Methods for detecting chromatin accessibility or DNA-binding protein footprints in cells based on a two-enzyme approach

Abstract

The present invention belongs to the field of biotechnology. In particular, the present invention relates to a method for detecting chromatin accessibility or DNA binding protein footprint in cells based on a two-enzyme method. More specifically, the present invention relates to a method for converting information on the openness of chromatin into information on mutations in DNA sequences by means of adenine methyltransferase and adenine deaminase, thereby quantitatively determining the chromatin accessibility of DNA molecules and the binding footprint of DNA binding proteins, such as transcription factors.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. Specifically, this invention relates to a method for detecting chromatin accessibility or DNA-binding protein footprint in cells based on a dual-enzyme method. More specifically, this invention relates to a method for quantitatively determining the chromatin accessibility of DNA molecules and the binding footprint of DNA-binding proteins, such as transcription factors, by converting chromatin accessibility information into DNA sequence mutation information based on adenine methyltransferase and adenine deaminase. Background Technology

[0002] The chromatin landscape encompasses epigenetic information such as the three-dimensional structure of chromatin, its accessibility, histone modifications, and the location of transcription factors (TFs). It regulates gene transcription, thereby determining cell fate and type. Although open regions in the genome account for only 2%–3% of the total DNA sequence, over 90% of transcription factors bind to these open regions. Transcription factors dynamically compete with histones and other chromatin-binding proteins, regulating nucleosome occupancy and promoting local accessibility to DNA; conversely, accessibility maps specific to certain cell types can also regulate transcription factor binding. Therefore, chromatin accessibility reflects both transcription factor binding and the regulatory potential of genetic loci. Efficiently and accurately locating open chromatin sites on the genome, exploring the dynamic changes in nucleosome positions, and precisely locating specific binding sites of transcription factors on the genome provide important clues and effective methods for successfully identifying genomic regulatory elements and even revealing the mechanisms of gene expression regulation.

[0003] Currently, there are four main methods for exploring chromatin accessibility: deoxyribonuclease I hypersensitive site sequencing (DNase-seq), micrococcal nuclease sequencing (MNase-seq), transposase-accessible chromatin sequencing (ATAC-seq), and nucleosome occupancy and methylome sequencing (NOMe-seq). These methods generally detect chromatin accessibility by quantifying its sensitivity to enzyme methylation or the cleavage of its constituent DNA, and then combine this with next-generation sequencing technology to detect open or protected regions of chromatin. In addition, a recent research team developed Fiber-seq, a chromatin landscape detection technology using the non-specific adenosine methyltransferase Hia5. Hia5 can methylate adenine in open DNA regions, and then third-generation sequencing captures the methylation sites to obtain information on chromatin accessibility. However, all of these technologies have certain limitations: 1) DNase-seq and MNase-seq, based on enzyme cleavage, exhibit sequence bias. To obtain good reproducibility and comparability results in different experiments, a large number of cells and strict enzymatic digestion conditions are required. 2) NOMe-seq suffers from low signal-to-noise ratio due to the severe pH and temperature conditions associated with bisulfite treatment, leading to significant DNA breakage and loss. It requires a relatively large number of sequencing reads to obtain sufficient depth to determine the accessibility level of the entire genome. 3) Fiber-seq, based on third-generation sequencing, is costly and cannot amplify the genome, making it unsuitable for detecting small numbers of cells. 4) These technologies only reflect the average state of multiple cells and cannot analyze differences between cell populations.

[0004] The traditional method for detecting transcription factor footprints is chromatin immunoprecipitation sequencing (ChIP-seq). With continuous technological advancements, more advantageous cut & run and cut & tag techniques have been developed. However, antibody-based detection techniques still have significant limitations, and data quality largely depends on antibody quality. Additionally, ATAC-seq can also be used to detect transcription factor footprints, but it suffers from poor stability and low resolution. Furthermore, the affinity of transposases for open sites can introduce biases into the analytical results.

[0005] Therefore, the development of more innovative and advantageous new detection technologies is still needed to more accurately and efficiently locate transcription factor binding sites and open chromatin regions at the single-cell and single-molecule level with high resolution and low cost, and even to analyze the dynamic structure of chromatin, the relationship between gene expression and disease, and to decipher important regulatory elements in gene transcription regulation. Summary of the Invention

[0006] This invention provides a method for systematically detecting chromatin accessibility and transcription factor footprint at the whole genome level using biological means, called A FOOT-seq (DNA A-deaminase-based assay for TFfootprint by sequencing).

[0007] This method combines DNA-nonspecific adenine methyltransferase and adenine deaminase to capture chromatin structural information across the entire genome. First, permeabilized cells or nuclei are treated with adenine methyltransferase. Based on the methyltransferase activity, adenine at open sites and accessible sites around transcription factor binding sites in chromatin is methylated. Then, the cellular genome is extracted and incubated with adenine deaminase. Utilizing the deamination activity of adenine deaminase, all unmethylated adenine A on naked DNA is deaminated to hypoxanthine I. During subsequent PCR amplification, hypoxanthine is recognized as guanine G, which pairs complementary with cytosine C. Ultimately, the adenine bases deaminated by the deaminase are replaced with guanine, resulting in an A-to-G mutation in the DNA. Adenine deaminase cannot demethylate adenine, thus it can distinguish methylation information on adenine. Only adenine at sites not attacked by adenine methyltransferase (chromatin non-open sites and transcription factor binding sites) undergoes an A to G mutation. This mutation can be detected by first-generation Sanger sequencing, second-generation sequencing (NGS), or single-molecule long-read sequencing. Therefore, using this dual-enzyme technique of adenine methyltransferase and adenine deaminase, cellular chromatin structural information can be converted into DNA sequence information. Through post-amplification sequencing and bioinformatics analysis, multi-dimensional whole-genome information can be captured and detected. This method requires a low initial cell quantity and can even achieve chromatin landscape detection of a single-cell genome.

[0008] Furthermore, by capturing specific sites within the genome using probes or CRISPR / Cas9, open regions or gene sites of interest can be specifically enriched, and chromatin accessibility and transcription factor footprints can be detected. In addition, this dual-enzyme technique can be combined with ChIP-seq and Hi-C techniques to simultaneously capture information on the three-dimensional structure of chromatin and gene regulatory elements at multiple levels and in high dimensions, exploring the relationship between chromatin accessibility, transcription factor binding, and the chromatin landscape.

[0009] This application therefore provides at least the following implementation schemes: Implementation Scheme 1. A method for detecting chromatin accessibility and / or DNA-binding protein footprints in a cellular genome, the method comprising: a) Permeabilizing at least one of the cells, or isolating the nucleus from at least one of the cells; b) Treat the permeable cells or isolated cell nuclei obtained in step a) with adenine methyltransferase, thereby allowing the adenine methyltransferase to fully react with the open regions of the genomic DNA in the cells; c) Isolate genomic DNA from the product of step b); d) Treat the genomic DNA obtained in step c) with adenine deaminase; e) Amplify and sequence the adenine deaminase-treated DNA or portions thereof obtained in step d); and f) Based on the sequencing results of step e), determine the chromatin accessibility and / or DNA-binding protein footprint of the cell genome.

[0010] Implementation Scheme 2. The method of Implementation Scheme 1, wherein cells are treated with a permeation buffer / nuclear extraction buffer containing a detergent to permeate at least one of the cells or to separate the nucleus from at least one of the cells, for example, the permeation buffer / nuclear extraction buffer contains approximately 10 mM Tris-HCl 7.4, approximately 10 mM NaCl, approximately 1% BSA, approximately 0.1% Tween-20, approximately 0.1% NP40, approximately 0.01% Digitonin, approximately 0.1 mM EDTA, approximately 3 mM MgCl2, and approximately 1xPIC.

[0011] Implementation Scheme 3. The method of Implementation Scheme 1 or 2, wherein the adenine methyltransferase is selected from Hia5, Dam, EcoGII, or a combination thereof; preferably, the adenine methyltransferase is Hia5, more preferably EcoGII.

[0012] Implementation Plan 4. The method of Implementation Plan 3, wherein The Hia5 contains the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:1; The Dam contains the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:2; or The EcoGII comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:3. Preferably, the EcoGII comprises the amino acid sequence shown in one of SEQ ID NO:3 or SEQ ID NO:9-12.

[0013] Implementation Scheme 5. The method of any one of Implementation Schemes 1-4, wherein the adenine deaminase is selected from ABE8e, ABE8.20, ABE7.1, or a combination thereof; preferably, the adenine deaminase is ABE8e.

[0014] Implementation Plan 6. The method of Implementation Plan 5, wherein... The ABE8e comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:4. The ABE8.20 comprises the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:5; or The ABE7.1 comprises the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:6.

[0015] Implementation Scheme 7. The method of any one of Implementation Schemes 1-6, wherein step b) is carried out in a methylation reaction system of about 10 µl to about 100 µl, preferably 50 µl.

[0016] Implementation Scheme 8. The method of Implementation Scheme 7, wherein the methylation reaction system of step b) contains a methyl donor molecule, such as S-adenosyl-methionine.

[0017] Implementation Scheme 9. The method of Implementation Scheme 7 or 8, wherein the methylation reaction system does not contain metal salts, preferably, the methylation reaction system does not contain NaCl and / or KCl.

[0018] Implementation Scheme 10. The method of any one of Implementation Schemes 7-9, wherein the methylation reaction system comprises approximately 15 mM Tris-HCl (pH 8.0), approximately 1 mM EDTA (pH 8.0), approximately 0.5 mM EGTA (pH 8.0), approximately 0.5 mM Spermidine, and approximately 0.8 mM S-adenosyl-methionine.

[0019] Implementation Scheme 11. The method of any one of Implementation Schemes 1-10, wherein the methylation reaction system of step b) comprises about 0.1 U / µl to about 100 U / µl, preferably about 0.2 U / µl to 4 U / µl of the adenine methyltransferase.

[0020] Implementation Scheme 12. The method of any one of Implementation Schemes 1-11, wherein in step b), the cells or cell nuclei are treated with adenine methyltransferase for about 5 minutes to about 60 minutes, preferably about 10 minutes to about 30 minutes, more preferably about 10 minutes.

[0021] Implementation Scheme 13. The method of any one of Implementation Schemes 1-12, wherein step b) is carried out at a temperature of about 4°C to about 40°C, for example about 30°C to about 40°C, preferably about 37°C.

[0022] Implementation Scheme 14. The method of any one of Implementation Schemes 1-13, wherein in step b), approximately 50,000 cells or cell nuclei from approximately 50,000 cells are treated with approximately 10 U or approximately 50 U of adenine methyltransferase at approximately 37°C for approximately 30 minutes in a reaction system of approximately 50 µl.

[0023] Implementation Scheme 15. The method of any one of Implementation Schemes 1-14, wherein in step d), the genomic DNA may be fragmented first, and then the fragmented DNA may be treated with adenine deaminase.

[0024] Implementation Scheme 16. The method of any one of Implementation Schemes 1-15, wherein step d) is carried out in a deamination reaction system of about 10 µl to about 100 µl, preferably 10 µl.

[0025] Implementation Scheme 17. The method of Implementation Scheme 16, wherein the deamination reaction system in step d) does not contain metal salts, such as KCl and / or MgSO4; and / or, the deamination reaction system further contains Triton X-100, such as about 0.1% Triton X-100.

[0026] Implementation Scheme 18. The method of Implementation Scheme 17, wherein the deamination reaction system comprises approximately 20 mM Tris-HCl (pH 7.5); approximately 5% glycerol; approximately 2 mM DTT; and approximately 0.1% Triton X-100.

[0027] Implementation Scheme 19. The method of any one of Implementation Schemes 1-18, wherein step d) involves treating the DNA with an adenine deaminase of about 1 U / µl to about 10 U / µl, preferably about 1 U / µl.

[0028] Implementation Scheme 20. The method of any one of Implementation Schemes 1-19, wherein in step d), the DNA is treated with adenine deaminase for about 30 minutes to about 180 minutes, preferably about 120 minutes.

[0029] Implementation Scheme 21. The method of any one of Implementation Schemes 1-20, wherein step d) is carried out at a temperature of about 4°C to about 50°C, preferably about 44°C.

[0030] Implementation Scheme 22. The method of any one of Implementation Schemes 1-21, wherein in step d), approximately 10 ng of DNA is treated with approximately 10 U of adenine deaminase at approximately 44°C for approximately 120 minutes in approximately 10 µl of reaction system.

[0031] Implementation Scheme 23. The method of any one of Implementation Schemes 1-22, wherein step d) further comprises the step of inactivating and / or removing adenine deaminase after treatment with adenine deaminase; for example, treating the product obtained after adenine deaminase treatment with a detergent such as SDS and / or a protease to inactivate and / or remove adenine deaminase; preferably, treating the product obtained after adenine deaminase treatment with about 0.1% SDS and about 0.2 mg / ml Protase K at about 50°C for about 30 minutes.

[0032] Implementation Scheme 24. The method of any one of Implementation Schemes 1-23, wherein step d) further comprises the step of isolating and / or purifying the treated DNA.

[0033] Implementation Scheme 25. The method of any one of Implementation Schemes 1-24, wherein step d) is performed two or more times, preferably twice.

[0034] Implementation Scheme 26. The method of any one of Implementation Schemes 1-25, wherein the amplification described in step e) is PCR amplification.

[0035] Implementation Scheme 27. The method of any one of Implementation Schemes 1-26, wherein step e) comprises amplifying a specific portion of genomic DNA from the deaminated DNA of step d) and sequencing it to determine the chromatin accessibility and / or DNA-binding protein footprint of the specific portion of the cell genome.

[0036] Implementation Scheme 28. The method of any one of Implementation Schemes 1-27, wherein step e) comprises constructing a DNA library from the deaminated DNA of step d) and sequencing the DNA library to determine the chromatin accessibility and / or DNA-binding protein footprint of the cell genome.

[0037] Implementation Scheme 29. The method of Implementation Scheme 28, wherein a DNA sequencing library is constructed from DNA by single-stranded DNA library construction.

[0038] Implementation Scheme 30. The method of Implementation Scheme 29, wherein the single-stranded DNA library is constructed using the EpiArt® DNA Methylation Library Kit for Illumina V3 NE103 kit.

[0039] Implementation Scheme 31. The method of any one of Implementation Schemes 1-30, wherein the presence of AT-to-GC transition in the cellular genomic DNA or its portion is determined by analyzing the presence of AT-to-GC transition in the cellular genomic DNA or its portion relative to control genomic DNA or its portion.

[0040] Implementation Scheme 32. The method of any one of Implementation Schemes 1-30, wherein the chromatin accessibility and / or DNA-binding protein footprint of the cellular genomic DNA or its portion is determined by analyzing the location and / or density and / or conversion rate of AT to GC transitions in the cellular genomic DNA or its portion relative to control genomic DNA or its portion.

[0041] Implementation Scheme 33. The method of any one of Implementation Schemes 1-32, wherein the cells are selected from animal cells, plant cells, and microbial cells.

[0042] Implementation Scheme 34. The method of Implementation Scheme 33, wherein the cell is a mammalian cell, including but not limited to human, mouse, rat, cat, dog, pig, or cow cell; or, the cell is a monocotyledonous or dicotyledonous plant cell, such as rice, corn, wheat, sorghum, soybean, potato, tomato, etc.; or, the cell is a fungal cell such as yeast cell.

[0043] Implementation Scheme 35. The method of Implementation Scheme 33 or 34, wherein the cell is a cell line cell; or, the cell is a primary cell from a different organ or tissue, for example, the cell is from blood, cerebrospinal fluid, biopsy tissue; for example, the cell may be hepatocytes, cardiomyocytes, neuronal cells, fibroblasts, epithelial cells; or, the cell is a tumor cell, stem cell such as embryonic stem cells or induced pluripotent stem cells.

[0044] Implementation Scheme 36. The method of any one of Implementation Schemes 33-35, wherein the cell is a cell treated with a specific substance or condition or a cell at a specific developmental stage, for example, the specific substance is a drug.

[0045] Implementation Scheme 37. A kit for detecting chromatin accessibility and / or DNA-binding protein footprint of a cellular genome by any one of the methods of Implementation Schemes 1-36, comprising adenine methyltransferase and adenine deaminase. Attached Figure Description

[0046] Figure 1 A-FOOT-seq flowchart.

[0047] Figure 2 A schematic diagram of the A-FOOT-seq library construction process.

[0048] Figure 3 A-FOOT-seq analysis of transcription start sites in HepG2 cells. Using a two-enzyme method, the changes in methylation rate of Hia5 in the genebody region and 1 kb upstream and downstream of the TSS under different enzyme concentrations were analyzed. Higher methylation rate at the TSS site indicates a more open location, and a clear nucleosome localization pattern can be seen in the right-hand figure.

[0049] Figure 4 Analysis of CTCF sites and chromatin accessibility in HepG2 cells using A-FOOT-seq. The top figure shows the methylation rate changes 1 kb and 100 bp upstream and downstream of CTCF, with binsizes of 20 bp and 1 bp, respectively. In the top left figure, the indentation at the center of CTCF is clearly visible, indicating that this is the CTCF binding site, while the bulges on both sides indicate openness, and the nucleosome localization pattern is clearly visible. The top right figure shows a protrusion in the center, indicating bias. The bottom figure shows the methylation rate changes 1 kb upstream and downstream of ATAC. The higher methylation rate at the center of ATAC indicates a more open site, demonstrating that the two-enzyme method can capture open sites in the genome.

[0050] Figure 5A-FOOT-seq was used to detect the footprint of DBP transcription factors in HepG2 cells. The changes in methylation rates 1 kb upstream and downstream of DBP transcription factors and 100 bp upstream and downstream are shown. Binsizes were 20 bp and 1 bp, respectively. A clear footprint is observed at the DBP center, indicating that this two-enzyme method can obtain transcription factor footprint information.

[0051] Figure 6 A-FOOT-seq was used to detect the footprint of the FOXA2 transcription factor in HepG2 cells. The changes in methylation rates 1 kb upstream and downstream of the FOXA2 transcription factor and 100 bp upstream and downstream are shown. Binsizes were 20 bp and 1 bp, respectively. A clear footprint was observed at the FOXA2 center, indicating that this two-enzyme method can obtain transcription factor footprint information.

[0052] Figure 7 A-FOOT-seq was used to detect the footprint of the GATA4 transcription factor in HepG2 cells. The changes in methylation rates 1 kb upstream and downstream of the GATA4 transcription factor and 100 bp upstream and downstream are shown. Binsizes were 20 bp and 1 bp, respectively. A clear footprint is observed at the GATA4 center, indicating that this two-enzyme method can obtain transcription factor footprint information.

[0053] Figure 8 A-FOOT-seq was used to detect the footprint of the HNF1A transcription factor in HepG2 cells. The changes in methylation rates 1 kb upstream and downstream of the HNF1A transcription factor and 100 bp upstream and downstream are shown. Binsizes were 20 bp and 1 bp, respectively. A clear footprint was observed at the HNF1A center, indicating that this two-enzyme method can obtain transcription factor footprint information.

[0054] Figure 9 A-FOOT-seq was used to detect the footprint of TEF transcription factors in HepG2 cells. The changes in methylation rates 1 kb upstream and downstream of TEF transcription factors and 100 bp upstream and downstream are shown. Binsizes were 20 bp and 1 bp, respectively. A clear footprint was observed at the TEF center, indicating that this two-enzyme method can obtain transcription factor footprint information.

[0055] Figure 10 Salt concentration affects the activity of adenine methyltransferase. NC is the negative control, and con is the original methylation reaction buffer.

[0056] Figure 11 Salt concentration, reaction temperature, and Triton X-100 affect adenine deaminase activity.

[0057] Figure 12A-FOOT-seq analysis of transcription start sites and nucleosome arrangement in HepG2 cells with different amounts of EcoGII enzyme was performed.

[0058] Figure 13 A-FOOT-seq analysis of chromatin accessibility in ATAC-rich regions of HepG2 cells with different amounts of EcoGII enzyme.

[0059] Figure 14 Different amounts of EcoGII were used to detect chromatin accessibility and nucleosome arrangement at the CTCF site in Hepg2 cells using A-FOOT-seq.

[0060] Figure 15 The footprints of transcription factors CTCF, ATF4, DBP, FOXC1, HNF1A, and HOXA10 were detected using EcoGII's A-FOOT-seq.

[0061] Figure 16 EcoGII and its homologs were subjected to aFOOT-seq experiments on HepG2 cells.

[0062] Figure 17 The results show that, under similar methylation efficiencies, EcoGII(a) and Hia5(b) have higher methylation efficiencies for the GA base environment than for the TA base environment, and EcoGII has a smaller preference for methylation efficiency than Hia5.

[0063] Figure 18 A heatmap analysis of trimethylation rates centered on A was used to compare the preferences of Hia5 and EcoGII. Detailed Implementation

[0064] I. Definition In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0065] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0066] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the stated sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still possessing the activities described in this invention. Furthermore, those skilled in the art will understand that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained in certain practical situations (e.g., when expressed in a specific expression system) without substantially affecting the polypeptide's function. Therefore, when describing a specific polypeptide amino acid sequence in this specification and claims, although it may not contain the methionine encoded by the start codon at the N-terminus, the sequence containing that methionine is still included, and correspondingly, its encoding nucleotide sequence may also contain the start codon; and vice versa.

[0067] Sequence “identity” has a recognized meaning in the art, and the percentage of sequence similarity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence similarity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule.

[0068] The term "genome," as used in this article, encompasses not only chromosomal DNA located in the cell nucleus but also organelle DNA located in subcellular components of the cell, such as mitochondria and plastids.

[0069] II. Methods for detecting chromatin accessibility and / or DNA-binding protein footprint of the cellular genome In one aspect, the present invention provides a method for detecting chromatin accessibility and / or DNA-binding protein footprints in a cellular genome, the method comprising: a) Permeabilizing at least one of the cells, or isolating the nucleus from at least one of the cells; b) Treat the permeable cells or isolated cell nuclei obtained in step a) with adenine methyltransferase, thereby allowing the adenine methyltransferase to fully react with the open regions of the genomic DNA in the cells; c) Isolate genomic DNA from the product of step b); d) Treat the genomic DNA obtained in step c) with adenine deaminase; e) Amplify and sequence the adenine deaminase-treated DNA or portions thereof obtained in step d); and f) Based on the sequencing results of step e), determine the chromatin accessibility and / or DNA-binding protein footprint of the cell genome.

[0070] In some embodiments, cells are treated with a permeation buffer / nuclear extraction buffer containing a detergent to permeate at least one of the cells or to isolate the nucleus from at least one of the cells. To maximize the preservation of chromatin accessibility and / or DNA-binding protein footprint information of the cell genome, the permeation buffer / nuclear extraction buffer should be mild, capable of disrupting or partially disrupting the cell wall / cell membrane, but not significantly disrupting the chromatin structure of the cell.

[0071] In some embodiments, the permeation buffer / nuclear extraction buffer contains NP40. In some preferred embodiments, the permeation buffer / nuclear extraction buffer contains NP40, Tween-20, and Digitonin. In some preferred embodiments, the permeation buffer / nuclear extraction buffer contains approximately 0.1% NP40, approximately 0.1% Tween-20, and approximately 0.01% Digitonin. In some embodiments, the permeation buffer / nuclear extraction buffer also contains Tris-HCl and / or a protease inhibitor such as PIC (a mixture of protease inhibitors). In some specific embodiments, the permeation buffer / nuclear extraction buffer contains approximately 10 mM Tris-HCl (pH 7.4), approximately 10 mM NaCl, approximately 1% BSA, approximately 0.1% Tween-20, approximately 0.1% NP40, approximately 0.01% Digitonin, approximately 0.1 mM EDTA, approximately 3 mM MgCl2, and approximately 1xPIC.

[0072] As used in this article, "adenine methyltransferase" refers to an enzyme that can catalyze the methylation of adenine bases in DNA using DNA as a substrate, and it usually uses S-adenosylmethionine as a methyl donor.

[0073] Exemplary adenine methyltransferases in this invention include, but are not limited to, Hia5, Dam, or EcoGII, or combinations thereof. Preferably, the adenine methyltransferase is Hia5. More preferably, the adenine methyltransferase is EcoGII. In some embodiments, the adenine methyltransferase may be a combination of Hia5 and EcoGII.

[0074] Hia5 adenine methyltransferase may contain the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:1.

[0075] Dam adenine methyltransferase may contain the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:2.

[0076] EcoGII adenine methyltransferase may comprise the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity with SEQ ID NO:3. In some embodiments, EcoGII adenine methyltransferase comprises any of the amino acid sequences shown in SEQ ID NO:3, 9-12.

[0077] As used in this article, "adenine deaminase" refers to a deaminase that can accept DNA (single-stranded or double-stranded, especially double-stranded) as a substrate and can catalyze the deamination of adenine (A) to hypoxanthine (I).

[0078] The exemplary adenine deaminases in this invention can be those that are already widely used in base editing. Exemplary adenine deaminases in this invention include, but are not limited to, ABE8e, ABE8.20, or ABE7.1, or combinations thereof. Preferably, the adenine deaminase is ABE8e.

[0079] ABE8e may contain the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:4.

[0080] ABE8.20 may contain the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:5.

[0081] ABE7.1 may contain the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:6.

[0082] In some embodiments, the adenine methyltransferase and / or adenine deaminase are recombinantly generated. In some embodiments, the adenine methyltransferase and / or adenine deaminase also contain a fusion tag, such as a tag for the isolation and / or purification of the enzyme. Methods for recombinantly generating proteins are known in the art. Furthermore, various tags that can be used for the isolation and / or purification of proteins are known in the art, including but not limited to His tags, SMT3 tags, GST tags, etc. Generally, these tags do not alter the activity of the target protein.

[0083] Preferably, the adenine methyltransferase and / or adenine deaminase are nonspecific, i.e., they do not have substrate sequence preference.

[0084] In some embodiments, step b) is carried out in a methylation reaction system, for example, in a methylation reaction system of about 10 µl to about 100 µl, preferably 50 µl. The methylation reaction system can be adjusted according to the number of cells or cell nuclei.

[0085] In some embodiments, the at least one cell is from one cell to about 50,000 cells or more, such as about 100, about 1,000, about 10,000, about 50,000 cells or more.

[0086] In some embodiments, the methylation reaction system of step b) comprises a methylation reaction buffer. For example, the methylation reaction buffer should contain a methyl donor molecule, such as S-adenosyl-methionine.

[0087] In some embodiments, the methylation reaction buffer may contain approximately 15 mM Tris-HCl (pH 8.0), approximately 15 mM NaCl, approximately 60 mM KCl, approximately 1 mM EDTA (pH 8.0), approximately 0.5 mM EGTA (pH 8.0), approximately 0.5 mM Spermidine, and approximately 0.8 mM S-adenosyl-methionine.

[0088] In some preferred embodiments, the methylation reaction system or the methylation reaction buffer does not contain metal salts. In some preferred embodiments, the methylation reaction system or the methylation reaction buffer does not contain NaCl and / or KCl.

[0089] In some preferred embodiments, the methylation reaction buffer may contain approximately 15 mM Tris-HCl (pH 8.0), approximately 1 mM EDTA (pH 8.0), approximately 0.5 mM EGTA (pH 8.0), approximately 0.5 mM Spermidine, and approximately 0.8 mM S-adenosyl-methionine.

[0090] In this article, the concentrations of various components in the reaction buffers refer to their final concentrations in the reaction system.

[0091] The various reaction buffers of the present invention may also contain protease inhibitors to prevent enzyme degradation. The protease inhibitors may be commercially available protease inhibitors, and those skilled in the art can use them according to the instructions or modify the dosage of the protease inhibitor according to the actual amount of enzyme used.

[0092] In some specific embodiments, the methylation reaction buffer may contain 2x of a commercially available PIC (protease inhibitor mixture). For example, the PIC described in this context may be cOmplete™ EDTA-free Protease Inhibitor Cocktail, available from Roche, trade number 04693132001.

[0093] In some embodiments, the methylation reaction system of step b) contains about 0.1 U / µl to about 100 U / µl, preferably about 0.2 U / µl to 4 U / µl of the adenine methyltransferase.

[0094] The enzyme activity unit U of adenine methyltransferase used in this article is defined as the amount of enzyme such as Hia5 that completely reacts 200 ng of substrate in a 10 µl volume at 37 °C for 1 h, wherein the substrate is a double-stranded DNA fragment (approximately 1.3 kb in length and containing two GATC sites) with the sequence shown in SEQ ID NO:7.

[0095] In some embodiments, in step b), the cells or cell nuclei are treated with adenine methyltransferase for about 5 minutes to about 60 minutes, preferably about 10 minutes to about 30 minutes, more preferably about 10 minutes.

[0096] In some embodiments, step b) is performed at a temperature of about 4°C to about 40°C, for example about 30°C to about 40°C, preferably about 37°C.

[0097] In some preferred embodiments, in step b), approximately 50,000 cells or cell nuclei from approximately 50,000 cells are treated with approximately 10 U or approximately 50 U of adenine methyltransferase at approximately 37°C for approximately 30 minutes in a reaction system of approximately 50 µl.

[0098] Various methods for isolating genomic DNA from cells or cell nuclei are known in the art, and all of them can be applied to this invention. An exemplary method is, for example, the conventional phenol:chloroform:isoamyl alcohol extraction method. Commercially available genomic DNA extraction kits can also be used.

[0099] In some implementations, step d) may involve first fragmenting the genomic DNA and then treating the fragmented DNA with adenine deaminase.

[0100] In some embodiments, step d) is carried out in a deamination reaction system, for example, in a deamination reaction system of about 10 µl to about 100 µl, preferably 10 µl. The deamination reaction system can be adjusted according to the amount of DNA.

[0101] In some embodiments, the deamination reaction system in step d) comprises a deamination reaction buffer. For example, the deamination reaction buffer may contain approximately 20 mM Tris-HCl (pH 7.5); approximately 100 mM KCl; approximately 5% glycerol; approximately 2.5 mM MgSO4; and approximately 2 mM DTT.

[0102] In some preferred embodiments, the deamination reaction system and / or the deamination reaction buffer does not contain metal salts, such as KCl and / or MgSO4. In some preferred embodiments, the deamination reaction buffer also contains Triton X-100, for example, about 0.1% Triton X-100. In some preferred embodiments, the deamination reaction buffer contains about 20 mM Tris-HCl (pH 7.5); about 5% glycerol; about 2 mM DTT; and about 0.1% Triton X-100.

[0103] In some embodiments, the DNA is treated with adenine deaminase at a concentration of approximately 0.1 U / µl to approximately 10 U / µl, preferably approximately 1 U / µl.

[0104] The enzyme activity unit U of adenine methyltransferase used in this article is defined as the amount of enzyme that completely reacts all A atoms on a 0.1 µM substrate in a 10 µL volume at 44 °C for 2 h, wherein the substrate is a single-stranded DNA with the sequence shown in SEQ ID NO:8. The substrate used to determine enzyme activity may also have a fluorescent group (such as FAM) at its 5' end.

[0105] In some embodiments, the DNA is treated with adenine deaminase in step d) for about 30 minutes to about 180 minutes, preferably about 120 minutes.

[0106] In some embodiments, step d) is carried out at a temperature of about 4°C to about 50°C, preferably about 44°C.

[0107] In some preferred embodiments, in step d), approximately 10 ng of DNA is treated with approximately 1 U / µl of adenine deaminase at approximately 44°C for approximately 120 minutes in approximately 10 µl of reaction system.

[0108] In some embodiments, step d) further includes the inactivation and / or removal of adenine deaminase after treatment. For example, the product obtained after adenine deaminase treatment can be treated with a detergent such as SDS and / or a protease. In some embodiments, step d) further includes treating the product obtained after adenine deaminase treatment with approximately 1% SDS and approximately 0.2 mg / ml Protase K at approximately 50°C for approximately 30 minutes. In some embodiments, step d) further includes the separation and / or purification of DNA, for example, by conventional phenol:chloroform:isoamyl alcohol extraction or using a commercially available DNA extraction / purification kit.

[0109] In some implementations, step d) is performed two or more times, preferably twice.

[0110] In some implementations, the amplification described in step e) is PCR amplification.

[0111] In some implementations, step e) includes enriching and / or amplifying specific portions of genomic DNA from the deaminated DNA of step d) and sequencing them to determine the chromatin accessibility and / or DNA-binding protein footprint of the specific portions of the cell genome.

[0112] In some implementations, step e) includes constructing a DNA library from the deaminated DNA of step d) and sequencing the DNA library to determine the chromatin accessibility and / or DNA-binding protein footprint of the cell genome (e.g., at the genome level).

[0113] Various methods known in the art for constructing DNA sequencing libraries from DNA can be used, including single-stranded DNA library construction and double-stranded DNA library construction. DNA sequencing libraries can be constructed using commercially available kits; for example, single-stranded library construction can use Novizan NE103, Swift Biosciences Cat. No. 33096, etc.; double-stranded library construction can use Tn5 library construction, such as Novizan TD202 kit; and WGBS library construction can use Zymo Research D5455 & D5456 kits.

[0114] In some preferred embodiments, DNA sequencing libraries are constructed from DNA using single-stranded DNA library construction.

[0115] In some specific implementations, the single-stranded DNA library is constructed using the EpiArt® DNA Methylation Library Kit for Illumina V3 NE103.

[0116] The sequencing described in this article can be first-generation sequencing such as Sanger sequencing, second-generation sequencing (NGS), or other high-throughput sequencing. For example, Illumina's second-generation sequencing can be used.

[0117] DNA in open chromatin regions is more readily methylated by adenine methyltransferases, while DNA bound to nucleosomes or DNA-binding proteins is protected and cannot be methylated. During subsequent treatment with adenine deaminase, unmethylated adenine (A) is deaminated to hypoxanthine (I). Hypoxanthine is then recognized by DNA polymerase as guanine (G) during subsequent PCR amplification, pairing complementary with cytosine (C). Ultimately, unmethylated adenine is replaced by guanine, resulting in mutations from A to G. Methylated adenine, however, is not deaminated and does not mutate after PCR amplification. Therefore, by analyzing the distribution of methylation and / or deamination sites, we can determine the openness of chromatin, the arrangement of nucleosomes, and the location and binding dynamics of DNA-binding proteins.

[0118] In some embodiments, the chromatin accessibility and / or DNA-binding protein footprint of the cellular genomic DNA or its portion are determined by analyzing the presence of AT-GC transitions (i.e., A to G and T to C) in the cellular genomic DNA or its portion relative to control genomic DNA or its portion.

[0119] The presence of AT-to-GC transitions in specific regions of the genome indicates that the chromatin in those regions is accessible and / or not bound by DNA-binding proteins.

[0120] In some embodiments, the chromatin accessibility and / or DNA-binding protein footprint of the cellular genomic DNA or its portion are determined by analyzing the location and / or density and / or conversion rate of AT to GC transitions relative to control genomic DNA or portions thereof.

[0121] The location and / or density of AT to GC transitions in the genome indicate the location and / or degree of openness of open chromatin regions.

[0122] The sequence of the control genomic DNA or a portion thereof may be a sequence of genomic DNA or a portion thereof from a public database. Alternatively, the sequence of the control genomic DNA or a portion thereof may also be a sequence of genomic DNA or a portion thereof from the same type of cells that have not been treated with the DNA deaminase.

[0123] The DNA-binding proteins described in this article can be transcription factors, histones, etc., preferably transcription factors such as CTCF, DBP, FOXA2, GATA4, HNF1A, TEF, HLF, HNF1a, etc.

[0124] The cells described herein can be animal cells, plant cells, or microbial cells. For example, the cells are mammalian cells, including but not limited to human, mouse, rat, cat, dog, pig, and cow cells; or, the cells are monocotyledonous or dicotyledonous plant cells, such as rice, corn, wheat, sorghum, soybean, potato, and tomato cells; or, the cells are fungal cells such as yeast cells.

[0125] The cells may be cell lines. Alternatively, the cells may be primary cells derived from different organs or tissues. For example, the cells may be derived from blood, cerebrospinal fluid, or biopsy tissue. The cells may be hepatocytes, cardiomyocytes, neurons, fibroblasts, epithelial cells, etc. The cells may also be tumor cells, stem cells such as embryonic stem cells, or induced pluripotent stem cells.

[0126] In some embodiments, the cells are cells treated with a specific substance (compound) or condition, or cells at a specific developmental stage. Therefore, the method of the present invention can determine the effect of this specific substance (compound) or condition, or specific developmental stage, on the cellular genome chromatin accessibility and / or DNA-binding protein footprint. For example, the specific substance may be a drug.

[0127] III. Reagent Kit In another aspect, the present invention provides a kit for detecting chromatin accessibility and / or DNA-binding protein footprints in a cellular genome, comprising at least the adenine methyltransferase and adenine deaminase described above herein.

[0128] In some embodiments, the kit is used to detect chromatin accessibility and / or DNA-binding protein footprint of a cell genome using the methods of the present invention.

[0129] In some embodiments, the kit further includes a permeation buffer and / or a nuclear extraction buffer for isolating cell nuclei. The permeation buffer and the nuclear extraction buffer are as defined above.

[0130] In some embodiments, the kit further includes a methylation reaction buffer, as defined above.

[0131] In some embodiments, the kit further includes a deamination reaction buffer, which is as defined above.

[0132] Those skilled in the art will understand that the concentration of the reaction buffer components in the kit may be higher than the final concentration in the reaction system, thereby facilitating the preparation of the final reaction system.

[0133] In some embodiments, the kit also includes reagents for amplifying the genomic DNA portion of interest, such as specific primers.

[0134] In some implementations, the kit also includes reagents for constructing a DNA library.

[0135] In some implementations, the kit also includes reagents for sequencing, such as Sanger sequencing or next-generation sequencing.

[0136] Kits typically also include labels indicating the intended use and / or method of use of the kit contents. Labels include any written or recorded material provided on or with the kit, or otherwise accompanying the kit.

[0137] Example The techniques used in the following embodiments, including sequencing library construction and high-throughput sequencing, molecular biology techniques such as cell fixation and DNA purification, and cell culture and detection techniques, are all conventional techniques known to those skilled in the art, unless otherwise specified. The instruments, equipment, reagents, and cell lines used, unless specifically noted in this specification, are generally available to researchers and those skilled in the art through public channels. In the examples listed in the specific implementation methods, the cell collection, processing, and single-cell isolation schemes, as well as some buffer systems, represent only a few of many feasible processing schemes.

[0138] Example 1: Detection of chromatin accessibility and transcription factor footprint based on the Hia5 and ABE8e dual-enzyme method This invention establishes a novel technique for detecting chromatin accessibility and transcription factor footprint based on a dual-enzyme method using Hia5 and ABE8e. Hia5 is used to methylate A in open chromatin regions and accessible sites around transcription factor binding sites. Then, ABE8e deaminates the unmodified A to I. I pairs with C during PCR, causing an A to G mutation. Since ABE8e cannot deaminate methylated A, it can distinguish A methylation information. Thus, by processing the extracted genome with ABE8e, chromatin structural information can be converted into DNA sequence information. After amplification, next-generation sequencing and bioinformatics analysis can yield multidimensional whole-genome information. This method is also known as A-FOOT-seq (DNA A-deaminase-based assay for TF footprint by sequencing), and a flowchart is shown below. Figure 1 .

[0139] Experimental methods: 1. Purification of Hia5 and ABE8e proteins After constructing a Hia5 fusion protein particle containing a 6×His tag, it was transformed into the Rosetta strain and cultured at 37°C until the OD value reached approximately 0.6-0.8. 500 μL of 1M IPTG was added to 1 L of bacterial culture to induce Hia5 protein expression in *E. coli*, and the expression was induced at 20°C for 4 hours. Subsequently, the bacterial cells were collected and lysed using an ultra-high pressure cell disruptor. The cell lysate supernatant was incubated with Ni-NTA magnetic beads for 3 hours, and the Hia5 protein was purified in vitro by affinity chromatography to obtain a high concentration of the enzyme.

[0140] After constructing an ABE8e fusion protein particle containing the Strep-Tactin tag, it was transformed into the Rosetta strain and cultured at 37°C until the OD value reached approximately 0.6-0.8. 500 µl of 1M IPTG was added to 1 L of bacterial culture to induce ABE8e protein expression in *E. coli*, and the expression was induced at 16°C for 12 hours. Subsequently, the bacterial cells were collected, and *E. coli* were lysed using an ultra-high pressure cell disruptor. The ABE8e protein was purified in vitro using affinity chromatography with desulfobiotin to obtain a high concentration of the enzyme.

[0141] 2. Detailed A-FOOT-seq process (1) Cell collection and nucleus extraction: After cell growth is satisfactory, add an appropriate amount of trypsin to digest the cells, neutralize the culture medium, count the cells, transfer them to 1.5 mL low-adsorption EP tubes, centrifuge at 300 × g for 4 min at room temperature, and discard the supernatant. Add 1 mL of cold PBS + 0.04% BSA to wash once, centrifuge at 300 × g for 4 min at 4 °C, and discard the supernatant. Repeat the process at 50 µl / 5 x 10⁻⁵ cells / tubes.4 Cells were resuspended in the corresponding volume of Nuclei Extraction Buffer (10 mM Tris-HCl 7.4, 10 mM NaCl, 1% BSA, 0.1% Tween-20, 0.1% NP40, 0.01% Digitonin, 0.1 mM EDTA, 3 mM MgCl2, 1xPIC), gently pipetted three times, and incubated on ice for 5 min. The reaction was terminated by adding 1 ml of Final Buffer (10 mM Tris-HCl 7.4, 10 mM NaCl, 1% BSA, 0.1 mM EDTA, 1xPIC), gently inverting to mix three times, incubating at 4°C, 700×g, for 5 min, centrifuging inverted mode for 1 min, removing the supernatant to approximately 100 µl, centrifuging again inverted mode for 1 min, and completely discarding the supernatant.

[0142] (2) Hia5 methylation reaction and optimization: Original methylation reaction steps: Add 50µl of 1x No Hia5 Reaction Buffer (15mM Tris-HCl pH 8.0, 15mM NaCl, 60mM KCl, 1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 0.5mM Spermidine, 0.8mM S-adenosyl-methionine, 2xPIC) to resuspend the precipitate, then incubate at 4°C, 700×g for 5 min, centrifuge for 1 min by inversion, remove the supernatant to about 100µl, centrifuge again by inversion for 1 min, and completely discard the supernatant. Add 50µl of deaminase reaction system (15mM Tris-HCl 8.0, 15mM NaCl, 60mM KCl, 1mM EDTA, 0.5mM EGTA pH8.0, 0.5mM Spermidine, 0.8mM S-adenosyl-methionine, 2xPIC, 10U / 30U / 50U / 100U / 150U / 200U Hia5), react at 37℃ for 10 min, and shake at 1000rpm on a hot plate.

[0143] Optimization of methylation reaction steps: Tests revealed that salt concentration affects the activity of adenine methyltransferase. Figure 10 Therefore, the optimized reaction system does not contain salt components, namely 15 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0; 0.5 mM EGTA, pH 8.0; 0.5 mM Spermidine; 0.8 mM S-adenosyl-methionine.

[0144] (3) Genomic DNA extraction Following this, a heat incubation was performed. 200 µl of gDNA Hot Elution Buffer (100 mM NaHCO3, 1% SDS) was added to a 50 µl reaction mixture, and the mixture was incubated at 65 °C and 1350 rpm for 1.5 h with gentle shaking. An equal volume (250 µl) of phenol:chloroform:isoamyl alcohol DNA extraction solution was added, and the mixture was thoroughly shaken to mix. The mixture was centrifuged at 13000 g for 5 min at room temperature. The supernatant was collected, and gDNA was precipitated with isopropanol. The concentration was determined using QUBIT.

[0145] (4) ABE8e deamination reaction and optimization: Take 10 ng of ssDNA for ABE8e deamination reaction. Dilute 10 ng of DNA with water to 7 µl, denature at 98 °C for 10 min, immediately place on ice for 2 min, then add 2 µl of 5xDL buffer (100 mM Tris-HCl pH=7.5, 500 mM KCl, 25% glycerol, 12.5 mM MgSO4; 10 mM DTT) and 1 µl of ABE8e (10 U / µl), react at 37 °C for 2 h, and denature at 85 °C for 15 min.

[0146] Deamination reaction optimization: The reaction buffer for primitive adenine deaminase ABE8e (final concentration in the reaction system) was: 20 mM Tris-HCl (pH 7.5); 100 mM KCl; 5% glycerol; 2.5 mM MgSO4; 2 mM DTT. Tests showed that 0.1% Triton X-100 significantly improved enzyme activity. Figure 11 A). Furthermore, it was found that salt concentration affects enzyme activity ( Figure 11 B), therefore, the concentrations of KCl and MgSO4 were reduced to 0. The optimized enzyme activity reaction system was thus: 20 mM Tris-HCl (pH 7.5); 5% glycerol; 2 mM DTT; 0.1% Triton X-100.

[0147] In addition, tests revealed that ( Figure 11 C) The enzyme activation reaction temperature was increased from 37℃ to 44℃, which also improved the enzyme activation effect.

[0148] Furthermore, it was found that ABE8e has the ability to bind DNA, therefore the deamination efficiency is affected by the occupancy of ABE8e on the DNA. Therefore, the reaction conditions were optimized. First, the extracted genomic DNA was reacted with ABE8e and then treated with 0.1% SDS and 0.2 mg / ml Protase K at 50℃ for 30 minutes. Subsequently, genomic DNA was extracted again (an equal volume of phenol:chloroform:isoamyl alcohol DNA extraction buffer was added, and the mixture was thoroughly shaken and centrifuged at 13000g for 5 minutes at room temperature; the supernatant was collected and isopropanol precipitated to obtain the first-round deamination DNA) and incubated a second time with ABE8e under the same conditions. Data showed that the deamination efficiency of ABE8e in the second round of deamination reaction was significantly improved, increasing from 83.38% to 95.56%.

[0149] 3. Document Library Establishment After the second round of deamination, single-stranded DNA library construction was performed directly using Novizan's EpiArt® DNA Methylation Library Kit for Illumina V3 NE103. (See detailed procedure below) Figure 2 ).

[0150] 4. Analyze A-FOOT-seq data to determine genome-wide chromatin accessibility and transcription factor footprint information. Bioinformatics analysis is used to analyze and calculate base transition sites and their quantities. Methylated sites attacked by Hia5 in cells are considered open regions, while sites that have not undergone transformation are considered closed regions or sites with protein binding. This allows us to determine chromatin accessibility sites, transcription factor footprints, and the status of proteins binding to specific sites.

[0151] Experimental results HepG2 cells were processed using the optimized method of this invention, followed by library construction, sequencing, and data analysis. The results are as follows: 1. Analysis of chromatin accessibility and nucleosome arrangement at transcription start sites in HepG2 cells using A-FOOT-seq A-FOOT-seq analysis was used to analyze the changes in methylation rate of Hia5 in the genebody region and 1 kb upstream and downstream of the TSS under different enzyme concentrations. Figure 3 It can be seen that the methylation rate is higher at the TSS site, indicating that this area is more open, and a clear nucleosome localization pattern can be seen in the right figure.

[0152] 2. Analysis of CTCF sites and chromatin accessibility in HepG2 cells using A-FOOT-seq A-FOOT-seq analysis of methylation rate changes in CTCF upstream and downstream of 1kb and 100bp ( Figure 4The binsize values ​​in the top images are 20bp and 1bp, respectively. In the top left image, the indentation at the center of CTCF is clearly visible, indicating this is the CTCF binding site. The bulges on both sides indicate openness, and the nucleosome localization pattern is clearly visible. In the top right image, the central protrusion indicates a bias. The bottom image shows the methylation rate change 1kb upstream and downstream of ATAC. The higher methylation rate at the ATAC center indicates a more open site, demonstrating that the two-enzyme method can capture open sites in the genome.

[0153] 3. Detection of DBP transcription factor footprint in HepG2 cells using A-FOOT-seq A-FOOT-seq technology was used to detect changes in methylation rates of DBP transcription factors 1 kb upstream and downstream and 100 bp upstream and downstream in HepG2 cells. Figure 5 The binsizes were 20bp and 1bp, respectively. A clear footprint can be seen at the DBP center, indicating that the dual-enzyme method can obtain transcription factor footprint information.

[0154] 4. Detection of the footprint of FOXA2 transcription factor in HepG2 cells using A-FOOT-seq A-FOOT-seq technology was used to detect changes in methylation rates of the FOXA2 transcription factor 1 kb upstream and downstream and 100 bp upstream and downstream in HepG2 cells. Figure 6 The binsizes were 20bp and 1bp, respectively. A clear footprint can be seen at the center of FOXA2, indicating that the dual-enzyme method can obtain transcription factor footprint information.

[0155] 5. Detection of GATA4 transcription factor footprint in HepG2 cells using A-FOOT-seq A-FOOT-seq technology was used to detect changes in methylation rates of GATA4 transcription factor 1 kb upstream and downstream and 100 bp upstream and downstream in HepG2 cells. Figure 7 The binsizes were 20bp and 1bp, respectively. A clear footprint can be seen at the center of GATA4, indicating that the dual-enzyme method can obtain transcription factor footprint information.

[0156] 6. Detection of HNF1A transcription factor footprint in HepG2 cells using A-FOOT-seq A-FOOT-seq technology was used to detect changes in methylation rates of HNF1A transcription factor 1 kb upstream and downstream and 100 bp upstream and downstream in HepG2 cells. Figure 8 The binsizes were 20bp and 1bp, respectively. A clear footprint can be seen at the HNF1A center, indicating that the dual-enzyme method can obtain transcription factor footprint information.

[0157] 7. Detection of TEF transcription factor footprint in HepG2 cells using A-FOOT-seq A-FOOT-seq technology was used to detect changes in methylation rates of TEF transcription factors 1 kb upstream and downstream and 100 bp upstream and downstream in HepG2 cells. Figure 9 The binsizes were 20bp and 1bp, respectively. A clear footprint can be seen at the TEF center, indicating that the dual-enzyme method can obtain transcription factor footprint information.

[0158] The experimental results above demonstrate that the method of this invention can simultaneously capture open chromatin regions of the cellular genome and detect transcription factor binding footprints within these regions, yielding highly reproducible experimental data. Compared to methods based on the methyltransferase Hia5 for whole-genome chromatin accessibility detection, next-generation sequencing reduces sequencing costs, amplifies genes, and captures transcription factor binding information even at lower sequencing depths, potentially enabling single-cell-level detection. By combining A-FOOT-seq with technologies such as ATAC-seq, it is hoped that the unique advantage of A-FOOT-seq in capturing transcription factor footprints can be leveraged at the single-cell level, thereby achieving higher throughput and capturing multi-omics information such as chromatin accessibility, transcription factor footprints, and transcriptome data. This is of significant importance for constructing gene regulatory networks in dynamic processes such as cell fate transitions, development, and carcinogenesis.

[0159] Example 2: Detection of chromatin accessibility and transcription factor footprint based on the EcoGII and ABE8e dual-enzyme method First, following the reaction conditions of Example 1, EcoGII was used instead of Hia5 and combined with ABE8e in HepG2 cells for aFOOT-seq experiments.

[0160] 1. Analysis of chromatin accessibility and nucleosome arrangement at transcription start sites in HepG2 cells using A-FOOT-seq A-FOOT-seq analysis was used to analyze the changes in methylation rate of EcoGII in the genebody region and 1 kb upstream and downstream of the TSS under different enzyme concentrations. Figure 12 It can be seen that the methylation rate is higher at the TSS site, indicating that this area is more open, and a clear nucleosome localization pattern can be seen in the right figure.

[0161] 2. Chromatin openness analysis of ATAC-rich regions in HepG2 cells using A-FOOT-seq Figure 13The figure shows the change in methylation rate 1 kb upstream and downstream of ATAC. The higher methylation rate at the ATAC center indicates that this area is more open, suggesting that the dual-enzyme method can capture open sites in the genome.

[0162] 3. Analysis of chromatin accessibility and nucleosome arrangement at the CTCF site in HepG2 cells using A-FOOT-seq Accessibility of the CTCF site was analyzed in Hepg2 cells using different amounts of EcoGII enzyme. The methylation rate changes at 1 kb upstream and downstream of the CTCF center, and at 0.1 kb upstream and downstream, are shown, with binsizes of 20 bp and 1 bp, respectively. Results are shown below. Figure 14 In the left image, the depression at the center of the CTCF is clearly visible, indicating that this is the CTCF binding site. The protrusions on both sides indicate openness, and the nucleosome localization pattern is clearly visible. In the right image, the protrusion in the center indicates a bias.

[0163] 4. Detection of transcription factor footprints in HepG2 cells using A-FOOT-seq Figure 15 This is an A-FOOT-seq analysis of the methylation rate changes of transcription factors CTCF, ATF4, DBP, FOXC1, HNF1A, and HOXA10 within 0.5 kb upstream and downstream, with a binsize value of 1 bp. A clear footprint can be seen at the center, indicating that this two-enzyme method can obtain transcription factor footprint information.

[0164] 5. aFOOT-seq of EcoGII and EcoGII homologs on HepG2 cells CTCF sites and chromatin accessibility were analyzed in HepG2 cells using EcoGII and its homologs (EcoGII291, EcoGII289, EcoGII284, and EcoGII242). Results are shown below. Figure 16 Similar to EcoGII, different EcoGII homologs can clearly detect CTCF binding sites and capture open sites in the genome.

[0165] Example 3: Preference Analysis of EcoGII and Hia5 Naked DNA was treated with EcoGII and Hia5, respectively, to analyze the A to G transformation efficiency and site methylation preference. Figure 17The Seplpgo plot is a superposition of all A sites, with the vertical axis height representing the information content (conservatism) at that position; the higher the height, the more conservative the site. It can be observed that, under similar methylation efficiencies (EcoGII methylation efficiency is 68.46%, and Hia5 methylation efficiency is 63.78%), EcoGII and Hia5 have higher methylation efficiencies for the GA base environment than for the TA base environment, and EcoGII shows less bias in methylation efficiency than Hia5.

[0166] Figure 18 A heatmap analysis of tribase methylation rates centered on A was performed. All tribases with A at the midpoint in the reference genome (5'-NCN-3', 16 combinations: AAA, AAC, AAG, AAT, CAA, CAC, CAG, CAT, GAA, GAC, GAG, GAT, TAA, TAC, TAG, TAT) were treated as a basic unit. The methylation ratio of adenine (A) at the midpoint within each unit was statistically analyzed, visualized using a heatmap, and its sequence preference was interpreted. In treatment groups with different overall methylation rates, both EcoGII and Hia5 showed poor methylation efficiency for base units such as TAA or AAA, while EcoGII exhibited a lower methylation preference than Hia5. aFOOT-seq preferred EcoGII.

[0167] This invention relates to partial sequence information: SEQ ID NO:1 Hia5 SEQ ID NO:2 Dam SEQ ID NO:3 EcoGII SEQ ID NO:4 ABE8e SEQ ID NO:5 ABE8.20 SEQ ID NO:6 ABE7.10 SEQ ID NO:7 Substrate sequence for adenine methyltransferase activity assay SEQ ID NO:8 Substrate sequence for adenine deaminase activity assay SEQ ID NO:9 EcoGII291 amino acid sequence SEQ ID NO:10 EcoGII289 amino acid sequence SEQ ID NO:11 EcoGII284 amino acid sequence SEQ ID NO:12 EcoGII242 amino acid sequence.

Claims

1. A method for detecting chromatin accessibility and / or DNA-binding protein footprint of a cellular genome, the method comprising: a) Permeabilizing at least one of the cells, or isolating the nucleus from at least one of the cells; b) Treat the permeable cells or isolated cell nuclei obtained in step a) with adenine methyltransferase, thereby allowing the adenine methyltransferase to fully react with the open regions of the genomic DNA in the cells; c) Isolate genomic DNA from the product of step b); d) Treat the genomic DNA obtained in step c) with adenine deaminase; e) Amplify and sequence the adenine deaminase-treated DNA or portions thereof obtained in step d); and f) Based on the sequencing results of step e), determine the chromatin accessibility and / or DNA-binding protein footprint of the cell genome.

2. The method of claim 1, wherein cells are treated with a permeation buffer / nuclear extraction buffer containing a detergent to permeate at least one of the cells or to separate the nucleus from at least one of the cells, for example, the permeation buffer / nuclear extraction buffer containing approximately 10 mM Tris-HCl 7.4, approximately 10 mM NaCl, approximately 1% BSA, approximately 0.1% Tween-20, approximately 0.1% NP40, approximately 0.01% Digitonin, approximately 0.1 mM EDTA, approximately 3 mM MgCl2, and approximately 1 x PIC.

3. The method of claim 1 or 2, wherein the adenine methyltransferase is selected from Hia5, Dam, EcoGII, or a combination thereof; preferably, the adenine methyltransferase is Hia5, more preferably EcoGII; For example, Hia5 comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:1; or The Dam contains the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:2; or The EcoGII comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:

3. Preferably, the EcoGII comprises the amino acid sequence shown in one of SEQ ID NO:3 or SEQ ID NO:9-12.

4. The method of any one of claims 1-3, wherein the adenine deaminase is selected from ABE8e, ABE8.20, ABE7.1, or a combination thereof; preferably, the adenine deaminase is ABE8e; For example, the ABE8e comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:4; or The ABE8.20 comprises the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:5; or The ABE7.1 comprises the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:

6.

5. The method of any one of claims 1-4, wherein step b) is carried out in a methylation reaction system of about 10 µl to about 100 µl, preferably 50 µl, preferably... The methylation reaction system in step b) contains a methyl donor molecule, such as S-adenosyl-methionine; and / or The methylation reaction system does not contain metal salts; preferably, the methylation reaction system does not contain NaCl and / or KCl; and / or The methylation reaction system described herein comprises approximately 15 mM Tris-HCl (pH 8.0), approximately 1 mM EDTA (pH 8.0), approximately 0.5 mM EGTA (pH 8.0), approximately 0.5 mM Spermidine, and approximately 0.8 mM S-adenosyl-methionine; and / or The methylation reaction system in step b) comprises approximately 0.1 U / µl to approximately 100 U / µl, preferably approximately 0.2 U / µl to 4 U / µl of the adenine methyltransferase; and / or In step b), the cells or cell nuclei are treated with adenine methyltransferase for approximately 5 minutes to approximately 60 minutes, preferably approximately 10 minutes to approximately 30 minutes, more preferably approximately 10 minutes; and / or Step b) is performed at a temperature of approximately 4°C to approximately 40°C, for example, approximately 30°C to approximately 40°C, preferably approximately 37°C; and / or In step b), approximately 50,000 cells or cell nuclei from approximately 50,000 cells are treated with approximately 10 U or approximately 50 U of adenine methyltransferase at approximately 37°C for approximately 30 minutes in a reaction system of approximately 50 µl.

6. The method of any one of claims 1-5, wherein in step d), the genomic DNA is first fragmented, and then the fragmented DNA is treated with adenine deaminase; and / or Step d) is carried out in a deamination reaction system of approximately 10 µl to approximately 100 µl, preferably 10 µl; and / or The deamination reaction system in step d) does not contain metal salts, for example, does not contain KCl and / or MgSO4; and / or, the deamination reaction system also contains Triton X-100, for example, about 0.1% Triton X-100; and / or The deamination reaction system described herein contains approximately 20 mM Tris-HCl (pH 7.5); approximately 5% glycerol; approximately 2 mM DTT; approximately 0.1% Triton X-100; and / or The step d) involves treating the DNA with approximately 1 U / µl to approximately 10 U / µl, preferably approximately 1 U / µl, adenine deaminase; and / or In step d), the DNA is treated with adenine deaminase for approximately 30 minutes to approximately 180 minutes, preferably approximately 120 minutes; and / or Step d) is carried out at a temperature of approximately 4°C to approximately 50°C, preferably approximately 44°C; and / or In step d), approximately 10 ng of DNA is treated with approximately 10 U of adenine deaminase at approximately 44°C for approximately 120 minutes in a reaction system of approximately 10 µl.

7. The method of any one of claims 1-6, wherein step d) further comprises the step of inactivating and / or removing adenine deaminase after treatment; for example, treating the product obtained after adenine deaminase treatment with a detergent such as SDS and / or a protease to inactivate and / or remove adenine deaminase; preferably, treating the product obtained after adenine deaminase treatment with about 0.1% SDS and about 0.2 mg / ml Protase K at about 50°C for about 30 minutes; and / or Step d) further includes the steps of isolating and / or purifying the treated DNA.

8. The method of any one of claims 1-7, wherein step d) is performed two or more times, preferably twice.

9. The method of any one of claims 1-8, wherein the amplification in step e) is PCR amplification.

10. The method of any one of claims 1-9, wherein step e) comprises amplifying a specific portion of genomic DNA from the deaminated DNA in step d) and sequencing it to determine the chromatin accessibility and / or DNA-binding protein footprint of the specific portion of the cell genome, or Step e) includes constructing a DNA library from the deaminated DNA of step d) and sequencing the DNA library to determine the chromatin accessibility and / or DNA-binding protein footprint of the cell genome. Preferably, the DNA sequencing library is constructed from DNA by single-stranded DNA library construction. More preferably, the single-stranded DNA library construction is performed using the EpiArt® DNA Methylation Library Kit for Illumina V3 NE103.

11. The method of any one of claims 1-10, wherein the presence of AT-to-GC transitions in the cellular genomic DNA or its portion is determined by analyzing the presence of AT-to-GC transitions relative to control genomic DNA or its portion; or The chromatin accessibility and / or DNA-binding protein footprint of the cellular genomic DNA or its portion are determined by analyzing the location and / or density and / or conversion rate of AT to GC transitions relative to control genomic DNA or its portion.

12. The method of any one of claims 1-11, wherein the cells are selected from animal cells, plant cells, or microbial cells. For example, the cells are mammalian cells, including but not limited to human, mouse, rat, cat, dog, pig, and cow cells; or the cells are monocotyledonous or dicotyledonous plant cells, such as rice, corn, wheat, sorghum, soybean, potato, tomato, etc.; or the cells are fungal cells such as yeast cells.

13. The method of claim 12, wherein the cell is a cell line; or, the cell is a primary cell from a different organ or tissue, for example, the cell is from blood, cerebrospinal fluid, or biopsy tissue; for example, the cell may be a hepatocyte, cardiomyocyte, neuronal cell, fibroblast, or epithelial cell; or, the cell is a tumor cell, stem cell such as embryonic stem cell or induced pluripotent stem cell.

14. The method of any one of claims 12-13, wherein the cell is a cell treated with a specific substance or condition or a cell at a specific developmental stage, for example, the specific substance being a drug.

15. A kit for detecting chromatin accessibility and / or DNA-binding protein footprint of a cellular genome by the method of any one of claims 1-14, comprising adenine methyltransferase and adenine deaminase.

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