aspect of the invention there is provided a method for detecting or diagnosing cancer in an animal or human subject comprising: (i) detecting or measuring DNA associated with cell-free chromatin fragments containing the transcription factor in a body fluid sample obtained from the subject; and (ii) using the level of associated DNA and / or the sequence of DNA detected in step (i) to identify the disease state in the subject.

[0225] In a further aspect of the invention there is provided a method for detecting or diagnosing an inflammatory disease in an animal or human subject comprising: (i) detecting or measuring DNA associated with cell-free chromatin fragments containing the transcription factor in a body fluid sample obtained from the subject; and (ii) using the level of associated DNA and / or the sequence of DNA detected in step (i) to identify an inflammatory disease state in the subject.

[0226] In one embodiment of the invention, the presence of cell-free chromatin fragments comprising transcription factors and DNA fragments in a sample is used to determine an optimal treatment regimen for a subject in need of such treatment.

[0227] A further aspect of the invention provides a method for assessing the suitability of an animal or human subject for a medical treatment, comprising: (i) detecting, measuring, or sequencing DNA associated with cell-free chromatin fragments containing the transcription factor in a bodily fluid sample obtained from the subject; and (ii) using the level of associated DNA and / or the sequence of DNA detected in step (i) as a parameter for selecting an appropriate treatment for the subject.

[0228] In a further aspect of the invention there is provided a method for monitoring treatment of an animal or human subject comprising: (i) detecting, measuring, or sequencing DNA associated with cell-free chromatin fragments that contain the transcription factor in a bodily fluid sample obtained from the subject; (ii) detecting, measuring, or sequencing DNA associated with cell-free chromatin fragments containing the transcription factor in the subject's body fluid one or more times; and (iii) using a change in the level and / or DNA sequence of the associated DNA detected in step (i) compared to that detected in step (ii) as a parameter for a change in the condition of the subject.

[0229] A change in the measured levels and / or sequences of DNA associated with cell-free chromatin fragments containing transcription factors detected in a test sample relative to the levels or sequences detected in a previous test sample taken earlier from the same test subject may indicate a beneficial effect of treatment on the disorder or suspected disorder, e.g., stabilization or amelioration. Furthermore, when treatment is completed, the methods of the invention may be repeated periodically to monitor for recurrence of the disease.

[0230] It will be appreciated that these aspects of the invention may be used in combination with the methods disclosed herein, for example step (i) comprising contacting the body fluid sample with a binding agent that binds to a transcription factor and then detecting or measuring DNA associated with the transcription factor.

[0231] In one embodiment, the cell-free chromatin fragments comprising the transcription factor and DNA fragments (i.e., DNA associated with the cell-free chromatin fragments comprising the transcription factor) are detected or measured as one of a panel of measurements, for example in combination with other cell-free chromatin transcription factor markers or in combination with other biomarkers.

[0232] In a further aspect of the invention, there is provided a method for detecting, measuring or sequencing cell-free chromatin fragments including transcription factors and DNA fragments, either alone or as part of a panel of measurements, for use in subjects actually afflicted with or suspected of having cancer or benign tumors, for the purposes of determining or assessing the suitability of a medical treatment in an animal or human subject, or for the purposes of monitoring the treatment of an animal or human subject.

[0233] It will be appreciated that measurements or assays performed by the methods of the invention may include the use of reference materials as calibration or positive controls to provide a standard against which the output of the assay can be compared or calibrated and / or to confirm or monitor that the assay chemistry is functioning correctly. Suitable reference materials may include biologically sourced chromatin fragments containing transcription factors or recombinant chromatin fragments containing recombinant transcription factor-DNA complexes, but are not limited to these.

[0234] The terms "detection" and "diagnosis" as used herein encompass the identification, confirmation, and / or characterization of the stage of a disease. The detection, monitoring, and diagnosis methods of the present invention are useful for confirming the presence of a disease, for monitoring the development of a disease by assessing onset and progression, or for assessing remission or regression of a disease. The detection, monitoring, and diagnosis methods are also useful in methods of clinical screening, prognosis, treatment selection, evaluation of treatment efficacy, i.e., drug screening and evaluation for pharmaceutical development.

[0235] It will be understood that detection and measurement includes sequencing. As used herein, the term "sequencing" encompasses the determination of the nucleoside base sequence (usually the adenine, guanine, thymine and cytosine base sequence) of all or part of a DNA fragment.

[0236] Efficient diagnostic and monitoring methods offer a very powerful "patient solution" with the potential to improve prognosis by establishing the correct diagnosis, allowing the most appropriate treatment to be rapidly identified (thus reducing unnecessary exposure to harmful drug side effects), and reducing recurrence rates.

[0237] It will be understood that the identification and / or quantification can be performed by any method suitable for identifying the presence and / or amount of a particular protein or DNA fragment sequence in a biological sample from a patient, or in a purification or extract of a biological sample, or in a dilution thereof. In the method of the invention, the quantification may be performed by sequencing the biomarker in one or more samples, or by measuring its concentration. Biological samples that can be examined in the method of the invention include those defined herein above. The sample can be prepared, if appropriate, for example by dilution or concentration, and can be stored in the usual manner.

[0238] Identification and / or quantification of a biomarker may be performed by detection of the biomarker or a fragment thereof, such as a C-terminally truncated or N-terminally truncated fragment, which fragment is suitably more than 4 amino acids in length, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0239] Biomarkers may be detected directly, for example by SELDI or MALDI-TOF. Alternatively, biomarkers may be detected directly or indirectly by interaction with one or more ligands, such as antibodies or biomarker-binding fragments thereof, or other peptides or ligands, e.g., aptamers or oligonucleotides, capable of specifically binding to the biomarker. The ligands or binding agents may comprise a detectable label, such as a luminescent, fluorescent, or radioactive label, and / or an affinity tag.

[0240] For example, detection and / or quantification can be performed by one or more method(s) selected from the group consisting of SELDI(-TOF), MALDI(-TOF), 1-D gel-based analysis, 2-D gel-based analysis, mass spectrometry (MS), reversed-phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC, and other LC or LC MS-based techniques. Suitable LC MS techniques include ICAT® (Applied Biosystems, California, USA), or iTRAQ® (Applied Biosystems, California, USA). Liquid chromatography (e.g., high performance liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC)), thin layer chromatography, NMR (nuclear magnetic resonance) spectroscopy can also be used.

[0241] It will be understood that detection and / or measurement of DNA may include, for example, hybridization or sequencing, as described herein.

[0242] The diagnostic or monitoring methods of the invention may involve analysis of samples by SELDI TOF or MALDI TOF to detect the presence or level of biomarkers. These methods are also suitable for clinical screening, prognosis, monitoring of treatment outcomes, identifying patients most likely to respond to a particular therapeutic treatment, drug screening and development, and identifying new targets for drug therapy.

[0243] Identification and / or quantification of an analyte biomarker may be performed using immunological methods involving antibodies or fragments thereof capable of specifically binding to the biomarker.

[0244] In a further aspect of the invention, there is provided a method for identifying cell-free chromatin fragments comprising a transcription factor and a DNA fragment as a combined biomarker for detecting or diagnosing a disease in an animal or human subject, comprising: (i) detecting and / or measuring and / or sequencing cell-free chromatin fragments comprising transcription factor and DNA fragment combination biomarkers in a body fluid sample from a disease subject; (ii) detecting and / or measuring and / or sequencing cell-free chromatin fragments comprising said transcription factor and DNA fragment combination biomarkers in a body fluid sample from a healthy or control subject; and (iii) using the detected differences in the levels and / or sequences of DNA between diseased subjects and healthy or control subjects to identify whether cell-free chromatin fragments comprising the transcription factor and DNA fragment combination biomarkers are useful as biomarkers for a disease state.

[0245] It will be appreciated that this aspect of the invention may be combined with the methods described herein, i.e. step (i) and / or step (ii) may be carried out using the methods defined herein.

[0246] In a further aspect of the invention, there is provided a biomarker or combination of biomarkers identified by the methods described herein.

[0247] A diagnostic or monitoring kit for carrying out the method of the present invention is provided herein. The kit for detecting and / or quantifying a biomarker or a combination of biomarkers suitably comprises a ligand or binder for a transcription factor, and optionally a reagent for amplifying and / or sequencing the DNA associated with the transcription factor, and optionally a ligand or binder for a nucleosome, optionally with instructions for use of the kit. Biomarker monitoring methods, biosensors and kits are also of great importance as patient monitoring tools, allowing physicians to determine whether relapse is due to a worsening of the disorder. If pharmacological treatment is assessed as inappropriate, therapy can be resumed or increased, and if appropriate, therapy can be changed. Biomarkers are sensitive to the state of the disorder, and therefore provide an indication of the impact of drug therapy.

[0248] In a further aspect of the invention there is provided a kit for detecting cell-free chromatin fragments comprising a transcription factor and a DNA fragment as a combined biomarker, said kit comprising a ligand or binder for said transcription factor, optionally together with reagents for amplifying and / or sequencing DNA associated with said transcription factor, and optionally a ligand or binder for nucleosomes, optionally together with instructions for use of the kit according to the methods described herein.

[0249] A further aspect of the present invention is a kit for detecting the presence of a disease stage comprising a biosensor capable of detecting and / or quantitating one or more biomarkers defined herein.

[0250] In a further aspect, there is provided the use of a kit as defined herein for the diagnosis of cancer. In a further aspect, there is provided the use of a kit as defined herein for the diagnosis of inflammatory disease. In a further aspect, there is provided the use of a kit as defined herein for the diagnosis of prenatal disease.

[0251] In a further aspect, there is provided a method of treating a disease in a subject in need thereof, comprising: (a) contacting a bodily fluid sample obtained from a human or animal subject with a binding agent that binds a transcription factor; (b) detecting, measuring, or sequencing DNA fragments associated with the transcription factor; and (c) using the presence, amount, or sequence of the DNA fragment as an indicator of the presence of disease in the subject; and (d) administering a treatment to the subject if the subject is determined to be afflicted with a disease in step (c).

[0252] In one embodiment, the disease is cancer. In another embodiment, the disease is an inflammatory disease. In a further aspect, there is provided a use of a kit as defined herein for the diagnosis of a prenatal disease in a fetus of a pregnant subject.

[0253] In one embodiment, the treatment administered is selected from surgery, radiation therapy, chemotherapy, immunotherapy, hormonal therapy, and biological therapy.

[0254] In a further aspect of the invention, there is provided a method of treating cancer in a subject in need thereof, comprising: (a) detecting or diagnosing cancer in said subject according to the methods described herein; followed by (b) administering to the individual an anti-cancer therapy, surgery, or a pharmaceutical agent.

[0255] In one embodiment, the subject is a human subject or an animal subject.

[0256] The invention will now be illustrated in the following examples. EXAMPLES

[0257] (Example) Example 1 An antibody that specifically binds to the transcription factor TTF-1 (also called NKX2-1) is coated onto magnetic beads (e.g., those commercially available as Dynabeads) for biomagnetic separation. TTF-1 is a homeobox helix-turn-helix transcription factor.

[0258] Anti-TTF-1 antibody-coated magnetic beads are added to EDTA plasma samples from human subjects diagnosed with stage IV lung cancer, human subjects diagnosed with stage IV thyroid cancer, and healthy subjects. After incubation (gently rotating to keep the magnetic particles suspended), the magnetic particles are removed from the plasma samples and washed with assay buffer. TTF-1-associated DNA fragments are isolated from the magnetic solid phase using Qiagen's QiaAMP Circulating Nucleic Acid Kit. Adapter oligonucleotides are ligated to the isolated DNA fragments, and a single-stranded DNA library of TTF-1-associated DNA sequences is created for each plasma sample by the library method described in Snyder et al., 2016 (incorporated herein by reference).

[0259] The fragment libraries generated for each subject are amplified by real-time quantitative PCR. The amplified libraries are sequenced using next-generation sequencing to compare the amount of DNA and associated sequences of each library. The coverage of the TTF-1 TFBS locus by small cfDNA fragments in the range of 35-80 bp will be low in healthy samples because the amount of TTF-1 associated DNA in healthy samples will be low or undetectable. In contrast, the coverage of the TTF-1 TFBS locus by small cfDNA fragments in the range of 35-80 bp will be high in cancer samples because the amount of TTF-1 associated DNA in stage IV lung cancer or stage IV thyroid cancer patients is high. The sequence of associated TTF-1 DNA determined in thyroid cancer samples will correlate with the known sequence of TTF-1-regulated gene promoters in thyroid cells. Similarly, the sequence of associated TTF-1 DNA determined in lung cancer samples will correlate with the known sequence of TTF-1-regulated gene promoters in thyroid cells. On this basis, most or all of the healthy samples, thyroid cancer samples, and lung cancer samples will be distinguishable from the data generated by this experiment.

[0260] Example 2 The experiment described in Example 1 is repeated, but anti-nucleosome antibody-coated magnetic beads are added to the plasma sample to preclear the sample of nucleosomes and nucleosome-bound DNA fragments before incubation with anti-TTF1 antibody-coated magnetic particles. After incubation (with gentle rotation to keep the magnetic particles suspended), the magnetic particles are removed from the plasma sample. The remaining sample is then used to complete the experiment as described in Example 1, with similar results obtained, except that the background level of DNA found in healthy samples will be even lower than that described in Example 1.

[0261] Example 3 Anti-TTF-1 antibody coated magnetic beads are added to EDTA plasma samples taken from human subjects with stage IV lung cancer, human subjects with stage IV thyroid cancer, and healthy subjects. After incubation (gentle rotation to keep the magnetic particles suspended), the magnetic particles are removed from the plasma samples and washed with assay buffer. TTF-1 associated DNA fragments are extracted from the magnetic solid phase using Qiagen QiaAMP Circulating Nucleic Acid Kit. To amplify DNA fragments of specific sequences associated with TTF-1 binding sites in the promoter regions of SPB, thyroid stimulating hormone receptor, and thyroperoxidase genes of the human genome, as well as their flanking DNA, specific sequence primers are designed using typical software known in the art for primer design. The primers are used to amplify the DNA fragments in real-time quantitative PCR. The amount of DNA present for each sequence in each plasma sample is measured. The results for samples taken from healthy subjects will be low or undetectable. Most samples taken from lung cancer patients will contain detectable amounts of SPB gene promoter sequence DNA fragments. Most samples taken from thyroid cancer patients will contain detectable amounts of thyroid stimulating hormone receptor and / or thyroperoxidase gene promoter sequence DNA fragments. Based on this, most or all of the healthy samples, thyroid cancer samples, and lung cancer samples will be distinguishable from the data generated by this experiment.

[0262] Example 4 The experiment described in Example 3 is repeated, but anti-nucleosome antibody-coated magnetic beads are added to the plasma sample prior to incubation with anti-TTF1 antibody-coated magnetic particles to pre-clear the sample of nucleosomes and nucleosome-bound DNA fragments. After incubation (with gentle rotation to keep the magnetic particles suspended), the magnetic particles are removed from the plasma sample. The remaining sample is then used to complete the experiment as described in Example 3, with similar results obtained, except that the background level of DNA found in healthy samples will be even lower than that described in Example 3.

[0263] Example 5 A similar experiment to that described in the above example is repeated by testing plasma samples taken from healthy men and from men diagnosed with stage IV prostate cancer for the helix-turn-helix transcription factor NKX3.1. The results for samples taken from healthy subjects will be low or undetectable. Most samples taken from prostate cancer patients will contain detectable amounts of NKX3.1 gene promoter sequence DNA fragments in the 35-80 bp size range. On this basis, most or all of the healthy samples and prostate cancer samples will be distinguishable from the data generated by this experiment.

[0264] Example 6 The same experiment as described in the above example is repeated by testing serum samples taken from healthy women and from women diagnosed with stage IV ovarian cancer for the zinc finger transcription factor WT1. The results of samples taken from healthy subjects will show low or undetectable coverage of the WT1 TFBS locus by WT1-associated cfDNA fragments in the 35-80 bp size range in healthy subjects. Most samples taken from ovarian cancer patients will show higher coverage of the WT1 TFBS locus by WT1-associated cfDNA fragments in the 35-80 bp size range as they contain detectable amounts of WT1 gene promoter sequence 35-80 bp cfDNA fragments. Based on this, most or all of the healthy samples and ovarian cancer samples will be distinguishable from the data generated by this experiment.

[0265] Example 7 Dynabeads M280, tosyl-activated magnetic beads, were coated with an antibody that binds to a histone H3 epitope located at amino acid positions 30 to 33. This antibody was selected from a large number of antibodies tested because it was observed to bind to both nucleosomes containing intact histone tails and nucleosomes with clipped histone tails.

[0266] Anti-H3 antibody-coated magnetic beads (1 mg) were added to a solution containing a range of recombinant mononucleosome concentrations (0.5 ml) purchased from Active Motif. The beads were incubated with the nucleosomes for 1 h at room temperature with gentle tube rotation to keep the beads suspended. The beads were magnetically isolated and washed. The nucleosomes adsorbed to the beads were then removed by elution and subjected to Western blot analysis. The results demonstrated that nucleosomes were adsorbed from the solution by the magnetic beads in a dose-dependent manner, as shown in Figure 3.

[0267] Example 8 Anti-H3 antibody-coated magnetic beads were prepared and used as described in Example 7. Anti-H3 antibody-coated magnetic beads and uncoated beads were added to solutions containing eight human EDTA plasma samples and a range of recombinant mononucleosome concentrations. The range of recombinant mononucleosome concentrations was chosen to include levels commonly observed in human clinical samples.

[0268] The presence of remaining nucleosomes in solution after incubation with magnetic beads was examined using a nucleosome ELISA with an optical density (OD) readout. The results shown in Figure 4 demonstrate that after adsorption with anti-H3 antibody-coated magnetic beads, the levels of recombinant mononucleosomes remaining in solution were undetectable (showing an OD similar to that of the control solution without nucleosomes), while the levels in solutions incubated with uncoated magnetic beads were unaffected, resulting in a normal ELISA dose-response curve. Similarly, the levels of nucleosomes remaining in solution of eight human plasma samples examined after adsorption with anti-H3 antibody-coated magnetic beads were also low or undetectable, but were not affected by incubation with uncoated magnetic beads. These results demonstrate the quantitative removal of nucleosomes from human plasma samples.

[0269] Example 9 Luminex beads of different colors are coated with antibodies that bind to the transcription factors TTF-1, NKX3.1, GATA-3, CDX-2, and GRHL2 according to the manufacturer's protocol. Plasma samples from healthy subjects and from subjects diagnosed with various cancers are contacted with a mixture of all beads. The amount or coverage of cfDNA in the 35-80 bp range covering each transcription factor TFBS bound to each bead-bound transcription factor is measured by PCR or by next generation sequencing. The results will show that the coverage of NKX3.1 and GRHL2 TFBS by 35-80 bp cfDNA bound to beads coated with antibodies that bind NKX3.1 and GRHL2 is elevated in samples from prostate cancer patients, while the coverage of transcription factors bound to other beads (coated with anti-TTF-1, GATA-3, or CDX-2 antibodies) is low. Similarly, the amount of short cfDNA fragments of 35-80 bp bound to beads coated with antibodies that bind to TTF-1 and GRHL2 will be elevated in samples taken from lung cancer patients, while the amount of transcription factors bound to other beads (coated with anti-NKX3.1, GATA-3 or CDX-2 antibodies) will be low. Similarly, the amount of short cfDNA fragments of 35-80 bp bound to beads coated with antibodies that bind to GATA-3 and GRHL2 will be elevated in samples taken from breast cancer patients, while the binding to other beads (coated with anti-TTF-1, NKX3.1 or CDX-2 antibodies) will be low. In contrast, the binding of short cfDNA fragments of 35-80 bp to all beads will be low in samples taken from healthy subjects.

[0270] Example 10 Magnetic beads are coated with an antibody that binds to RNA polymerase II according to the manufacturer's protocol. Plasma samples taken from healthy subjects and from subjects diagnosed with various cancers are contacted with the beads. The beads are washed to remove unbound chromatin fragments.

[0271] The DNA bound to the beads is extracted, attached to adaptor oligonucleotides, and the library is sequenced to obtain a set of active genes present in the subject's sample. The results will show that the active genes present in the sample taken from the healthy subject represent genes active in hematopoietic cells. The same sequences are also present in samples taken from cancer patients, but these samples were found to additionally contain RNA polymerase II-associated DNA sequences representing genes that are not active in hematopoietic cells, but are active in cells of diseased tissues, including genes that are typically active in cells (healthy or diseased) of the tissue of interest and / or are upregulated in cancer cells.

[0272] Example 11 Magnetic beads are coated with an antibody that binds to RNA polymerase II according to the manufacturer's protocol. Plasma samples from healthy subjects and from subjects diagnosed with various cancers are contacted with the beads. The beads are washed to remove unbound chromatin fragments.

[0273] The DNA bound to the beads is analyzed for the presence of specific DNA sequences using PCR primers to amplify the sequences. Sequences that are specifically associated with colorectal cancer are selected for analysis. The results will show that the sequence is present in samples taken from colorectal cancer subjects, but not present in samples taken from healthy subjects or from subjects with other cancers.

[0274] Example 12 EDTA plasma samples were collected from six women diagnosed with ovarian cancer, two women diagnosed with ER-negative breast cancer, and eight women diagnosed with ER-positive breast cancer (four women diagnosed with an ER score of 7 and four women diagnosed with an ER score of 8). EDTA plasma samples were assayed for ERα using a commercially available ERα ELISA kit. The quantitative detection range of the ELISA kit used was 3-200 pg / ml, with an ERα lower detection limit of 0.8 pg / ml. The mean ERα levels measured were low in ER-negative subjects and high in subjects diagnosed with ovarian cancer or ER-positive breast cancer. Furthermore, the mean levels measured in subjects diagnosed with ER-positive breast cancer were higher in women with high ER scores (Figure 5). It is concluded that the presence of ERα in EDTA plasma samples prepared from whole blood samples collected from women is useful as a biomarker for gynecological diseases, including gynecological cancers.

[0275] (Example 13) The progesterone receptor status of breast cancer as PR positive or PR negative is equally important in the diagnosis and treatment of gynecological cancers. It is further concluded that the measurement of progesterone receptor (PR) levels in EDTA plasma samples prepared from whole blood samples collected from women is equally useful as a biomarker for gynecological diseases, including gynecological cancers.

[0276] Example 14 Androgen receptor status in prostate cancer is important in the diagnosis and treatment of prostate cancer as well. It is concluded that measurement of androgen receptor (AR) levels in EDTA plasma samples prepared from whole blood samples collected from men is useful as a biomarker for prostate disease, including prostate cancer.

[0277] Example 15 Background levels of proteins adsorbed from plasma samples to nonspecifically (nonspecifically) mouse IgG-coated magnetic particles were assessed by Western blot using Coomassie Blue staining for color development. Background was assessed after washing the particles five times with regular immunochemical washing buffer containing 0.1% Tween 20 detergent or with washing buffer containing a high concentration of 1.2% of a mixture of detergents containing 1% octylphenoxypolyethoxyethanol detergent, 0.1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate. The results (Figure 6) show that the background staining was significantly reduced when a strong detergent was used.

[0278] The same experiment was applied to proteins specifically adsorbed to mouse anti-poly ADP antibodies (which bind to poly ADP-parylated proteins of any size), where staining was not significantly affected, indicating that washing removes non-specifically bound proteins but does not (or only slightly) affect specifically bound proteins attached to the antibody.

[0279] (Example 16) A monoclonal antibody that specifically binds to the transcription factor CTCF was coated onto magnetic beads (MyOne Tosyl-activated Dynabeads™) using standard methods. Briefly, 0.86 mg of monoclonal antibody was incubated with 29 mg of magnetic beads (30 μg antibody / mg beads) in 2.9 ml of 0.1 M borate buffer pH 9.5 containing 1 M ammonium sulfate for 18 hours at 37° C. with gentle rotation of the test vessel to keep the beads suspended. The beads were allowed to settle and the supernatant was decanted. The beads were resuspended and incubated in 2.9 mL of blocking buffer of phosphate buffered saline pH 7.4 (PBS) containing 0.1% Tween 20 and 1% bovine serum albumin (BSA) for 1 hour at 37° C. The beads were then pelleted, washed twice with 3 mL of PBS containing 0.1% Tween 20 and 1% BSA, and stored in 2.9 mL of PBS containing 0.1% Tween 20, 1% BSA, and preservative. Nonspecific mouse IgG was similarly coated onto magnetic beads as a nonspecific control reagent.

[0280] Chromatin immunoprecipitation (ChIP) of CTCF-DNA fragments was performed on four pools of cross-linked EDTA plasma samples (1.6 mL collected with Streck Cell-Free DNA BCT) obtained from cancer patients. Each pooled sample was diluted with 0.4 mL of commercial radioimmunoprecipitation assay buffer and 1 mg of anti-CTCF-coated magnetic particles was added. This mixture was incubated for 1 h at room temperature with rotation to keep the beads suspended. The beads were then sedimented and washed five times with a strong detergent washing solution containing a mixture of 1% Triton X-100 detergent, 0.1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate, and stored in 0.1 mL of buffer. In parallel, a control experiment was performed by incubating 1.6 mL of each pooled plasma sample with nonspecific mouse IgG-coated magnetic beads.

[0281] After incubation of the magnetic particles with the pooled plasma samples, the proteins bound to the magnetic particles were suspended in a denaturing 1% sodium dodecyl sulfate (SDS) buffer, and the denatured proteins were analyzed by Western blot using anti-CTCF and a detectably labeled anti-mouse antibody. In Western blot experiments, the presence of CTCF is indicated by the presence of a band at 130-140 kD (Klenova et al., 1997). The results of the Western blot analysis are shown in Figure 7. Briefly, a protein band of approximately 140 kD, corresponding to the presence of the CTCF transcription factor, was visible in all four samples when exposed to magnetic particles coated with anti-CTCF antibodies (anti-CTCF). In contrast, no band was visible in any of the same four samples exposed to magnetic particles coated with nonspecific mouse IgG (NS-IgG). This indicates that the employed ChIP method was able to selectively isolate the circulating transcription factor CTCF from all four pooled samples tested. It was also demonstrated that the employed washing regimen created a clean background.

[0282] (Example 17) CTCF is a zinc finger transcription factor. Chromatin immunoprecipitation (ChIP) of CTCF-DNA fragments was performed on cross-linked EDTA plasma samples (2.4 mL collected with Streck cell-free DNA BCT) obtained from subjects diagnosed with breast cancer. ChIP was performed as described in Example 16, except that 2.4 mL of sample was diluted with 0.6 mL of radioimmunoprecipitation assay buffer and 1.5 mg of anti-CTCF-coated magnetic particles were added. In a parallel control experiment, 2.4 mL of cross-linked EDTA plasma sample was incubated with magnetic beads coated with non-specific mouse IgG. The magnetic beads were fractionated into two fractions. One fraction was used for Western blot analysis to confirm the presence of CTCF protein on the beads using sheared chromatin from MCF7 breast cancer cells as a positive control.

[0283] The second fraction of beads (test and control) was used for DNA extraction and analysis. Cross-linking of the chromatin fragments associated with the magnetic beads to the associated DNA was reversed by heating at 95°C for 15 min. The DNA associated with the magnetic beads was then extracted using a commercial DNA extraction kit (Qiagen, QIAamp DSP Circulation NA Kit) according to the manufacturer's instructions.

[0284] The extracted cfDNA was amplified using a commercial kit (Claret Bio, SRSLY NGS Library Preparation Kit) according to the manufacturer's instructions, using 16 amplification cycles to generate single-stranded libraries for sequencing. The amplified test and non-specific cfDNA fragment libraries were analyzed by electrophoresis using a bioanalyzer instrument. The results (Figure 8) show that the amplified cfDNA library obtained from the specific anti-CTCF coated magnetic particles contained small fragments in the 35-80 bp range. Note that the sharp peak at approximately 140 bp in the electropherogram represents the adapter dimer, so the adapter-ligated fragments of 175-220 bp represent cfDNA fragments of 35-80 bp. The major peak of adapter-ligated cfDNA fragments was observed at approximately 190 bp, which corresponds to a cfDNA fragment of approximately 50 bp in length. The amplified cfDNA library contained small fragments in the 35-80 bp range, but not all of these fragments bound to CTCF in the sample, because the extract amplification from the solid support coated with nonspecific mouse IgG also yielded small DNA fragments. However, the specific peak (1000 fluorescent units (FU)) obtained by ChIP with specific anti-CTCF antibodies was higher than the nonspecific IgG peak (80 FU). This sample was sent for sequencing.

[0285] (Example 18) Amplified cfDNA libraries were generated from cross-linked EDTA plasma samples (collected at the Streck cfDNA BCT) from patients diagnosed with colorectal cancer (CRC) by anti-CTCF immunoprecipitation as described above in Example 17. Amplified cfDNA libraries isolated using anti-CTCF immunoprecipitation were sequenced by Illumina NovaSeq next generation sequencing.

[0286] Read sequences representing cfDNA fragments were aligned to the human reference genome GRCh38 / hg38 using the Illumina DRAGEN bioinformatics pipeline (https: / / emea.illumina.com / products / by-type / informatics-products / dragen-bio-it-platform.html). Non-aligned reads were discarded. The resulting aligned BAM files were used to create subsets of different fragment sizes (35–80 bp, 135–155 bp, 156–180 bp) using sequence alignment / map SAMtools (Li et al., 2009). Read coverage (the number of fragments found to cover a particular locus) was calculated using a bin size of 1 bp (highest resolution possible). Read coverage was normalized to the total number of reads mapped to the human genome in RPGC (reads per genome coverage) using bamCoverage from deepTools. Coverage profile plots (Figures 9 and 10) were generated for each fragment size using plotProfile in deepTools (Ramirez et al., 2016).

[0287] Coverage of 9780 published CTCF binding site loci (Kelly et al., 2012) by short 35-80 bp fragments associated with CTCF compared to coverage by longer cfDNA fragments that match the size predicted for circular mononucleosome association (135-155 bp and 156-180 bp) is shown in Figure 9(a). Coverage is shown over a 5000 bp range that includes 2500 bases upstream and downstream of the location of the CTCF binding site. A strong peak of coverage by small 35-80 bp cfDNA fragments binding was observed at the exact same genomic location of the CTCF TFBS locus reported by Kelly et al., 2012. Because the sequenced library was created directly from cfDNA attached to CTCF protein isolated on anti-CTCF-coated magnetic beads with low background, this cfDNA library contained few nucleosomes and low signals at nucleosome positions. This feature creates a clear 35-80 bp signal and eliminates the need for deconvolution of competitive signals in mixed samples (e.g., samples containing mixed cfDNA fragments originating from hematopoietic and cancer tissues). In contrast, the cfDNA library obtained for binding with nonspecific mouse IgG showed no peaks at CTCF TFBS loci (Figure 9(b)).

[0288] Numerous proteins may bind to or proximal to TFBS, including transcription factors, or any combination of various transcription factors, transcription enhancers, repressors, or other regulatory proteins that are co-operatively linked.The major advantage of the method of the present invention is that the small cfDNA fragment coverage of CTCF TFBS locus is found to only relate to cfDNA fragments that are associated with CTCF.In contrast, methods in the art, such as the fragmentomics method of Snyder et al., 2016 and Ulz et al., 2019, map all cfDNA fragments of all sizes extracted from EDTA plasma and infer that protein binding has or has not occurred at any particular genomic location. It was not known which proteins were involved because the first step in all such methods is extraction of cfDNA, which entails disassociation of all nuclear protein-chromatin fragments in the sample (including nucleosomes and transcription factor-DNA complexes), thus destroying the direct information linking specific cfDNA sequences to specific transcription factors or other proteins.

[0289] Peak calling of the cfDNA fragment sequences relative to the input nonspecific control revealed that CTCF was the transcription factor with the most TFBS sequence fragments. Peak calling was performed on the BAM files using MACS2 (Zhang et al., 2008) narrow peaks. The peak files were used to detect transcription factor binding sites using the findMotifGenome tool in the Homer software package (Heinz et al., 2010).

[0290] We next repeated the analysis for enrichment of 1041 CTCF TFBSs occupied in immortalized cancer cells (Liu et al., 2017). The results shown in Figure 10(a) show that there were clear peaks of CTCF-associated cfDNA fragments of 35-80 bp bound to the 1041 cancer-specific CTCF TFBS sequences. Unlike fragmentomics, the cfDNA fragments contributing to this analysis were solely attributable to CTCF-DNA complexes and not other transcription factor-DNA complexes or cofactor-DNA complexes in the absence of CTCF. This indicates CTCF occupancy of cancer-specific loci and therefore also the tumor cell origin of those cfDNA fragments and the CTCF-DNA complexes from which they were derived. There were no peaks of longer (nucleosome-sized) cfDNA fragments. The cfDNA library obtained for binding to non-specific mouse IgG showed no peaks (Figure 10(b)).

[0291] The demonstration that CTCF-associated cfDNA fragments bound to cancer-specific TFBS loci in body fluids by ChIP-Seq indicates the presence of cancer disease in the subjects studied and can be used as a biomarker in this fashion. It is concluded that the method of the present invention is successful in ChIP-Seq of transcription factors in plasma and as a biomarker for disease.

[0292] (Example 19) The androgen receptor (AR) is a zinc finger transcription factor that has been of interest in prostate cancer. The same method described for CTCF in Example 17 was applied to AR. Mouse anti-AR antibody was used to immunoprecipitate AR from cross-linked EDTA plasma samples (collected with Streck cfDNA BCT) from eight subjects diagnosed with prostate cancer. Western blot analysis of proteins isolated on solid support using AR by ChIP method was performed with LnCAP prostate cancer cell line cells as a positive control. The results in Figure 11 show that a protein band with a molecular weight of approximately 100 kD corresponding to AR was present in all eight samples and at high levels in two samples (lanes 2 and 3 in Figure 11). The band at approximately 50 kD corresponds to the binding of the labeled anti-mouse IgG antibody to the heavy chain of the mouse anti-AR antibody used in ChIP. DNA was then extracted from the solid support, the extracted DNA fragments were ligated to adapter oligonucleotides, and the DNA present was amplified. The results (Figure 12) showed that in all eight samples, the amplified cfDNA libraries contained small fragments in the 35-80 bp range (adapter-ligated fragments of 175-220 bp). Although the amplified cfDNA libraries contained small fragments in the 35-80 bp range, not all of these fragments bound to AR in the samples, because amplification of extracts from solid supports coated with nonspecific mouse IgG also yielded small DNA fragments. Amplified cfDNA libraries from the two samples with the highest levels of AR observed by Western blot were sequenced by next-generation sequencing. (References) [Table 1] TIFF2024501063000007.tif199170

Claims

1. 1. A method for detecting cell-free chromatin fragments, including transcription factors and DNA fragments, in a body fluid sample obtained from a human or animal subject, comprising: (i) contacting the bodily fluid sample with a binding agent that binds to the transcription factor; (ii) detecting or measuring the DNA fragment associated with the transcription factor; and (iii) using the presence or amount of the DNA fragment as a measure of the amount of cell-free chromatin fragment containing the transcription factor in the sample.

2. 2. The method of claim 1, further comprising isolating the transcription factor bound in step (i) from the remaining body fluid sample prior to detecting the associated DNA fragment in step (ii).

3. The method of claim 1 or 2, wherein step (ii) comprises sequencing the DNA fragment associated with the transcription factor.

4. The method according to any one of claims 1 to 3, further comprising the step of extracting the DNA fragment associated with the transcription factor.

5. The method of claim 4, additionally comprising a step of amplifying the extracted DNA fragments, for example by PCR.

6. The method according to any one of claims 1 to 5, wherein the DNA fragment associated with the transcription factor is detected and / or measured by real-time PCR.

7. The method according to any one of claims 1 to 6, further comprising the step of removing cell-free nucleosomes from the body fluid sample.

8. 8. The method of claim 7, further comprising, prior to step (ii), contacting the body fluid sample with a binding agent that binds to nucleosomes or components thereof, and removing the sample bound to the binding agent.

9. The method according to any one of claims 1 to 8, wherein the cell-free chromatin fragment consists of the transcription factor and a DNA fragment.

10. The method according to any one of claims 1 to 9, wherein the transcription factor bound by the binding agent in step (i) is washed with a buffer containing a detergent at a concentration of at least 1% before detecting the associated DNA fragment in step (ii).

11. 1. A method for detecting a disease in a human or animal subject, comprising: (i) contacting a bodily fluid sample obtained from the human or animal subject with a binding agent that binds to a transcription factor; (ii) detecting or measuring DNA associated with the transcription factor; and (iii) using the presence or amount of said DNA as an indicator of the presence of disease in said subject.

12. 12. The method of claim 11, comprising using the sequence of said transcription factor and said associated DNA as a combined biomarker indicative of the presence of said disease in said subject.

13. 1. A method for detecting diseased tissue in a human or animal subject, comprising: (i) contacting a body fluid sample obtained from the human or animal subject with a binding agent that binds a transcription factor; (ii) sequencing the DNA associated with the transcription factor; and (iii) using the presence of said transcription factor and the sequence of said associated DNA as a combined biomarker for determining tissue affected by said disease in said subject.

14. The method of claim 13, wherein the diseased tissue is an organ of development.

15. The method according to any one of claims 11 to 14, wherein the disease is cancer or an inflammatory disease.

16. The method of any one of claims 1 to 15, wherein the binding agent that binds to the transcription factor is an antibody or a fragment thereof.

17. The method according to any one of claims 1 to 16, wherein the body fluid sample is a blood, serum or plasma sample.

18. 18. The method of any one of claims 1 to 17, wherein the body fluid sample is a plasma sample obtained by: (1) contacting a whole blood sample with a cross-linking agent; (2) contacting the cross-linked sample with a calcium ion chelator; and (3) isolating plasma from the sample.

19. 1. A method for assessing the suitability of an animal or human subject for a medical treatment, comprising: (i) detecting, measuring, or sequencing DNA associated with cell-free chromatin fragments containing the transcription factor in a bodily fluid sample obtained from the subject; and (ii) using the level and / or sequence of the associated DNA detected in step (i) as a parameter for selecting an appropriate treatment for the subject.

20. 1. A method for monitoring treatment of an animal or human subject, comprising: (i) detecting, measuring, or sequencing DNA associated with cell-free chromatin fragments containing the transcription factor in a bodily fluid sample obtained from the subject; (ii) repeating the step of detecting, measuring or sequencing DNA associated with cell-free chromatin fragments containing the transcription factor in a bodily fluid sample obtained from the subject one or more times; and (iii) using the change in the level and / or DNA sequence of the associated DNA detected in step (i) compared to step (ii) as a parameter for a change in the condition of the subject.

21. 21. The method of claim 20, wherein the treatment is for the treatment of cancer.

22. The method of any one of claims 1 to 21, wherein the DNA associated with cell-free chromatin fragments comprising the transcription factor is detected or measured as one of a panel of measurements.

23. 23. A kit for detecting cell-free chromatin fragments comprising a transcription factor and a DNA fragment as a combined biomarker, comprising a ligand or binder for said transcription factor, optionally together with reagents for amplifying and / or sequencing DNA associated with said transcription factor, and / or comprising a ligand or binder for nucleosomes, and / or comprising instructions for using the kit according to the method of any one of claims 1 to 22.

24. 1. A method for detecting a disease in a human or animal fetus, comprising: (i) obtaining a body fluid sample from a pregnant human or animal subject; (ii) contacting the bodily fluid sample with a binding agent that binds to a transcription factor; (iii) detecting, measuring, or sequencing DNA associated with the transcription factor; and (iv) using the presence, sequence, or amount of said DNA as an indicator of the presence of disease in the fetus.

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