Methods and compositions relating to cell cycle RNA

Abstract

The present disclosure relates to compositions and methods for treating cancer in a subject in need thereof, which relate to specific non-coding RNA (ncRNA)-S phase early RNA (SPEAR) or inhibitors thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 180,756, filed April 28, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present technology relates to methods and compositions relating to specific non-coding RNA (ncRNA)-S phase early RNA (SPEAR).

[0003] Government Interests This invention was made with Government support under Grants CA197697 and CA211304 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0004] Description of electronically submitted text files This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on or about April 18, 2022, is named "BID-011PC_110304-5011_Sequence_Listing_ST25.txt" and is approximately 19,732 bytes in size. [Background technology]

[0005] Functional noncoding RNAs (ncRNAs) are essential components of chromatin structure shaped by epigenetic marks such as DNA methylation, histone modifications, nucleosome positioning, and incorporation of histone variants into nucleosomes. Despite their importance, many questions remain unanswered about the biology of ncRNAs.

[0006] Aberrant epigenetic signatures are associated with deleterious conditions such as cancer and other genetic disorders, however, little is known about active versus inactive epigenetic marks and how they influence deleterious conditions.

[0007] A more complete understanding of ncRNA and epigenetic regulation is needed to enable effective therapies. Summary of the Invention

[0008] Thus, in some embodiments, the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising: (i) administering to the subject an effective amount of one or more inhibitors of S phase early RNA (SPEAR); or (ii) administering an effective amount of cells from the subject that have been contacted with an effective amount of one or more inhibitors of SPEAR, wherein the cancer is characterized by epigenetic dysregulation.

[0009] In embodiments, the disclosure provides a method for preventing the onset or progression of cancer in a subject in need thereof, the method comprising: (i) administering to the subject an effective amount of one or more inhibitors of SPEAR; or (ii) administering an effective amount of cells from the subject that have been contacted with an effective amount of one or more inhibitors of SPEAR, wherein the subject is characterized by a precancerous condition involving epigenetic dysregulation.

[0010] In embodiments, the disclosure provides a method for treating a genetic disease or disorder associated with epigenetic dysregulation in a subject in need thereof, comprising (i) administering to the subject an effective amount of one or more inhibitors of SPEAR, or (ii) administering an effective amount of cells from the subject that have been contacted with an effective amount of one or more inhibitors of SPEAR.

[0011] In embodiments, the disclosure provides a method for preventing the onset or progression of a genetic disease or disorder associated with epigenetic dysregulation in a subject in need thereof, comprising (i) administering to the subject an effective amount of one or more inhibitors of SPEAR, or (ii) administering an effective amount of cells from the subject that have been contacted with an effective amount of one or more inhibitors of SPEAR.

[0012] In several embodiments, the disclosure provides a method for resetting the formation of active histone marks in cancer or precancerous cells, the method comprising (i) administering to a subject an effective amount of one or more SPEARs or one or more inhibitors of SPEAR, or (ii) contacting a cell derived from a subject with an effective amount of one or more SPEARs or one or more inhibitors of SPEAR.

[0013] In embodiments, the disclosure provides a method for restoring a replication origin complex associated with a pre-disease state in a cell characterized by a genetic disease or disorder, the method comprising (i) administering to a subject an effective amount of one or more SPEARs or one or more inhibitors of SPEAR, or (ii) contacting a cell derived from a subject with an effective amount of one or more SPEARs or one or more inhibitors of SPEAR.

[0014] In any of the above aspects, the inhibitor causes modulation of the expression level of one or more genes controlled by SPEAR. In some embodiments, the modulation of the expression level of one or more genes controlled by SPEAR is upregulation of the genes. In some embodiments, the modulation of the expression level of one or more genes controlled by SPEAR is downregulation of the genes. In some embodiments, the modulation of the expression level of one or more genes controlled by SPEAR is restoration of the level of the one or more genes compared to an untreated state. In some embodiments, the one or more genes are oncogenes or proto-oncogenes. In some embodiments, the gene is a myc gene. In some embodiments, the myc gene is selected from c-MYC (MYC), l-myc (MYCL), and n-myc (MYCN). In some embodiments, the gene is a tumor suppressor gene.

[0015] In some embodiments, one or more SPEARs are overexpressed to generate an artificial origin of replication complex. In some embodiments, one or more SPEARs are overexpressed to control the direction of replication. In some embodiments, one or more SPEARs are overexpressed and one or more SPEAR inhibitors (i) slow or prevent the progression of the deleterious direction of replication, activate the opposite direction of replication, and / or (ii) modulate the site of a trinucleotide repeat, optionally reducing the size or reversing the expression of the trinucleotide repeat. In some embodiments, one or more SPEARs are overexpressed and one or more SPEAR inhibitors treat or prevent a triplet disease ("TRD"). In some embodiments, the TRD is fragile X syndrome, fragile X-E syndrome, Huntington's disease (HD), spinocerebellar ataxia, movement disorder, dentatorubral-pallidoluysian atrophy, or autism. In some embodiments, the TRD is a polyglutamine (PolyQ) disease and / or a non-polyglutamine disease. In some embodiments, the polyglutamine disease is DRPLA (dentatorubral-pallidoluysian atrophy), HD (Huntington's disease), SBMA (spinal-bulbar muscular atrophy or Kennedy disease), SCA1 (Spinocerebellar ataxia type 1), SCA2 (Spinocerebellar ataxia type 2), SCA3 (Spinocerebellar ataxia type 3 or Machado-Joseph disease), SCA6 (Spinocerebellar ataxia type 6), SCA7 (Spinocerebellar ataxia type 7), or SCA17 (Spinocerebellar ataxia type 17). In some embodiments, the non-polyglutamine disease is FXS (Fragile X Syndrome), FXTAS (Fragile X-associated Tremor / Ataxia Syndrome), FRAXE (Fragile XE Mental Retardation), FRDA (Friedreich's Ataxia), DM (Myotonic Dystrophy), SCA8 (Spinocerebellar Ataxia Type 8), SCA12 (Spinocerebellar Ataxia Type 12), and Premature Ovarian Failure (POF).

[0016] In some embodiments, one or more SPEARs are overexpressed and one or more SPEAR inhibitors treat or prevent TRD by reversing the expansion of trinucleotide repeats.

[0017] In some embodiments, the trinucleotide repeat is selected from CAG, CTG, CGG, and GAA.

[0018] In some embodiments, the inhibitor reduces or substantially eliminates the activity of an epigenetic mark associated with SPEAR.

[0019] In some embodiments, the inhibitor reduces or substantially eliminates the formation and / or recycling of epigenetic marks.

[0020] In some embodiments, an inhibitor reduces or substantially eliminates activation of a gene.

[0021] In some embodiments, the inhibitor causes activation of the gene.

[0022] In some embodiments, the inhibitor reduces or substantially eliminates one or more of DNA methylation, histone modification, and nucleosome remodeling. In some embodiments, the histone modification is selected from one or more of histone acetylation, phosphorylation, methylation, ubiquitination, and proteolysis, and chromatin remodeling modifications. In some embodiments, the histone modification is histone acetylation. In some embodiments, the inhibitor causes regulation of pathogenic nucleotide expansion controlled by SPEAR.

[0023] In some embodiments, the inhibitor reduces or substantially eliminates the interaction between SPEAR and one or more histones or histone-associated proteins. In some embodiments, the histones or histone-associated proteins are one or more of H1, H2A, H2B, H3, and H4 proteins, or variants thereof. In some embodiments, the histones or histone-associated proteins are one or more of H2A.Z or variants thereof, and H3.3 or variants thereof. In some embodiments, the histones or histone-associated proteins are histone acetyltransferases. In some embodiments, the histone acetyltransferases are TIP60 or variants thereof.

[0024] In some embodiments, the inhibitor reduces or substantially eliminates the interaction between SPEAR and one or more components of ORC, hi some embodiments, the one or more components of ORC are selected from one or more of ORC1, ORC2, ORC3, ORC4, and ORC5, or mutants thereof.

[0025] In some embodiments, the epigenetic dysregulation is a dysregulation of one or more epigenetic marks.

[0026] In some embodiments, the epigenetic dysregulation of one or more epigenetic marks comprises activation of additional epigenetic marks compared to a non-disease state and / or inactivation of epigenetic marks compared to a non-disease state. In some embodiments, the epigenetic dysregulation is an altered origin of replication. In some embodiments, the altered origin of replication comprises activation of additional origins of replication compared to a non-disease state and / or inactivation of an origin of replication compared to a non-disease state.

[0027] In some embodiments, the subject is afflicted with a cancer associated with epigenetic dysregulation.

[0028] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological cancer.

[0029] In some embodiments, the cancer is selected from the group consisting of basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, cancer of the peritoneum, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatoma, intraepithelial neoplasia, kidney or renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (lips, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, cancer), testicular cancer, thyroid cancer, uterine or endometrial cancer, cancer of the urinary system, vulvar cancer, Hodgkin and non-Hodgkin lymphomas, and lymphomas including B-cell lymphomas (low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, giant mass disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia (CMYK), as well as other carcinomas and sarcomas, and post-transplant lymphoproliferative disorders (PTLDs), as well as one or more of phacomatosis, abnormal blood vessel proliferation associated with edema, and Meigs' syndrome.

[0030] In some embodiments, SPEAR is a non-coding RNA. In some embodiments, SPEAR is a long non-coding RNA (lncRNA). In some embodiments, SPEAR is about 200 nucleotides or more. In some embodiments, SPEAR is encoded in a region adjacent to the promoter of an active gene. In some embodiments, SPEAR is induced in early S phase of the cell cycle. In some embodiments, SPEAR comprises one or more motifs selected from 3, 5, and 9. In some embodiments, SPEAR comprises one or more RM9A motifs. In some embodiments, SPEAR comprises one or more stem-loop-like structures.

[0031] In some embodiments, the inhibitor is a small molecule. In some embodiments, the small molecule directly or indirectly modulates the interaction of SPEAR with a histone or histone-associated protein or ORC.

[0032] In some embodiments, the inhibitor is a nucleic acid. In some embodiments, the nucleic acid is RNA or DNA. In some embodiments, the nucleic acid directly or indirectly regulates the interaction of SPEAR with histone or histone-associated protein or ORC. In some embodiments, the nucleic acid comprises a sequence that is at least partially complementary to a portion of SPEAR.

[0033] In some embodiments, one or more nucleotides of the inhibitor are chemically modified. In some embodiments, the chemical modification is selected from locked nucleic acid (LNA), phosphorothioate, 2'-O-methyl, 2'-O-methoxyethyl, 2'-O-alkyl-RNA unit, 2'-OMe-RNA unit, 2'-amino-DNA unit, 2'-fluoro-DNA unit, peptide nucleic acid (PNA) unit, hexitol nucleic acid (HNA) unit, INA unit, and 2'-O-(2-methoxyethyl)-RNA (2'MOE RNA) unit.

[0034] In some embodiments, the nucleic acid is an antisense oligonucleotide or a small interfering RNA (siRNA).

[0035] In some embodiments, the inhibitor modulates the expression and / or activity of SPEAR.

[0036] In some embodiments, the cells from the subject are derived from a biological sample. In some embodiments, the biological sample comprises a biopsy, a tissue, or a bodily fluid. In some embodiments, the biological sample comprises one or more of tumor cells, cultured cells, stem cells, and differentiated cells.

[0037] In some embodiments, the method disclosed herein further comprises administering one or more epigenetic drugs or contacting the cells with one or more epigenetic drugs. In some embodiments, the epigenetic drug is a DNA methyltransferase inhibitor, optionally selected from azacitidine, escitabine, zebularine, panobinostat, belinostat, dacinostat, xinostat, tefinostat, acediline, entinostat, mocetinostat, chidamide, butyric acid, pivanex, phenylbutyric acid, and valproic acid. In some embodiments, the epigenetic drug is a histone deacetylase inhibitor, optionally selected from vorinostat, romidepsin, trichostatin A, and trapoxin A.

[0038] In embodiments, the disclosure provides methods of making an epigenetic modulating agent, the method comprising: (a) identifying an epigenetic modulating agent by: (i) determining whether the agent binds to or interacts with one or more SPEARs; (ii) classifying the agent as epigenetic modulating based on its ability to bind to or interact with one or more SPEARs; and (b) formulating the agent for use in a therapy selected from the treatment or prevention of cancer associated with epigenetic dysregulation or a genetic disease or disorder associated with epigenetic dysregulation.

[0039] In some embodiments, the agent reduces or substantially eliminates the activity of epigenetic marks associated with SPEAR. In some embodiments, the agent reduces or substantially eliminates the formation and / or recycling of epigenetic marks. In some embodiments, the agent reduces or substantially eliminates gene activation. In some embodiments, the agent causes gene activation. In some embodiments, the agent reduces or substantially eliminates one or more of DNA methylation, histone modification, and nucleosome remodeling. In some embodiments, the histone modification is selected from one or more of histone acetylation, phosphorylation, methylation, ubiquitination, and proteolysis, and chromatin remodeling modifications. In some embodiments, the histone modification is histone acetylation. In some embodiments, the agent causes the regulation of pathogenic nucleotide expansion controlled by SPEAR. In some embodiments, the agent reduces or substantially eliminates the interaction between SPEAR and one or more histones or histone-associated proteins. In some embodiments, the histone or histone-associated protein is one or more of H1, H2A, H2B, H3, and H4 proteins, or variants thereof. In some embodiments, the histone or histone-associated protein is one or more of H2A.Z or variants thereof, and H3.3 or variants thereof. In some embodiments, the histone or histone-associated protein is a histone acetyltransferase. In some embodiments, the histone acetyltransferase is TIP60 or variants thereof. In some embodiments, the epigenetic dysregulation is a dysregulation of one or more epigenetic marks. In some embodiments, the epigenetic dysregulation of one or more epigenetic marks comprises activation of additional epigenetic marks compared to a non-disease state, and / or inactivation of epigenetic marks compared to a non-disease state.

[0040] In some embodiments, the epigenetic dysregulation is an altered origin of replication.

[0041] In some embodiments, SPEAR is a non-coding RNA. In some embodiments, SPEAR is a long non-coding RNA (lncRNA). In some embodiments, SPEAR is about 200 nucleotides or more. In some embodiments, SPEAR is encoded in a region adjacent to the promoter of an active gene. In some embodiments, SPEAR is induced in early S phase of the cell cycle. In some embodiments, SPEAR comprises one or more motifs selected from 3, 5, and 9. In some embodiments, SPEAR comprises one or more RM9A motifs. In some embodiments, SPEAR comprises one or more stem-loop-like structures.

[0042] In some embodiments, the agent comprises a small molecule. In some embodiments, the small molecule directly or indirectly modulates the interaction of SPEAR with a histone or histone-associated protein or ORC.

[0043] In some embodiments, the agent comprises nucleic acid. In some embodiments, the nucleic acid is RNA or DNA. In some embodiments, the nucleic acid directly or indirectly regulates the interaction of SPEAR with histone or histone-associated protein or ORC. In some embodiments, the nucleic acid comprises a sequence that is at least partially complementary to a portion of SPEAR.

[0044] In some embodiments, one or more nucleotides of the agent are chemically modified. In some embodiments, the chemical modification is selected from locked nucleic acid (LNA), phosphorothioate, 2'-O-methyl, 2'-O-methoxyethyl, 2'-O-alkyl-RNA unit, 2'-OMe-RNA unit, 2'-amino-DNA unit, 2'-fluoro-DNA unit, peptide nucleic acid (PNA) unit, hexitol nucleic acid (HNA) unit, INA unit, and 2'-O-(2-methoxyethyl)-RNA (2'MOE RNA) unit.

[0045] In some embodiments, the nucleic acid is an antisense oligonucleotide or a small interfering RNA (siRNA). In some embodiments, the agent is capable of modulating the expression and / or activity of SPEAR.

[0046] In several embodiments, the disclosure provides methods for assessing a subject's response to an epigenetic modulation therapy, comprising assessing one or more levels of SPEAR in a biological sample from the subject, where (i) a reduction in one or more levels of SPEAR compared to a pre-treatment state indicates a response to the therapy, and / or (ii) an increase or substantially unchanged level of SPEAR compared to a pre-treatment state indicates no or poor response to the therapy.

[0047] In embodiments, the disclosure provides methods for predicting a subject's likely response to an epigenetic modulation therapy, comprising assessing one or more levels of SPEAR in a biological sample from the subject, where (i) a higher level or levels of SPEAR indicates a higher likelihood of response to the therapy, and / or (ii) a lower level or levels of SPEAR indicates a lower likelihood of response to the therapy.

[0048] In some embodiments, SPEAR is a non-coding RNA. In some embodiments, SPEAR is a long non-coding RNA (lncRNA). In some embodiments, SPEAR is about 200 nucleotides or more. In some embodiments, SPEAR is encoded in a region adjacent to the promoter of an active gene. In some embodiments, SPEAR is induced in early S phase of the cell cycle. In some embodiments, SPEAR comprises one or more motifs selected from 3, 5, and 9. In some embodiments, SPEAR comprises one or more RM9A motifs. In some embodiments, SPEAR comprises one or more stem-loop-like structures.

[0049] In some embodiments, the biological sample comprises a biopsy, a tissue, or a bodily fluid. In some embodiments, the biological sample comprises one or more of tumor cells, cultured cells, stem cells, and differentiated cells.

[0050] The details of one or more embodiments of the present disclosure are described in the following description. Other features or advantages of the present disclosure will become apparent from the following drawings, detailed description of some embodiments, and also from the appended claims. The details of the present disclosure are described in the following accompanying description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods and materials are described herein. Other features, objects, and advantages of the present disclosure will become apparent from the description and claims. In this specification and the appended claims, the singular form includes the plural form unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. [Brief description of the drawings]

[0051] [Figure 1A]Graphs and images characterizing CEBPA and c-MYC SPEAR are shown. The upper panel shows a diagram of the CEBPA locus. Lower panel: levels of the coding (mRNA), ecCEBPA (DNMT1-interacting), and UpTr (SPEAR) transcripts immediately after release from double thymidine block are shown. Induction of CEBPA SPEAR (UpTr) preceded and exceeded the expression of ecCEBPA and CEBPA mRNA (qRT-PCR; bars indicate mean ± s.d.). [Figure 1B] Graphs characterizing CEBPA and c-MYC SPEAR are shown. It is a graph showing a genome-wide alignment of nascent RNA (nasRNA). It is a binary Venn diagram showing that SPEAR-specific peaks overlap with transcriptional elements identified in the HL-60 total RNA-Seq library. [Figure 1C] Graphs characterizing CEBPA and c-MYC SPEAR are shown. It is a graph showing the correlation between the levels of SPEAR and the transcriptional status of their respective coding genes. The violin plot ranks into four separate groups according to the global gene expression level (HL-60 total RNA-Seq library) (very high: log2RPKM > 8; high: 4 < log2RPKM < 8; moderate: 2 < log2RPKM < 4, low: log2RPKM < 2) and shows the distribution of SPEAR expression (HL-60 nasRNA-Seq library). Statistically significant differences between different SPEAR expression groups were computed by the Mann-Whitney-Wilcoxon test (**P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). [Figure 1D] Graphs characterizing CEBPA and c-MYC SPEAR are shown. It is an image showing a snapshot of the c-MYC locus. See PU.1 in Figure 8D as an example of another locus. [Figure 1E]Figure 1 shows graphs and images characterizing CEBPA and c-MYC SPEAR. Figure 2 shows levels of c-MYC SPEAR immediately after release from a double thymidine block. Induction of c-MYC SPEAR preceded and was overexpressed by c-MYC mRNA (qRT-PCR; bars show mean ± sd). [Figure 2A] Graphs showing the interaction of SPEAR with H2A.Z, acH2A.Z, and TIP60. Graphs showing c-MYC SPEAR immunoprecipitated with anti-H2A.Z, anti-acH2A.Z, and anti-TIP60 antibodies (qRT-PCR, bars show mean + / - sd). [Figure 2B] Figure 1 shows a graph of SPEAR interactions with H2A.Z, acH2A.Z, and TIP60. Figure 2 shows RIP-Seq peak intensity histograms for enrichment of acH2A.Z, H2A.Z, and TIP60 at coding region boundaries across the genome. The density of aligned RIP-Seq tags is normalized per base pair and averaged over all genes in the genome. [Figure 2C] 9B and 9C show graphs of SPEAR interaction with H2A.Z, acH2A.Z, and TIP60. Graphs showing SPEAR-producing loci overlap with TIP60 / H2A.Z (20%) and TIP60 / acH2A.Z (32%) datasets. Venn diagrams show the overlap of total SPEAR with peaks of its corresponding loci significantly enriched in H2A.Z (upper panel), acH2A.Z (lower panel), and TIP60. Genes from all four SPEAR expression groups (light yellow circles) intersect with the H2A.Z and acH2A.Z (green circles), TIP60 (blue circles), datasets. Venn diagrams showing the overlap of H2A.Z, acH2A.Z, and TIP60 peaks with individual SPEAR expression groups are shown in FIG. 9B and FIG. 9C. [Figure 2D]Shows graphs of the interactions of SPEAR with H2A.Z, acH2A.Z, and TIP60. Shows scatter plots of the correlations between global gene expression (HL-60, total RNA-Seq library), SPEAR expression (HL-60, nasRNA-Seq library), and acH2A.Z peak intensity at the boundaries of the TSSs of active genes. The intensity and number of acH2A.Z peaks at the boundaries of the TSSs of genes are plotted against the expression of genes within each SPEAR expression group (very high: log2RPKM > 8, high: 4 < log2RPKM < 8, moderate: 2 < log2RPKM < 4, low: log2RPKM < 2). [Figure 3-1] Shows images identifying the SPEAR common binding motif. A shows a schematic flowchart of the SPEAR motif discovery pipeline. B shows the sequences of the common binding motif "9" (RNA oligonucleotide RM9A) within c-MYC SPEAR, unrelated RNA oligonucleotides UR1, UR2, and UR3, and the corresponding DNA oligonucleotides. [Figure 3-2] Shows graphs identifying the SPEAR common binding motif. C shows the distribution of the RM9A motif in the peak regions where the RIP-Seq of acH2A.Z and TIP60 overlap. Sequence logo, significance, and enrichment rate (%) are shown. D shows, in the left panel, that c-MYC SPEAR with the identified RM9A binding motif forms a complex with histone H2A.Z. In the right panel, the same is shown, but using an acetylated peptide from the N-terminal sequence of H2A.Z. [Figure 3-3]A graph is shown identifying the SPEAR consensus binding motif. B shows that c-MYC SPEAR with the identified RM9A binding motif, but not the upstream RNA and DNA motifs, forms a complex with TIP60 protein. Both RNA and DNA probes were used at equal molar concentrations. This corresponds to twice the counts / min for double-stranded DNA oligonucleotides versus single-stranded RNA oligonucleotides. C shows that c-MYC SPEAR with the identified binding motif must fold into a stem-loop structure in addition to the primary sequence requirements. RNA secondary structure was predicted with RNAfold. [Figure 4A] Image showing how global downregulation of SPEAR results in decreased occupancy of acH2A.Z at the TSS of linked genes. Schematic diagram showing synchronization of HL-60 cells by double thymidine block followed by treatment with RNA polymerase inhibitors. Upon release from the block, cells were treated with 0.05% DMSO (control); ActD, 0.8 μM; and DRB, 200 μM. EU analogs were also added to cells for downstream Click-iT transformation. After 2 h, cells were treated with Ficoll and crosslinked chromatin and RNA were collected. [Figure 4B] Figure 1 shows how global downregulation of SPEAR results in decreased occupancy of acH2A.Z at the TSSs of linked genes. Scatter plots highlighting the correlation between gene expression and ChIP-Seq intensity of acH2A.Z from cells treated with transcription inhibitors DRB (light blue) and ActD (light red) as well as DMSO-treated controls (green). The upper subplot shows the density of ChIP-Seq tag counts independent of gene expression. The rightmost subplot shows the density of gene expression profiles independent of ChIP-Seq tag counts. These data imply a causal relationship between acH2A.Z and gene expression, since treatment with transcription inhibitors causes a significant reduction in acH2A.Z signal and a shift in the density profile towards lower values. [Figure 4C]Figure 1 shows how global downregulation of SPEAR results in decreased occupancy of acH2A.Z at the TSSs of linked genes. Comparison of enrichment of H2A.Z and acH2A.Z ChIP-Seq signals around gene TSS loci (±2kb) after treatment with DRB and ActD. Enrichment was calculated as the area under the curve around all gene TSSs and transformed using the hyperbolic arcsine function. This allowed comparison of ChIP-Seq occupancy of control (DMSO) samples versus DRB and ActD samples. It is shown as a scatter plot with a color gradient. In these scatter plots, each dot represents an individual TSS. Regions with high concentrations of TSS appear in red, while blue represents the absence of TSS. The different scales on the plots indicate different antibody properties specific to the H2A.Z and acH2A.Z antibodies. Changes in enrichment are shown. [Figure 4D] 10A-10E are graphs showing how global downregulation of SPEAR results in a decrease in acH2A.Z occupancy at the TSSs of linked genes. The top and middle panels are ChIP-Seq snapshots of acH2A.Z at the c-MYC and PU.1 loci, demonstrating a large decrease in the acH2A.Z peak compared to total H2A.Z after addition of transcription inhibitors. Full snapshots are shown in FIG. 10D and FIG. 10E. The bottom panel shows the MYB locus, which does not show a reduction in the acH2A.Z peak after addition of transcription inhibitors because MYB SPEAR escapes the action of ActD and DRB. Full snapshots are shown in FIG. 10D and FIG. 10E. [Figure 4E] FIG. 13 shows how global downregulation of SPEAR leads to decreased occupancy of acH2A.Z at the TSSs of linked genes. ChIP-qPCR validation of ChIP-Seq results for the c-MYC locus. Double arrows indicate the position of the qRT-PCR amplicon. (qRT-PCR, bars show mean ± sd). [Figure 4F]Figure 8 shows how global downregulation of SPEAR results in decreased occupancy of acH2A.Z at the TSS of linked genes. c-MYC and PU.1 Figure 8 shows how DRB-induced downregulation of SPEAR results in decreased occupancy of H2A.Z, acH2A.Z, and TIP60 at the TSS of c-MYC and PU.1 genes. HL-60 cells were released into S phase and treated with DRB for 2 h (as above). The medium was supplemented with EdU DNA analog to allow collection of nascent DNA. Chromatin was collected and ChIP assays were performed with antibodies against H2A.Z, acH2A.Z, TIP60, and IgG. Nascent DNA was isolated from immunoprecipitated chromatin (see Methods below and Figure 8A for details). Isolated nascent DNA was analyzed by qPCR (qPCR, bars indicate mean ± sd). [Figure 5A] Figure 1 shows a graph of how RNAi-mediated downregulation of c-MYC SPEAR leads to a decrease in acH2A.Z occupancy at the TSS of the c-MYC gene. Figure 2 shows how siRNA-mediated downregulation of c-MYC SPEAR (approximately 75%) leads to approximately 70% downregulation of c-MYC mRNA (qRT-PCR, bars show mean ± s.d.). [Figure 5B] Graphs are shown of how RNAi-mediated downregulation of c-MYC SPEAR results in decreased occupancy of acH2A.Z at the TSS of the c-MYC gene. In the left panel, snapshots of ChIP-Seq results of acH2A.Z at the c-MYC locus are shown, demonstrating a decrease in the acH2A.Z peak. In the right panel, the PU.1 locus showed no significant change in occupancy of acH2A.Z (negative control). Full snapshots are shown in Figure 10F and Figure 10G. [Figure 5C]Figure 1 shows a graph of how RNAi-mediated downregulation of c-MYC SPEAR results in decreased occupancy of acH2A.Z at the TSS of the c-MYC gene. ChIP-qPCR validation of ChIP-Seq results of RNA interference for the c-MYC locus. Double arrows indicate the position of the qRT-PCR amplicon. qRT-PCR, bars indicate mean + / - sd. [Figure 6A] Figure 1 shows how SPEAR regulate the expression of the mRNAs they link via the TIP60 / acH2AZ pathway. Figure 2 shows the response of total c-MYC mRNA and SPEAR to TIP60 / HAT inhibitors. c-MYC mRNA is downregulated by MG149 and TH1834, whereas c-MYC SPEAR is unaffected. HL-60 cells were released from double thymidine block and treated with MG149 (200 μM) and TH1834 (500 μM) for 2 h. Control (mock) treatments were supplemented with DMSO (0.05%) or water, respectively. SPEAR and mRNA were quantified using qRT-PCR and strand-specific qRT-PCR. Bars indicate mean ± sd. [Figure 6B] Images showing how SPEARs control the expression of the mRNAs they link via the TIP60 / acH2AZ pathway. Schematic diagram outlining the experimental design. HL-60 cells were released into S phase and treated with DMSO / DRB for 2 h (as described in Fig. 4A). After 2 h, DRB-treated cells were washed and incubated for an additional 2 h with or without TIP60 inhibitors. The medium was supplemented with Eu RNA and EdU DNA analogs to allow collection of nascent RNA / DNA. Chromatin was collected and ChIP assays were performed with antibodies against H2A.Z, acH2A.Z, TIP60, and IgG. Nascent DNA and RNA were isolated from immunoprecipitated chromatin and total RNA, respectively (see Methods below and Fig. 8A for details). Isolated nascent DNA and RNA were analyzed by qPCR and qRT-PCR / strand-specific qRT-PCR, respectively. [Figure 6C]FIG. 1 shows how SPEARs regulate the expression of the mRNAs they link via the TIP60 / acH2AZ pathway. Graphs of the differential response of nascent c-MYC SPEAR and mRNA to TIP60 / HAT inhibitors are shown: c-MYC mRNA is downregulated, whereas c-MYC SPEAR is unaffected. Quantification was performed by qRT-PCR (for mRNA) and strand-specific qRT-PCR (for SPEAR). Bars indicate mean ± sd. [Figure 6D] Graphs showing how SPEARs control the expression of the mRNAs they link via the TIP60 / acH2AZ pathway. Graphs of nascent ChIP-qPCR (using antibodies against H2A.Z, acH2A.Z, and TIP60, with IgG control) for the c-MYC locus are shown. These experiments demonstrate that: (i) DRB treatment (downregulating c-MYC SPEAR) results in loss of acH2A.Z (middle panel) and TIP60 (RH panel) and partial loss of H2A.Z (LH panel); (ii) restoring c-MYC SPEAR (after reversal of DRB) results in reappearance of H2A.Z, AC2H2A.Z, and TIP60; and (iii) inhibiting the activity of TIP60 with MG149 and TH1834 prevents the recovery of acH2A.Z, but only slightly affects the recovery of TIP60 and H2A.Z distribution. Quantification by qPCR. Bars indicate mean ± sd (n = 2). [Figure 7] Image showing a non-limiting model for the establishment of active epigenetic acetylation marks on histone H2A.Z (not wishing to be bound by theory). During early S phase, SPEAR interacts with both histone H2A.Z and the acetyltransferase TIP60. The physical proximity of H2A.Z and TIP60 results in a high local effective protein concentration that favors acetylation of H2A.Z and its exchange with the canonical H2A form within the nucleosome. In this active chromatin conformation, the RNAPII complex binds to the site and gene expression is initiated. [Figure 8-1]Images identifying early S-phase RNA (SPEAR) are shown. It is a schematic diagram showing synchronization of HL-60 cells by double thymidine block. At the release from double thymidine block, an EU analog was added to the cells for downstream Click-iT conversion. One hour later, total RNA and nuclear RNA were collected. The RNA was processed according to the manufacturer's recommendations, an RNA-seq library was generated, sequenced, and analyzed. [Figure 8-2] Graphs identifying early S-phase RNA (SPEAR) are shown. Enrichment plots of SPEAR expression at the loci of genes belonging to four different expression groups (very high: log2RPKM > 8; high: 4 < log2RPKM < 8; moderate: 2 < log2RPKM < 4; low: log2RPKM < 2) are shown. Genes were classified into four different expression groups according to the total RNA-Seq library of HL-60, and lead count enrichment in regions covering the upstream regulatory region, gene body, and non-coding downstream region was calculated using SPEAR expression measured according to the nasRNA-Seq library of HL-60. [Figure 8-3] Graphs identifying early S-phase RNA (SPEAR) are shown. An enrichment heatmap of SPEAR expression in genes belonging to four different expression groups (very high: log2RPKM > 8; high: 4 < log2RPKM < 8; moderate: 2 < log2RPKM < 4; low: log2RPKM < 2) is shown. For individual genes across the genome, SPEAR expression in the upstream regulatory region, gene body, and downstream non-coding region was plotted, showing the relative positional enrichment of different genomic regions within the locus of each gene. [Figure 8-4] Graphs identifying early S-phase RNA (SPEAR) are shown. A snapshot of the locus of the PU.1 gene showing each SPEAR is shown. [Figure 8-5]E shows an image identifying S-phase early RNA (SPEAR). Diagram of the transcriptome of the c-MYC locus. Vertical arrows indicate the TSS and TES, and dashed arrows indicate the position of the primers in the primer extension experiment. F shows a graph identifying S-phase early RNA (SPEAR). Graph showing the copy number of c-MYC SPEAR (experimental details are available in Methods below). [Figure 8-6] G shows an image identifying the S-phase early RNA (SPEAR). Primer extension experiment. Radioautographs of the primer extension reaction for c-MYC SPEAR are shown; radioautographs for SPEAR corresponding to loci CEBPA, CTCF, and PU.1 are not shown. The black arrow indicates the longest extension product. The dashed arrow indicates the "hard stop" of the extension reaction (experimental details are available in "Methods" below). H shows an image identifying the S-phase early RNA (SPEAR). 5'3'Rapid Amplification of cDNA Ends ("RACE") is shown. The "longest" isoform of c-MYC SPEAR is as follows: the TSS is at -2160 nt relative to the TSS of c-MYC mRNA and the TES is at -38 nt relative to the TSS of c-MYC mRNA (nucleotide positions indicated by solid arrows). The TSS and TES of the "shorter" isoform are indicated by dashed arrows (see "Methods" below for details). [Figure 8-7]G shows an image identifying the S-phase early RNA (SPEAR). Primer extension experiment. Radioautographs of the primer extension reaction for c-MYC SPEAR are shown; radioautographs for SPEAR corresponding to loci CEBPA, CTCF, and PU.1 are not shown. The black arrow indicates the longest extension product. The dashed arrow indicates the "hard stop" of the extension reaction (experimental details are available in "Methods" below). H shows an image identifying the S-phase early RNA (SPEAR). 5'3'Rapid Amplification of cDNA Ends ("RACE") is shown. The "longest" isoform of c-MYC SPEAR is as follows: the TSS is at -2160 nt relative to the TSS of c-MYC mRNA and the TES is at -38 nt relative to the TSS of c-MYC mRNA (nucleotide positions indicated by solid arrows). The TSS and TES of the "shorter" isoform are indicated by dashed arrows (see "Methods" below for details). [Figure 9A] (a) Images showing the SPEAR-H2A.Z-acH2A.Z-TIP60 interaction. (b) Diagram depicting the protocol used to generate biotinylated SPEAR probes and pull down SPEAR-containing RNA-protein complexes (SPEAR-RNPs). Collected soluble SPEAR-RNPs were separated by 5% PAGE and subjected to mass spectrometry (experimental details are available in Methods below). [Figure 9B]A graph showing the interaction of SPEAR-H2A.Z-acH2A.Z-TIP60. A graph showing the RIP-Seq analysis of H2A.Z and TIP60. A Venn diagram of the overlap between the gene set containing significant H2A.Z and TIP60 peaks in the promoter region and each individual SPEAR expression group (the upper circles in each graph). Gene datasets stratified into four expression groups (Group A - very high: log2RPKM > 8, Group B - high: 4 < log2RPKM < 8, Group C - moderate: 2 < log2RPKM < 4, Group D - low: log2RPKM < 2) were intersected with gene sets having H2A.Z marks (lower right circle), TIP60 (lower left circle), or marks for both TIP60 and H2A.Z. (Details of the experiment are available in the "Methods" section below). [Figure 9C] A graph showing the interaction of SPEAR-H2A.Z-acH2A.Z-TIP60. A graph showing the RIP-Seq analysis of acH2A.Z and TIP60. A Venn diagram of the overlap between the gene set containing significant acH2A.Z and TIP60 peaks in the promoter region and each individual SPEAR expression group (the upper circles in each graph). Gene datasets stratified into four expression groups (Group A - very high: log2RPKM > 8, Group B - high: 4 < log2RPKM < 8, Group C - moderate: 2 < log2RPKM < 4, Group D - low: log2RPKM < 2) were intersected with gene sets having acH2A.Z marks (lower right circle), TIP60 (lower left circle), or marks for both TIP60 and acH2A.Z. (Details of the experiment are available in the "Methods" section below). [Figure 10A]Images showing how downregulation of SPEAR reduces occupancy of acH2A.Z at the TSS of the respective genes. Schematic of pilot experiments showing synchronization of HL-60 cells by double thymidine block followed by treatment with RNA polymerase inhibitors. Upon release from double thymidine block, cells were treated with DMSO; actinomycin D (ActD; RNA polymerase I, II, and III inhibitor), 0.8 μM; and 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside inhibitor (DRB; RNAPII inhibitor), 200 μM. Cells were also added with analogs EU and / or EdU for downstream Click-iT conversion. Cells were harvested at different time points and total proteins were subjected to Western blot analysis. Shown are blots with proteins isolated after 2 h of treatment. These experiments revealed that the overall global content of H2A.Z and TIP60 was not affected by drug treatment. [Figure 10B] A graph showing how downregulation of SPEAR reduces acH2A.Z occupancy at the TSS of each gene. Genome-wide transcription inhibition upon DRB and ActD treatment. The figure shows violin plots of the distribution of gene expression in control (DMSO) and cells treated with transcription inhibitors DRB and ActD. Comparing gene expression distributions, DRB treatment shows significant difference from control and ActD treatment shows large significant difference from control in the Mann-Whitney-Wilcoxon test (p<0.05). The ratio of gene expression reduction between distributions is DRB / DMSO=0,45 and ActD / DMSO=0,21. With DRB and ActD treatment, the average gene expression reduction was 55% and 79%, respectively. [Figure 10C] Graph showing how downregulation of SPEAR reduces acH2A.Z occupancy at the TSS of each gene. Individual SPEAR inhibition upon DRB and ActD treatment. qRT-PCR quantification of the effects of DRB and ActD on c-MYC, PU.1, and MYB SPEAR. [Figure 10D]Figure 1 shows how global downregulation of SPEAR reduces acH2A.Z occupancy at the TSS of each gene. Figure 2 shows how global downregulation of SPEAR reduces acH2A.Z occupancy at the TSS of each gene. Figure 3 shows full H2A.Z ChIP-Seq snapshots of unmodified H2A.Z peaks upon DRB and ActD treatment. [Figure 10E] Figure 1 shows how global downregulation of SPEAR reduces acH2A.Z occupancy at the TSS of each gene. Figure 2 shows how global downregulation of SPEAR reduces acH2A.Z occupancy at the TSS of each gene. Figure 3 shows full ChIP-Seq snapshots of acH2A.Z at gene loci demonstrating reduced acH2A.Z peaks (genes: c-MYC and PU.1) and at gene loci where acH2A.Z peaks remain unchanged (gene: MYB) upon DRB and ActD treatment. [Figure 10F] Figure 1 shows how global downregulation of SPEAR reduces acH2A.Z occupancy at the TSS of the respective genes. Figure 2 shows that RNAi-mediated downregulation of c-MYC SPEAR reduces acH2A.Z occupancy, but not H2A.Z occupancy, at the TSS of the c-MYC gene. Figure 3 shows a complete ChIP-Seq snapshot of H2A.Z at the targeted (c-MYC) and non-targeted (PU.1) gene loci, demonstrating that H2A.Z peaks remain unchanged after siRNA-induced downregulation of c-MYC SPEAR. [Figure 10G]Figure 1 shows how global downregulation of SPEAR reduces acH2A.Z occupancy at the TSS of the respective genes. Figure 2 shows that RNAi-mediated downregulation of c-MYC SPEAR reduces acH2A.Z occupancy, but not H2A.Z occupancy, at the TSS of the c-MYC gene. Figure 3 shows a complete ChIP-Seq snapshot of acH2A.Z at the targeted (c-MYC) gene locus, showing a reduction in acH2A.Z peaks after siRNA-induced downregulation of c-MYC SPEAR, whereas no effect is observed at the non-targeted gene locus (PU.1). [Figure 11A] Images and graphs showing how SPEAR controls the expression of respective mRNAs through recruitment / loading of TIP60 / acH2AZ. Graph of blot with isolated proteins after 2 hours of treatment. Upon release from double thymidine block, cells were treated with DMSO; MG149, either 100 or 200 μM; and TH1834, 250 or 500 μM. Cells were harvested at different time points and total proteins were subjected to Western blot analysis. Reduction of acH2A.Z was observed with MG149 at 200 μM and TH1834 at 500 μM. Quantification of bands was obtained using ImageJ; levels of acH2A.Z were normalized with levels of H2A.Z. [Figure 11B] Images and graphs showing how SPEAR controls the expression of respective mRNAs through recruitment / loading of TIP60 / acH2AZ. Shown is how downregulation of c-MYC and c-MYC SPEAR (after DRB treatment) leads to loss of enrichment of acH2A.Z and TIP60 ("DRB" bar) (middle and right panels), and restoration of c-MYC and c-MYC SPEAR (after DRB reversal). qRT-PCR and strand-specific qRT-PCR; bars indicate mean + / - sd. [Figure 11C](a) Graph showing how SPEAR controls expression of respective mRNAs via recruitment / loading of TIP60 / acH2AZ. (b) Nascent ChIP-qPCR for gene desert loci showing no change in enrichment levels for H2A.Z, acH2A.Z, and TIP60. (c) qPCR, bars indicate mean + / -sd (n=2). [Figure 11D] FIG. 13 shows how SPEAR controls the expression of the respective mRNAs through recruitment / loading of TIP60 / acH2AZ. FIG. 14 shows the differential response of total PU.1 SPEAR and mRNA to TIP60 / HAT inhibitors: PU.1 mRNA is downregulated, whereas PU.1 SPEAR is unaffected. qRT-PCR and strand-specific qRT-PCR; bars indicate mean + / - sd. [Figure 11E] Graph showing how SPEAR controls the expression of respective mRNAs via recruitment / loading of TIP60 / acH2AZ. Nascent ChIP-qPCR for the PU.1 locus is shown, demonstrating that: (i) downregulation of PU.1 SPEAR (after DRB treatment) results in loss of acH2A.Z and TIP60 enrichment ("DRB" bar) (middle and right panels); (ii) restoration of PU.1 SPEAR (after DRB reversal) results in re-emergence of acH2A.Z and TIP60 enrichment ("DRB REV" bar) (middle and right panels); (iii) inhibition of TIP60 / HAT activity (after MG149 and TH1834 treatment) prevents the restoration of acH2A.Z enrichment without affecting the restoration of TIP60 enrichment (TH1834 and MG149 bars) (middle and right panels); qPCR, bars indicate mean + / -sd (n=2). [Figure 12]1A-C are images showing how induction of SPEAR-like transcription affects the size of trinucleotide repeats. In A, as a non-limiting example (without wishing to be bound by theory), sample #1 refers to a model of a "myocyte" MyoD-generated from fibroblasts isolated from a subject with myotonic dystrophy type 1 (DM1), which, without wishing to be bound by theory, results in induction of bidirectional transcription within the DMPK locus. Sample #2 refers to untreated fibroblasts isolated from a DM1 subject. B and C show how genomic DNA was extracted from sample #1 and sample #2 and PCR was performed. The primer pairs flank the CTGn repeat region. C shows the interpretation of the PCR bands. Sample #2 shows how the wild-type DMPK allele produces a 150 nucleotide (nt) band (black dot / black arrow) and the mutant DMPK allele produces a 450 nt band (red dot / red arrow). In sample #1, similar bands as seen in sample #2 are present, but an additional band at 450 nt is present because the mutant DMPK allele produces bands at approximately 800 nt (expanded mutant allele; blue arrow) and approximately 350 nt (shortened mutant allele; purple arrow). D shows Sanger sequencing of the PCR bands with CTGn shown in C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Prior to this disclosure, the mechanism by which epigenetic modifications are established in gene regulatory regions of active genes remained unclear. This disclosure demonstrates, inter alia, that the establishment of a major epigenetic mark, an acetylated form of the substituted histone H2A.Z, is controlled by cell cycle-specific long non-coding RNAs encoded in regions adjacent to the promoters of active genes. These transcripts, termed SPEAR (S Phase EArly RNA), are induced in early S phase and their expression precedes that of the downstream genes they control. SPEAR brings the substituted histones together with the histone acetyltransferase TIP60, thereby setting the stage for the modification and loading of the acetylated form of histone H2A.Z. As disclosed herein, this extensive bimodal interaction constitutes a novel RNA-mediated mechanism for the establishment of epigenetic marks and cell-specific epigenetic profiles, providing a unified mechanistic explanation for the precision and persistence of epigenetic marks on chromatin.

[0053] The present disclosure is based in part on the discovery of compositions and methods relating to treating cancer in a subject in need thereof, comprising (i) administering to the subject an effective amount of one or more inhibitors of S-phase early RNA (SPEAR), or (ii) administering to the subject an effective amount of cells that have been contacted with an effective amount of one or more inhibitors of SPEAR. In some embodiments, the cancer is characterized by epigenetic dysregulation.

[0054] In various embodiments, disclosed herein are methods for preventing the onset or progression of cancer in a subject in need thereof, comprising: (i) administering to the subject an effective amount of one or more inhibitors of SPEAR; or (ii) administering an effective amount of cells from the subject that have been contacted with an effective amount of one or more inhibitors of SPEAR, wherein the subject is characterized by a precancerous condition involving epigenetic dysregulation.

[0055] In various embodiments, disclosed herein are methods for treating a genetic disease or disorder associated with epigenetic dysregulation in a subject in need thereof, comprising (i) administering to the subject an effective amount of one or more inhibitors of SPEAR, or (ii) administering an effective amount of cells from the subject that have been contacted with an effective amount of one or more inhibitors of SPEAR.

[0056] In various embodiments, disclosed herein are methods for preventing the onset or progression of a genetic disease or disorder associated with epigenetic dysregulation in a subject in need thereof, comprising (i) administering to the subject an effective amount of one or more inhibitors of SPEAR, or (ii) administering an effective amount of cells from the subject that have been contacted with an effective amount of one or more inhibitors of SPEAR.

[0057] In various embodiments, disclosed herein are methods for resetting the formation of active histone marks in cancer or precancerous cells, comprising (i) administering to a subject an effective amount of one or more SPEARs or one or more inhibitors of SPEAR, or (ii) contacting cells derived from a subject with an effective amount of one or more SPEARs or one or more inhibitors of SPEAR.

[0058] In various embodiments, disclosed herein are methods for restoring a replication origin complex associated with a non-disease state in cells characterized by a genetic disease or disorder, comprising (i) administering to a subject an effective amount of one or more SPEARs or one or more inhibitors of SPEARs, or (ii) contacting a cell derived from a subject with an effective amount of one or more SPEARs or one or more inhibitors of SPEARs. For example, administering to a subject an effective amount of one or more SPEARs or one or more inhibitors of SPEARs restores a replication origin complex associated with a non-disease state in cells characterized by a genetic disease or disorder by restoring the expression level of one or more SPEARs, by restoring the replicative capacity of the replication origin complex, and / or by re-emerging histones or histone-associated proteins (e.g., histone acetyltransferase, H2A.Z, H3.3, or variants thereof). In some embodiments, one or more SPEARs are overexpressed to restore the replication origin complex. In some embodiments, the method includes contacting a cell from a subject with an effective amount of one or more SPEARs or one or more inhibitors of SPEARs, and restoring an origin of replication complex by administering an effective amount of one or more SPEARs and / or by re-expressing histones or histone-associated proteins (e.g., histone acetyltransferase, H2A.Z, H3.3, or variants thereof). In some embodiments, one or more SPEARs are overexpressed to restore an origin of replication complex or generate an artificial origin of replication complex. In some embodiments, one or more SPEARs are overexpressed to control the direction of replication. In some embodiments, one or more SPEARs are overexpressed and one or more SPEAR inhibitors (i) slow or prevent the progression of a deleterious direction of replication, activate the opposite direction of replication, and / or (ii) modulate the site of a trinucleotide repeat, optionally reducing the size or reversing the expression of the trinucleotide repeat.In some embodiments, one or more SPEARs are overexpressed and one or more SPEAR inhibitors treat or prevent triplet disease ("TRD"; e.g., Huntington's disease (HD), spinocerebellar ataxia, movement disorders, autism) by reversing the expansion of trinucleotide repeats ("TNRs", including CAG, CTG, CGG, and GAA) that occur during replication and repair. In some embodiments, ChIP assays using antibodies against histones or histone-associated proteins, as well as PCR and qRT-PCR, detect restoration of origin complexes associated with a non-disease state in cells characterized by a genetic disease or disorder (e.g., by measuring and quantifying the expression levels of one or more SPEARs and / or the reappearance of histones or histone-associated proteins). In some embodiments, qRT-PCR and strand-specific qRT-PCR assays detect restoration of origin complexes associated with a non-disease state in cells characterized by a genetic disease or disorder (e.g., by measuring and quantifying the expression levels of one or more SPEARs and / or the reappearance of histones or histone-associated proteins).

[0059] In some embodiments, during early S phase, the formation of major "active" epigenetic marks is driven by the action of a cell cycle-specific non-coding RNA ("ncRNA") called "SPEAR" that is encoded adjacent to the promoters of actively transcribed genes. In some embodiments, locally induced SPEAR binds to replacement histone H2A.Z and a nuclear factor, histone acetyltransferase TIP60, resulting in the loading / acetylation of replacement histone H2A.Z. In this active chromatin conformation, RNAPII complexes bind to the site and gene expression is initiated.

[0060] In various embodiments, the sequencing assay identifies SPEARs encoded adjacent to the promoters of actively transcribed genes. In some embodiments, to identify SPEARs, nascent RNA is captured and sequenced (e.g., nasRNA-Seq) in a high-throughput sequencing assay to identify transcripts, for example, by mapping RNA-Seq reads to the genome or by de novo assembling reads into contigs and then mapping the contigs to the transcriptome. In nasRNA-Seq, cells are first synchronized and labeled for 1 hour upon release into S phase. The collected RNA is then biotinylated by click chemistry, followed by isolation with streptavidin beads and deep sequencing to generate a nasRNA library. SPEARs are identified by correlating gene expression levels with transcripts close to the transcription start sites (TSS) of coding genes.

[0061] In some embodiments, epigenetic dysregulation is a change or modification of epigenetic control of gene expression. In some embodiments, epigenetic dysregulation is a change or modification of epigenetic mark (e.g., histone modification such as histone acetylation, histone methylation, etc.) on the DNA of a cell. In some embodiments, epigenetic dysregulation leads to expression or silencing of a gene. In some embodiments, the epigenetic mark is a histone modification selected from one or more of histone acetylation, phosphorylation, methylation, ubiquitination, and proteolysis, and chromatin remodeling modification. In some embodiments, the histone modification is histone acetylation.

[0062] Two components of gene expression typically include DNA methylation and chromatin modification. DNA methylation is a reversible process that downregulates gene activity by adding a methyl group to the 5-carbon of cytosine base, while chromatin modification is carried out by several mechanisms, resulting in either upregulation or downregulation of associated genes. In some embodiments, to assess changes in DNA methylation, bisulfite modification or bisulfite sequencing (e.g., by using DNA sequencing, single-base primer extension, and / or methylation-sensitive primer (MSP)) is disclosed herein to assay changes in DNA methylation. In some embodiments, to assay for epigenetic dysregulation in cancer, chromatin immunoprecipitation (ChIP) assays are used to assess epigenetic changes as well as the effects of epigenetic modifications of histones (e.g., native ChIP (nChIP), real-time PCR (Q-ChIP), DNA methylation ChIP (ChIP-MSP). In some embodiments, the ChIP assay is ChIP sequencing (ChIP-Seq), which is used to identify epigenetic marks including DNA binding proteins, histone modifications, or nucleosomes. , which combines ChIP and DNA sequencing. In some embodiments, ChIP assays are combined with nasChIP and / or RNAi to identify epigenetic marks and gene expression levels. In some embodiments, epigenetic dysregulation is assessed using DNase I hypersensitive methods. DNase I hypersensitive sites are typically located in or around promoter regions, allowing for mapping of transcriptionally active versus inactive chromatin. In various embodiments, DNA methylation and chromatin modifications are assessed using assays such as those described in, for example, DeAngelis, J. et al., Mol Biotechnol. 2008 Feb;38(2):179-183, or Gul, S. Clin Epigenet 9,41(2017), the entire contents of which are incorporated herein by reference.

[0063] As disclosed herein, histones are altered in cancer. For example, cancer cells generally show loss of lysine 16 acetylation and lysine 20 methylation. In some embodiments, epigenetic dysregulation is local changes at histone acetylation and methylation loci from ChIP assays or global changes in the genome in cancer cells or precancerous cells compared to normal or healthy cells. In some embodiments, epigenetic dysregulation characterizes cancer. In some embodiments, changes in histone acetylation and methylation from ChIP assays (e.g., ChIP-Seq) are used to predict the outcome of cancer treatment in a subject or to predict the likelihood of a subject's response to epigenetic modulation therapy.

[0064] In some embodiments, a ChIP assay is disclosed herein for evaluating epigenetic dysregulation in genomic sequences from cancer. A ChIP assay typically refers to a process that includes: (1) isolation from cells of the chromatin to be analyzed; (2) immunoprecipitation of the chromatin using an antibody; and (3) DNA analysis. In a ChIP assay, fragments of the DNA-protein complexes that package DNA in living cells (i.e., chromatin) are prepared to preserve the specific DNA-protein interactions that characterize each living cell. These chromatin (i.e., protein-DNA complex) fragments can then be immunoprecipitated using an antibody against the protein of interest. The isolated chromatin fraction can then be treated to separate DNA and protein components, and the identity of the isolated DNA fragments associated with a particular protein (i.e., the protein against which the antibody used for immunoprecipitation is directed) can then be determined by polymerase chain reaction (PCR) or other techniques used to identify DNA fragments of defined sequences.

[0065] In some embodiments, the ChIP assay disclosed herein is used to assay any kind of epigenetic modification of any gene or region of genome of any cell type of interest. Examples of epigenetic marks that can be caused by the modification of DNA in a sample include histone protein modification, non-histone protein modification, and DNA methylation. In some embodiments, the epigenetic mark is a histone modification selected from one or more of histone acetylation, phosphorylation, methylation, ubiquitination, and proteolysis, and chromatin remodeling modification. In some embodiments, the histone modification is histone acetylation.

[0066] Thus, for example, the antibody used in the immunoprecipitation step may be immunospecific for a non-histone protein, such as a transcription factor, or other DNA binding protein. For example, the antibody may be immunospecific for any of histones H1, H2A, H2B, H3, and H4, and their various post-translationally modified isoforms and variants (e.g., H2A.Z). In some embodiments, the histone or histone-associated protein is one or more of H1, H2A, H2B, H3, and H4 proteins, or variants thereof. In some embodiments, the histone or histone-associated protein is one or more of H2A.Z or variants thereof, and H3.3 or variants thereof. In some embodiments, the antibody may be immunospecific for an enzyme involved in the modification of chromatin, such as a histone acetylase or deacetylase, or a DNA methyltransferase. In some embodiments, the histone or histone-associated protein is a histone acetyltransferase. In some embodiments, the histone acetyltransferase is TIP60 or a variant thereof. Furthermore, histones may be post-translationally modified in vivo by defined enzymes, e.g., by acetylation, methylation, phosphorylation, ADP-ribosylation, sumoylation, and ubiquitination, and thus the antibody may be immunospecific for any of these post-translational modifications.

[0067] Following the immunoprecipitation step, the method generally includes purifying the DNA from the isolated protein / DNA fraction, which may be accomplished, for example, by standard techniques of phenol-chloroform extraction, or by any other purification method known to those of skill in the art.

[0068] Following the purification step, the isolated DNA fragments associated with the protein are analyzed by PCR. For example, the analysis step may include the use of suitable primers that will result in the amplification of a certain length of nucleic acid during PCR. Those skilled in the art will understand that the method according to the invention can be applied to analyze epigenetic modifications of any gene or any region of the genome for which specific PCR primers have been prepared. For example, the ChIP assay uses formaldehyde to crosslink DNA and proteins, followed by immunoprecipitation of the DNA-protein complex. After reversing the crosslinking, the recovered DNA can then be analyzed, for example, by PCR or real-time PCR, to measure the amount of DNA that binds to a particular protein. In some embodiments, the ChIP assay uses micrococcal nuclease digestion instead of formaldehyde to prepare chromatin for analysis.

[0069] In some embodiments, disclosed herein are bisulfite modifications or bisulfite sequencing to assay for changes in DNA methylation to assess changes in DNA methylation.

[0070] In some embodiments, epigenetic dysregulation is assessed using DNase I hypersensitivity methods. DNase I hypersensitive sites are typically located within or surrounding promoter regions, allowing mapping of transcriptionally active versus inactive chromatin.

[0071] In some embodiments, epigenetic dysregulation in DNA methylation is evaluated by DNA bisulfite modification.Bisulfite modification converts unmethylated cytosine to uracil, which is then converted to thymine during DNA amplification by PCR, while methylated cytosine is protected from bisulfite modification.In some embodiments, bisulfite sequencing is used to analyze bisulfite-treated DNA and generate a comprehensive "methylome" (e.g., the pattern of methylated DNA in genome) map.As disclosed herein, sequencing analysis of bisulfite-modified DNA reveals the methylation state of specific cytosines.

[0072] In some embodiments, the combination of bisulfite modifications disclosed herein with ChIP assays allows for the assessment of methylation status and chromatin structure from one biological sample.

[0073] In some embodiments, the methods disclosed herein include (i) administering to a subject an effective amount of one or more inhibitors of SPEAR, or (ii) administering an effective amount of cells from the subject that have been contacted with an effective amount of one or more inhibitors of SPEAR, thereby treating a cancer characterized by epigenetic dysregulation in a subject and / or treating a genetic disease or disorder associated with epigenetic dysregulation in a subject.

[0074] In some embodiments, the methods disclosed herein prevent the onset or progression of cancer by comprising (i) administering to a subject an effective amount of one or more inhibitors of SPEAR, or (ii) administering an effective amount of cells from a subject that have been contacted with an effective amount of one or more inhibitors of SPEAR, wherein the subject is characterized by a precancerous condition involving epigenetic dysregulation.

[0075] As a non-limiting example, the prevention, presence, and / or progression of cancer in a subject can be evaluated according to the Tumor / Nodes / Metastases (TNM) classification system (International Union Against Cancer, 6th edition, 2002), or the Whitmore-Jewett staging system (American Urological Association). Typically, cancer is staged using a combination of physical examination, blood tests, and medical imaging. If tumor tissue is obtained by biopsy or surgery, examination of the tissue under a microscope can also provide pathological staging. In some embodiments, the stage or grade of cancer determines the prognosis of the cancer and assists physicians in selecting appropriate epigenetic modification therapy.

[0076] In some embodiments, prevention of cancer onset or progression is assessed using overall staging, non-limiting examples of which are as follows: stage I cancer is localized to a part of the body, typically a small area; stage II cancer is locally advanced and has grown into nearby tissues or lymph nodes, as is stage III cancer. Whether a cancer is designated as stage II or stage III may depend on the particular type of cancer. The specific criteria for stages II and III may vary depending on the diagnosis. Stage IV cancers often have metastasized or spread to other organs or the entire body. Cancer onset or progression can be assessed using conventional methods available to those skilled in the art, such as physical examinations, blood tests, and imaging scans (e.g., X-rays, MRIs, CT scans, ultrasounds, etc.).

[0077] As disclosed herein, administering or administering a treatment / therapy refers to a treatment / therapy in which the subject receives a beneficial effect, such as reducing, decreasing, attenuating, decelerating, stabilizing, ameliorating, suppressing, inhibiting, or halting the development or progression of a cancer and / or genetic disease or disorder, or symptoms thereof.

[0078] In some embodiments, the methods disclosed herein prevent the onset or progression of cancer characterized by epigenetic dysregulation, and / or the onset or progression of a genetic disease or disorder associated with epigenetic dysregulation in a subject in need thereof.

[0079] In some embodiments, the treatment / therapy received by a subject or the prevention of the onset of a cancer and / or genetic disease or disorder results in at least one or more of the following effects: (1) reduction or amelioration of the severity of the cancer and / or genetic disease or disorder, and / or symptoms associated therewith; (2) reduction in the duration of symptoms associated with the cancer and / or genetic disease or disorder; (3) prevention of the recurrence of symptoms associated with the cancer and / or genetic disease or disorder; (4) regression of the cancer and / or genetic disease or disorder, and / or symptoms associated therewith; (5) reduction in the hospitalization of the subject; (6) reduction in the length of hospitalization; (7) increase in the survival rate of the subject; (8) inhibition of the progression of the cancer and / or genetic disease or disorder, and / or symptoms associated therewith; (9) enhancement or improvement of the therapeutic efficacy of other therapies; (10) reduction or elimination of the cancer cell population and / or cell population associated with the genetic disease or disorder; (11) reduction in the growth of a tumor or neoplasm; (12) reduction in tumor size. (13) reducing the formation of tumors; (14) eradicating, removing, or controlling primary, regional, and / or metastatic cancer; (15) reducing the number or size of metastases; (16) reducing mortality; (17) increasing the cancer-free survival rate of a subject; (18) increasing recurrence-free survival rate; (19) increasing the number of subjects in remission; (20) reducing hospitalization rates; (21) maintaining and not increasing in size, or increasing in size by less than 5% or 10% after administration of the therapy, as measured by conventional methods available to one of skill in the art, e.g., X-ray, MRI, CT scan, ultrasound, and the like; (22) preventing the development or onset of cancer and / or a genetic disease or disorder, and / or symptoms associated therewith; (23) increasing the length of remission in a subject; (24) reducing the number of symptoms associated with cancer and / or a genetic disease or disorder; (25) increasing symptom-free survival in a subject with cancer and / or a subject associated with a genetic disease or disorder; and / or (26) limiting or reducing metastases. In some embodiments, the treatment / therapy that the subject receives does not cure the cancer, but prevents the progression or worsening of the disease. In certain embodiments, the treatment / therapy that the subject receives does not prevent the onset / progression of cancer, but can prevent the onset of symptoms of cancer.

[0080] In some embodiments, "preventing" the onset or progression of cancer in a subject in need thereof, or "preventing" the onset or progression of a genetic disease or disorder associated with epigenetic dysregulation in a subject in need thereof, is inhibiting or blocking the cancer or genetic disease or disorder. In some embodiments, the methods disclosed herein prevent or inhibit cancer or genetic disease or disorder at any amount or level. In some embodiments, the methods disclosed herein prevent or inhibit cancer or genetic disease or disorder by at least about 10% inhibition (e.g., at least about 20% inhibition, at least about 30% inhibition, at least about 40% inhibition, at least about 50% inhibition, at least about 60% inhibition, at least about 70% inhibition, at least about 80% inhibition, at least about 90% inhibition, at least about 95% inhibition, at least about 98% inhibition, or at least about 100% inhibition).

[0081] In some embodiments, the subject is characterized by a precancerous condition that includes epigenetic dysregulation. In some embodiments, the precancerous condition is associated with epigenetic dysregulation of a genetic locus, or a mutation or mutations. In some embodiments, the precancerous condition includes abnormal cells that are at increased risk of developing into cancer. The precancerous condition can be evaluated in a variety of ways. For example, screening such as physical exams, blood tests, and imaging scans (e.g., X-rays, MRIs, CT scans, ultrasounds, etc.) can check for the presence of cancer in subjects not previously known to have cancer. In some embodiments, characterizing a precancerous subject includes checking for cancer in those who have suggestive features of cancer (e.g., symptoms or other positive tests) or have a certain "pathological level." The "pathological level" can refer to a pathological level associated with a pathogen, which can be as described above for cancer. If the cancer is associated with a pathogen, the level of cancer can be a certain pathological level.

[0082] In some embodiments, any gene indicative of the occurrence of cancer is used to determine whether a subject is characterized by a precancerous condition. In several embodiments, any of the following genes are used to determine whether a subject is characterized by a precancerous condition: [Table 1]

[0083] In some embodiments, the methods disclosed herein prevent the onset or progression of a genetic disease or disorder associated with epigenetic dysregulation in a subject in need thereof, comprising (i) administering to the subject an effective amount of one or more inhibitors of SPEAR, or (ii) administering an effective amount of cells from the subject that have been contacted with an effective amount of one or more inhibitors of SPEAR. Although most diseases or disorders have a genetic component, identification of a genetic disease or disorder typically requires clinical analysis, including: 1) a physical examination, 2) evaluation of a medical family history, and / or 3) clinical and laboratory tests. For example, the occurrence of the same condition in two or more family members (e.g., first-degree relatives), multiple miscarriages, stillbirths, and childhood deaths suggest the presence, development, or progression of a genetic disease or disorder. In some embodiments, a family history of a common adult condition (e.g., heart disease, cancer, dementia) occurring in two or more relatives at a relatively young age may also suggest a genetic predisposition. In some embodiments, other clinical symptoms suggesting the presence or onset or progression of a genetic disease or disorder may include developmental / mental retardation and congenital anomalies. Often, dysmorphisms, including cardiac and facial, and growth problems, suggest genetic diseases caused by inherited mutations, spontaneous mutations, exposure to teratogens, or unknown factors. Some genetic conditions appear in childhood, while others appear in adolescence or adulthood. In many cases, genetic diseases or disorders may remain undetected for years until events such as puberty or pregnancy trigger the onset of symptoms or the accumulation of toxic metabolites that appear in the disease or disorder.

[0084] In some embodiments, genetic testing includes cytogenetic and / or biochemical / molecular testing to detect abnormalities in chromosome structure, protein function, or DNA sequence. Cytogenetics generally involves examining and staining entire chromosomes for abnormalities, which can reveal distinct bands for each chromosome to show chromosome structure. Biochemical / molecular testing includes detecting: (1) whether a protein is being made; (2) whether too much or too little protein is being made; (3) whether a protein is misfolded; (4) whether an active site or other critical region is altered; (5) whether a protein is mismodified; (6) whether a protein is mislocalized (whether a protein is misaccumulated); (7) and / or whether a protein is misassembled.

[0085] In some embodiments, the methods disclosed herein reset the formation of active histone marks in cancer or pre-cancer cells, and include (i) administering an effective amount of one or more SPEARs or one or more inhibitors of SPEARs to a subject, or (ii) contacting a cell from a subject with an effective amount of one or more SPEARs or one or more inhibitors of SPEARs. In some embodiments, the reset of the formation of active histone marks is, for example, by histone exchange, e.g., replacing existing nucleosomes with newly synthesized histones. In some embodiments, the histone exchange results in the removal or dilution of existing histone modification marks. In some embodiments, a genome-wide ChIP-chip assay is used to identify the reset of the formation of active histone marks in cancer or pre-cancer cells. In some embodiments, as a non-limiting example, histone exchange rapidly delivers epigenetic marks of histone acetylation on a genome-wide scale. In some embodiments, the formation of active histone marks in cancer or precancerous cells is reset by (i) administering an effective amount of one or more SPEARs or one or more inhibitors of SPEAR to a subject, or (ii) contacting a cell from a subject with an effective amount of one or more SPEARs or one or more inhibitors of SPEAR, thereby allowing histones to be exchanged and replaced with active histone marks. For example, administering an effective amount of one or more SPEARs or one or more inhibitors of SPEAR to a subject resets the formation of active histone marks in cancer or precancerous cells by restoring the expression level of one or more SPEARs, restoring the replicative capacity of the origin of replication complex, and / or reappearing histones or histone-associated proteins (e.g., histone acetyltransferase, H2A.Z, H3.3, or variants thereof). In some embodiments, the formation of active histone marks in cancer or precancerous cells is reset by overexpressing one or more SPEARs.In some embodiments, the method includes contacting cells from a subject with an effective amount of one or more SPEARs or inhibitors of one or more SPEARs, where the formation of active histone marks in the cancer or precancerous cells is reset by administration of an effective amount of one or more SPEARs and / or by the reappearance of histones or histone-associated proteins (e.g., histone acetyltransferase, H2A.Z, H3.3, or variants thereof). In some embodiments, one or more SPEARs are overexpressed to reset the formation of active histone marks. In some embodiments, ChIP assays using antibodies against histones or histone-associated proteins, as well as PCR and qRT-PCR, detect the resetting of the formation of active histone marks associated with the cancer or precancerous cells (e.g., by measuring and quantifying the expression levels of one or more SPEARs and / or by the reappearance of histones or histone-associated proteins). In some embodiments, qRT-PCR and strand-specific qRT-PCR assays detect the resetting of active histone mark formation associated with cancer or precancerous cells (e.g., by measuring and quantifying the expression levels of one or more SPEAR and / or the reappearance of histones or histone-associated proteins).

[0086] In some embodiments, one or more SPEARs are overexpressed to restore origin of replication complexes or generate artificial origin of replication complexes. In some embodiments, one or more SPEARs are overexpressed to control the direction of replication. In some embodiments, one or more SPEARs are overexpressed and one or more SPEAR inhibitors (i) slow or prevent the progression of the deleterious direction of replication, activate the opposite direction of replication, and / or (ii) modulate the site of a trinucleotide repeat, optionally reducing the size or reversing the expression of the trinucleotide repeat. In some embodiments, one or more SPEARs are overexpressed and one or more SPEAR inhibitors treat or prevent triplet disease ("TRD"; e.g., Huntington's disease (HD), spinocerebellar ataxia, movement disorder, autism) by reversing the expansion of trinucleotide repeats ("TNR", including CAG, CTG, CGG, and GAA) that occur during replication and repair. In some embodiments, the TRD is a polyglutamine (PolyQ) disease and / or a non-polyglutamine disease. In some embodiments, the polyglutamine disease is DRPLA (dentatorubral-pallidoluysian atrophy), HD (Huntington's disease), SBMA (spinal-bulbar muscular atrophy or Kennedy disease), SCA1 (Spinocerebellar ataxia type 1), SCA2 (Spinocerebellar ataxia type 2), SCA3 (Spinocerebellar ataxia type 3 or Machado-Joseph disease), SCA6 (Spinocerebellar ataxia type 6), SCA7 (Spinocerebellar ataxia type 7), or SCA17 (Spinocerebellar ataxia type 17). In some embodiments, the non-polyglutamine disease is FXS (Fragile X Syndrome), FXTAS (Fragile X-associated Tremor / Ataxia Syndrome), FRAXE (Fragile XE Mental Retardation), FRDA (Friedreich's Ataxia), DM (Myotonic Dystrophy), SCA8 (Spinocerebellar Ataxia Type 8), SCA12 (Spinocerebellar Ataxia Type 12), and Premature Ovarian Failure (POF).

[0087] In some embodiments, the methods disclosed herein restore an origin of replication complex associated with a pre-disease state in a cell characterized by a genetic disease or disorder, and include (i) administering to a subject an effective amount of one or more SPEAR or one or more inhibitors of SPEAR, or (ii) contacting a cell from the subject with an effective amount of one or more SPEAR or one or more inhibitors of SPEAR. In some embodiments, the origin of replication complex is restored by (i) administering to a subject an effective amount of one or more SPEAR or one or more inhibitors of SPEAR, or (ii) contacting a cell from the subject with an effective amount of one or more SPEAR or one or more inhibitors of SPEAR.

[0088] In some embodiments, the inhibitor causes the regulation of the expression level of one or more genes controlled by SPEAR. For example, the inhibitor causes the regulation of the level of one or more genes selected from RARB2, MSH2, ESR1B, AKR1B1, COL6A2, GPX7, HIST1H3C, HOXB4, RASGRF2, TM6SF1, ARHGEF7, TMEFF2, RASSF1, BRCA1, STRATIFIN, and RASSF1A, which are associated with breast cancer. In some embodiments, the inhibitor causes the regulation of the level of one or more genes selected from RUNX3, CDKN2A, and APC, which are associated with gastric cancer, liver cancer, and esophageal cancer, respectively. In some embodiments, the inhibitor causes the regulation of the level of one or more genes selected from SEPT9, hMLH1, CDKN2A / p16, HTLF, ALX4, TMEFF2 / HPP1, NGFR, SFRP2, NEUROG1, RUNX3, and UBE2Q1, which are associated with colorectal cancer. In some embodiments, the inhibitor causes modulation of the levels of one or more genes associated with lung cancer selected from RARB2, RASSF1A, CHFR, STRATI-FIN, SHOX2, RASF1A, and APC1. In some embodiments, the inhibitor causes modulation of the levels of one or more genes selected from RARB2, MSH2, ESR1B, AKR1B1, COL6A2, GPX7, HIST1H3C, HOXB4, RASGRF2, TM6SF1, ARHGEF7, TMEFF2, RASSF1, BRCA1, STRATIFIN, RASSF1A, RUNX3, CDKN2A, APC, SEPT9, hMLH1, CDKN2A / p16, HTLF, ALX4, TMEFF2 / HPP1, NGFR, SFRP2, NEUROG1, RUNX3, UBE2Q1, RARB2, RASSF1A, CHFR, STRATI-FIN, SHOX2, RASSF1A, and APC1.In some embodiments, promoters associated with RARB2, MSH2, ESR1B, AKR1B1, COL6A2, GPX7, HIST1H3C, HOXB4, RASGRF2, TM6SF1, ARHGEF7, TMEFF2, RASSF1, BRCA1, STRATIFIN, RASSF1A, RUNX3, CDKN2A, APC, SEPT9, hMLH1, CDKN2A / p16, HTLF, ALX4, TMEFF2 / HPP1, NGFR, SFRP2, NEUROG1, RUNX3, UBE2Q1, RARB2, RASSF1A, CHFR, STRATI-FIN, SHOX2, RASSF1A, and APC1 are hypermethylated. In some embodiments, the modulation of the expression level of one or more genes controlled by SPEAR is upregulation of the genes. In some embodiments, the upregulation of one or more genes regulated by SPEAR is selected from RARB2, MSH2, ESR1B, AKR1B1, COL6A2, GPX7, HIST1H3C, HOXB4, RASGRF2, TM6SF1, ARHGEF7, TMEFF2, RASSF1, BRCA1, STRATIFIN, RASSF1A, RUNX3, CDKN2A, APC, SEPT9, hMLH1, CDKN2A / p16, HTLF, ALX4, TMEFF2 / HPP1, NGFR, SFRP2, NEUROG1, RUNX3, UBE2Q1, RARB2, RASSF1A, CHFR, STRATI-FIN, SHOX2, RASSF1A, and APC1.

[0089] In some embodiments, the modulation of the expression level of one or more genes regulated by SPEAR is down-regulation of the gene. In some embodiments, the down-regulation of the expression of one or more genes regulated by SPEAR is selected from RARB2, MSH2, ESR1B, AKR1B1, COL6A2, GPX7, HIST1H3C, HOXB4, RASGRF2, TM6SF1, ARHGEF7, TMEFF2, RASSF1, BRCA1, STRATIFIN, RASSF1A, RUNX3, CDKN2A, APC, SEPT9, hMLH1, CDKN2A / p16, HTLF, ALX4, TMEFF2 / HPP1, NGFR, SFRP2, NEUROG1, RUNX3, UBE2Q1, RARB2, RASSF1A, CHFR, STRATI-FIN, SHOX2, RASSF1A, and APC1.

[0090] In some embodiments, the modulation of the expression level of one or more genes controlled by SPEAR is restoration of the level of one or more genes compared to an untreated state. In some embodiments, the gene is an oncogene or a proto-oncogene. In some embodiments, the oncogene is selected from HER2 / neu, RAS, MYC, SRC, BCL2, EGFR, FGFR1, NCOA4, BCL2, FUS, NTRK1, BRCA1, MSH2, WT1, BCL3, GOLGA5, NUP214, BRCA2, NF1, BCL6, GOPC, PAX8, CARS, NF2, BCR, HMGA1, PDGFB, CBFA2T3, NOTCH1, IL2, TNFAIP3, ABL2, EWSR1, MYCL1, ARHGEF12, JAK2, TP53, AKT1, FEV, MYCN, ATM, MAP2K4, and TSC1. In some embodiments, the proto-oncogene is selected from RAS, HER2, MYC, cyclin D, cyclin E, BRAF, and BCR-ABL.

[0091] In some embodiments, the gene is a myc gene. In some embodiments, the myc gene is selected from c-MYC (MYC), l-myc (MYCL), and n-myc (MYCN).

[0092] In some embodiments, the gene is a tumor suppressor gene, hi some embodiments, the tumor suppressor gene is selected from Rb, p53, VHL, APC, BRCA2, NF1, and / or PTCH.

[0093] In some embodiments, the inhibitor reduces or substantially eliminates the activity of an epigenetic mark associated with SPEAR.

[0094] In some embodiments, the inhibitor reduces or substantially eliminates the formation and / or recycling of epigenetic marks.

[0095] In some embodiments, an inhibitor reduces or substantially eliminates activation of a gene.

[0096] In some embodiments, the inhibitor causes activation of the gene.

[0097] In some embodiments, the inhibitor reduces or substantially eliminates one or more of DNA methylation, histone modification, and nucleosome remodeling.

[0098] In some embodiments, the inhibitor causes modulation of pathogenic nucleotide expansions controlled by SPEAR.

[0099] In some embodiments, an inhibitor reduces or substantially eliminates the interaction between SPEAR and one or more histones or histone-associated proteins.

[0100] In some embodiments, the inhibitor reduces or substantially eliminates the interaction between SPEAR and one or more components of ORC, hi some embodiments, the one or more components of ORC are selected from one or more of ORC1, ORC2, ORC3, ORC4, and ORC5, or mutants thereof.

[0101] In some embodiments, the epigenetic dysregulation is a dysregulation of one or more epigenetic marks. In some embodiments, the epigenetic dysregulation of one or more epigenetic marks comprises activation of additional epigenetic marks compared to a non-disease state and / or inactivation of epigenetic marks compared to a non-disease state. In some embodiments, the epigenetic dysregulation is an altered origin of replication. In some embodiments, the altered origin of replication comprises activation of additional origins of replication compared to a non-disease state and / or inactivation of origins of replication compared to a non-disease state.

[0102] In some embodiments, the subject is afflicted with a cancer associated with epigenetic dysregulation. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological cancer.

[0103] In some embodiments, the cancer is selected from the group consisting of basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, cancer of the peritoneum, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatoma, intraepithelial neoplasia, kidney or renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (lips, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, cancer), testicular cancer, thyroid cancer, uterine or endometrial cancer, cancer of the urinary system, vulvar cancer, Hodgkin and non-Hodgkin lymphomas, and lymphomas including B-cell lymphomas (low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, giant mass disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia (CMYK), as well as other carcinomas and sarcomas, and post-transplant lymphoproliferative disorders (PTLDs), as well as one or more of phacomatosis, abnormal blood vessel proliferation associated with edema, and Meigs' syndrome.

[0104] In some embodiments, SPEAR is a non-coding RNA. In some embodiments, SPEAR is a long non-coding RNA (lncRNA). In some embodiments, SPEAR is about 200 nucleotides or more. In some embodiments, SPEAR is about 200-10,000 nucleotides long, or about 200-5000 nucleotides long, or about 200-1000 nucleotides long, or about 200-500 nucleotides long, or about 5000-10000 nucleotides long, or about 1000-10000 nucleotides long, or about 500-10000 nucleotides long. In some embodiments, SPEAR is about 500 nucleotides long or more. In some embodiments, SPEAR is about 1,000 nucleotides long or more. In some embodiments, SPEAR is about 1,500 nucleotides long or more. In some embodiments, SPEAR is about 2,000 nucleotides long or more. In some embodiments, SPEAR is about 2,500 nucleotides long or more. In some embodiments, SPEAR is about 3,000 nucleotides or more in length. In some embodiments, SPEAR is about 3,500 nucleotides or more in length. In some embodiments, SPEAR is about 4,000 nucleotides or more in length. In some embodiments, SPEAR is about 4,500 nucleotides or more in length. In some embodiments, SPEAR is about 5,000 nucleotides or more in length. In some embodiments, SPEAR is about 5,500 nucleotides or more in length. In some embodiments, SPEAR is about 6,000 nucleotides or more in length. In some embodiments, SPEAR is about 6,500 nucleotides or more in length. In some embodiments, SPEAR is about 7,000 nucleotides or more in length. In some embodiments, SPEAR is about 7,500 nucleotides or more in length. In some embodiments, SPEAR is about 8,000 nucleotides or more in length. In some embodiments, SPEAR is about 8,500 nucleotides or more in length. In some embodiments, SPEAR is about 9,000 nucleotides or more in length.In some embodiments, SPEAR is about 9,500 nucleotides or more in length. In some embodiments, SPEAR is about 10,000 nucleotides or more in length.

[0105] In some embodiments, SPEAR is encoded in the region adjacent to the promoter of an active gene. In some embodiments, SPEAR is induced in early S phase of the cell cycle. In some embodiments, SPEAR is induced in early S phase of the cell cycle and detected by flow cytometry. In various embodiments, detection includes the method described on the world wide web at biotech.illinois.edu / sites / biotech.illinois.edu / files / uploads / cb0804.pdf, the entire contents of which are incorporated herein by reference.

[0106] In some embodiments, during early S phase, the formation of major "active" epigenetic marks is driven by the action of a cell cycle specific non-coding RNA ("ncRNA") disclosed herein as "SPEAR", which is encoded adjacent to the promoters of actively transcribed genes. In some embodiments, locally induced SPEAR binds to replacement histone H2A.Z and a nuclear factor, histone acetyltransferase TIP60, resulting in loading / acetylation of replacement histone H2A.Z. In this active chromatin conformation, RNAPII complexes bind to the site and gene expression is initiated.

[0107] In some embodiments, motif discovery analysis is performed on SPEAR to analyze common binding motifs. In various embodiments, the motif discovery analysis is a computational method as described in Achar, A. et al., Biol Direct 10, 61 (2015), the entire contents of which are incorporated herein by reference. In some embodiments, promoter loci are subjected to coverage calculations and filtered based on expression levels. In some embodiments, SPEAR comprises one or more motifs selected from 3, 5, and 9. In some embodiments, motif 9 corresponds to the RNA oligonucleotide RM9A. In some embodiments, SPEAR comprises one or more RM9A motifs. In some embodiments, SPEAR comprises one or more motifs selected from FIG. 3F or variants thereof.

[0108] In some embodiments, a SPEAR comprises one or more stem-loop-like structures. In some embodiments, the inhibitor is a small molecule. In some embodiments, the small molecule directly or indirectly regulates the interaction of SPEAR with histones or histone-associated proteins or ORC. In some embodiments, the inhibitor is a nucleic acid. In some embodiments, the nucleic acid is RNA or DNA. In some embodiments, the nucleic acid directly or indirectly regulates the interaction of SPEAR with histones or histone-associated proteins or ORC. In some embodiments, the nucleic acid comprises a sequence that is at least partially complementary to a portion of SPEAR.

[0109] In some embodiments, one or more nucleotides of the inhibitor are chemically modified. In some embodiments, the chemical modification is selected from locked nucleic acid (LNA), phosphorothioate, 2'-O-methyl, 2'-O-methoxyethyl, 2'-O-alkyl-RNA unit, 2'-OMe-RNA unit, 2'-amino-DNA unit, 2'-fluoro-DNA unit, peptide nucleic acid (PNA) unit, hexitol nucleic acid (HNA) unit, INA unit, and 2'-O-(2-methoxyethyl)-RNA (2'MOE RNA) unit.

[0110] In some embodiments, the nucleic acid is an antisense oligonucleotide or a small interfering RNA (siRNA).

[0111] In some embodiments, the inhibitor modulates the expression and / or activity of SPEAR.

[0112] In some embodiments, the cells from the subject are derived from a biological sample.

[0113] In some embodiments, the biological sample comprises a biopsy, tissue, or bodily fluid.

[0114] In some embodiments, the biological sample comprises one or more of tumor cells, cultured cells, stem cells, and differentiated cells.

[0115] In some embodiments, the method further comprises administering one or more epigenetic drugs or contacting the cells with one or more epigenetic drugs. In some embodiments, the epigenetic drug is a DNA methyltransferase inhibitor, optionally selected from azacitidine, escitabine, zebularine, panobinostat, belinostat, dacinostat, xinostat, tefinostat, acediline, entinostat, mocetinostat, chidamide, butyric acid, pivanex, phenylbutyric acid, and valproic acid. In some embodiments, the epigenetic drug is a histone deacetylase inhibitor, optionally selected from vorinostat, romidepsin, trichostatin A, and trapoxin A.

[0116] In some embodiments, disclosed herein are methods of making an epigenetic modulating agent, comprising: (a) identifying an epigenetic modulating agent by: (i) determining whether the agent binds to or interacts with one or more SPEARs; (ii) classifying the agent as epigenetic modulating based on its ability to bind to or interact with one or more SPEARs; and (b) formulating the agent for use in a therapy selected from the treatment or prevention of cancer associated with epigenetic dysregulation or a genetic disease or disorder associated with epigenetic dysregulation.

[0117] In some embodiments, disclosed herein are methods for assessing a subject's response to an epigenetic modulation therapy, comprising assessing one or more levels of SPEAR in a biological sample from the subject, where (i) a reduction in one or more levels of SPEAR compared to a pre-treatment state indicates a response to the therapy, and / or (ii) an increase or substantially unchanged one or more levels of SPEAR compared to a pre-treatment state indicates a lack or poor response to the therapy. In some embodiments, the epigenetic modulation therapy includes drugs designed to target epigenetic machinery, such as histone deacetylases (HDACs), DNA methyltransferases (DNMTs), enhancer of zeste homolog 2 (EZH2), bromodomain and extra terminal domain proteins (BETs), protein arginine N-methyltransferases (PRMTs), and isocitrate dehydrogenases (IDHs). In some embodiments, the epigenetic modulation therapy comprises a drug selected from vorinostat, romidepsin, panobinostat, belinostat, azacitidine, decitabine, enacinib, and ivosidenib.

[0118] In some embodiments, assessing one or more levels of SPEAR in a biological sample from the subject includes ChIP assays, sequencing (e.g., ChIP sequencing, RNA sequencing, next generation sequencing (e.g., high throughput sequencing, deep sequencing), PCR, and qRT-PCR. In some embodiments, assessing one or more levels of SPEAR in a biological sample from the subject includes capturing nascent RNA and sequencing the captured RNA in a high throughput sequencing assay (e.g., nasRNA-Seq), e.g., to map RNA-Seq reads to a genome or decode reads into contigs. Transcript levels are identified by assembling contigs in vitro and subsequently mapping them to the transcriptome. In nasRNA-Seq, cells are first synchronized and labeled for 1 h upon release into S phase. Collected RNA is then biotinylated by click chemistry, followed by isolation with streptavidin beads and deep sequencing to generate a nasRNA library. SPEAR levels are assessed by correlating gene expression levels with transcripts close to the transcription start sites (TSS) of coding genes.

[0119] In various embodiments, biological sample refers to a sample obtained or derived from a source of interest (e.g., cells) as described herein. In certain embodiments, the source of interest includes an organism, such as an animal or human. In certain embodiments, the biological sample is a biological tissue or liquid. Non-limiting examples of biological samples include bone marrow, blood, blood cells, ascites, (tissue or fine needle) biopsy samples, bodily fluids containing cells, free floating nucleic acid, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymphatic fluid, gynecological fluids, swabs (e.g., skin swabs, vaginal swabs, oral swabs, and nasal swabs), washings or lavages, such as ductal or bronchoalveolar lavages, aspirates, scrapings, specimens (e.g., bone marrow specimens, tissue biopsy specimens, and surgical specimens), feces, other bodily fluids, secretions, and / or excretions, and cells therefrom, etc.

[0120] In various embodiments, "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, and non-human animals, including, but not limited to, non-human primates, dogs, cats, rodents, horses, cows, pigs, mice, rats, hamsters, rabbits, etc. (e.g., those that will be the recipient of a particular treatment or from which cells are harvested). In some embodiments, the subject is a human.

[0121] It will also be understood that terms such as "first", "second" and the like may be used herein to describe various elements, but these elements should not be limited by these terms. These terms are merely used to distinguish one element from another. For example, a first subject could be referred to as a second subject, and similarly, a second subject could be referred to as a first subject, without departing from the scope of the present disclosure. Although a first subject and a second subject are both subjects, they are not the same subject. Furthermore, "subject", "user", and "patient" are used interchangeably herein.

[0122] As used herein, the word "include" and variations thereof are intended to be open-ended, such that the description of items in a list does not exclude other similar items that may also be useful in the materials, compositions, devices, and methods of the present technology. Similarly, the terms "can" and "may" and variations thereof are intended to be open-ended, such that a description that an embodiment can or may include certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features. The open-ended term "comprising" as a synonym for terms such as including, containing, or having is used herein to describe and claim the present disclosure, although the present technology or embodiments thereof may instead be described using more restrictive terms such as "consisting of" or "consisting essentially of" the listed components.

[0123] Unless otherwise defined, all technical and scientific terms in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, preferred methods and materials are described herein. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.

[0124] The present disclosure is further illustrated by the following non-limiting examples. EXAMPLES

[0125] Example 1: SPEAR expression correlates with expression of neighboring genes In this experiment, we investigated whether the epigenetic balance between acetylated and unmodified forms of H2A.Z is maintained by a type of locally guided ncRNA similar to DNMT1-interacting RNAs ("DiRs") that control cell-type specific DNA methylation patterns. By mapping RNAs originating from the CCAAT / enhancer-binding protein alpha ("CEBPA") locus, we defined a transcript upstream of the CEBPA DiR (ecCEBPA), which we call Upper Transcript (UpTr) (Figure 1A). Expression of UpTr precedes expression of both ecCEBPA and CEBPA mRNA during early S phase (Figure 1A). Since most active genes undergo replication during early S phase, followed by the formation of proper active chromatin, in this experiment we investigated whether UpTr (and similar transcripts from other loci) play a role in chromatin reassembly, thereby controlling the expression of their respective mRNAs.

[0126] To establish the global occurrence of similar RNAs, newly named herein as "S-phase early RNAs" ("SPEAR"), nascent RNAs were captured and sequenced (nasRNA-Seq). Synchronized human HL-60 cells were labeled with the ribonucleotide homolog 5-ethynyluridine (EU) for 1 h upon release into S phase. Collected RNA was then biotinylated by click chemistry, isolated with streptavidin beads, and deep sequenced to generate nasRNA libraries (Figure 8A and Figure 1B). Four distinct groups of SPEAR were ranked by expression level (Figure 8A, Figure 8B, Figure 8C). Analysis of transcripts from all four groups revealed that SPEAR initiates near the transcription start sites (TSS) of coding genes and correlates with their expression levels (Figure 1C, Figure 8B, and Figure 8C). Among the genes regulated by SPEAR, c-MYC, the most frequently altered oncogene in cancer, was identified. Examples of SPEAR arising from the c-MYC locus ("c-MYC SPEAR") and SPEAR arising from the PU.1 locus ("PU.1 SPEAR") are shown in Figures 1D and 8D, respectively. c-MYC SPEAR demonstrated a similar expression pattern to UpTr (Figure 1E) and was shown to be represented by approximately 13 copies in the nuclei of HL-60 cells (Figures 8E and 8F). They were mapped by primer extension and 5',3'-RACE (Figures 8E, 8G, and 8H).

[0127] Example 2: SPEAR interacts with H2A.Z, acH2A.Z, and TIP60 Given the close proximity of TSS and SPEAR, in this experiment we analyzed the possible association of SPEAR expression with that of H2A.Z / acH2A.Z (variant histones with acetylation marks). This association was investigated by: (i) ribonucleoprotein (RNP) pull-down experiments followed by mass spectrometry; and (ii) RNA immunoprecipitation followed by RNA sequencing (RIP-Seq). Five SPEAR sequences identified by nasRNA-Seq, corresponding to the loci c-MYC, PU.1, MYB, CEBPA, and CTCF (Figures 8A-8H), were verified by primer extension and 5'3'RACE (Figures 8G and 8H). Approximately 500 nt long uninterrupted sequence segments (Figures 9A, 9B, and 9C) were cloned under the T7 RNA polymerase promoter to express biotinylated sense and antisense SPEAR probes for RNP pull-down experiments. SPEAR-containing RNPs can be identified by probes from both strands; the antisense probe by directly base-pairing with native SPEAR and the sense probe by displacing them (see, e.g., FIG. 9A). Recovered RNPs interacting with both antisense and sense SPEAR probes, as well as a negative control (D-biotin), were analyzed by mass spectrometry. Some of the peptides pulled down by the SPEAR probes corresponded to H2A.FZ / H2A.FV (FIGS. 10A, 10B, 10C, 10D, 10E, 10F, and 10G). The highly homologous forms H2A.FZ and H2A.FV could not be distinguished, and peptides containing acetylation sites were not detected due to the relatively low fragment coverage. Other peptides pulled down by the SPEAR probes corresponded to histones H2A, H2B, H3, H4, and H1. No peptides corresponding to H2A / H2A.Z or other histones were detected by the negative control probes (Figures 10A, 10B, 10C, 10D, 10E, 10F, and 10G).

[0128] In this experiment, we also investigated whether SPEAR exerts its function through direct interaction with H2A.Z and the histone acetyltransferase (HAT) TIP60, an enzyme that acetylates H2A.Z. To directly test the interaction of SPEAR with H2A.Z, acH2A.Z, and TIP60, we performed RIP-Seq using antibodies against H2A.Z (total form), acetylated H2A.Z (acH2A.Z), and TIP60 (Figure 2A). Importantly, significant overlap of RIP-Seq peaks was observed in immunoprecipitations using all three antibodies; i.e., significant colocalization of the three data sets was observed in the region surrounding the TSS (Figure 2B). To estimate the magnitude of SPEAR's interactions with H2A.Z, acH2A.Z, and TIP60, we aligned nasRNA-Seq libraries with the RIP-Seq databases of TIP60, H2A.Z, and acH2A.Z (Fig. 2C, D, A, B). H2AZ and acH2A.Z overlapped with 3120 expressed loci, demonstrating the high likelihood that SPEAR is globally involved in cooperating with TIP60 and H2A.Z / acH2A.Z in establishing active expression modes at the corresponding genes. Thus, these alignments demonstrate a direct correlation between SPEAR expression levels and those of the corresponding genes, and importantly, the occupancy of acH2A.Z at each locus (Fig. 2D; axes are "RNA-Seq expression" and "acH2A.Z peak score," respectively). Comparison of the H2A.Z and acH2A.Z data implies that it is the acetylated form of H2A.Z that is largely associated with SPEAR.

[0129] Taken together, the experiments in this example, including pull-down and RIP experiments, demonstrate a dual interaction of SPEAR with H2A.Z, acH2A.Z, and TIP60.

[0130] Example 3: SPEAR has a common binding motif To further test the SPEAR-H2A.Z / TIP60 relationship, motif discovery analysis was performed on SPEAR to search for common binding motifs. Briefly, approximately 14000 promoter loci (Broad HMM) were subjected to coverage calculations and filtered according to expression levels. Prior to motif discovery analysis, 5' and 3' SPEAR boundaries were inferred from nasRNA-Seq and the corresponding sequences obtained (see Methods below for details; Fig. 3A). 25 motif candidates were identified (Fig. 11A, 11B, 11C, 11D, 11E). Among the promising candidates, three motifs, 3, 5, and 9, exhibited the most significant enrichment across SPEAR-regulated loci. Importantly, similar motifs were not found in the largest class of nuclear RNAs, i.e., transcripts arising from short and long interspersed repeat elements (SINEs and LINEs), or in ribosomal RNAs (see Methods below for details). Among the top three candidates, we ranked motif 9 (corresponding to RNA oligonucleotide RM9A; Fig. 3B) as the most strongly enriched motif in c-MYC SPEAR sequences. This motif was enriched in the overlapping peak of both acH2A.Z RIP-Seq and TIP60 RIP-Seq (52% and 13%, respectively) and was located near the center of the peak (Fig. 3C). Consistent with these results, we used RNA electrophoretic mobility shift assays (REMSA) to show that RM9A can form RNPs in vitro with H2A.Z and with TIP60 (Fig. 3D, lanes 3, 9, and 13; and Fig. 3E, lane 4). After incubation of RM9A with a synthetic peptide corresponding to the N-terminal sequence of H2A.Z, both unmodified and acetylated at lysine 7 (K7), a migration shift was observed (Fig. 3D; right panel). Acetylation of the H2A.Z tail peptide did not abrogate binding, thereby ruling out the possibility that simple charge-charge interactions are involved in the SPEAR-H2A.Z or SPEAR-acH2A.Z complexes.Interestingly, the presence of a predicted stem-loop-like structure (RNAfold, Fig. ​(Fig.3F)3) appears to be required for RM9A to bind H2A.Z and TIP60 (Fig.3D). Indeed, mutation of RM9A that abolished the predicted stem-loop structure altered the intensity and mobility of the RNA-TIP60 complex (Mut RM9A; Fig.3E, lane 5). Unrelated oligonucleotides lacking the common binding motif (UR1, UR2, and UR3) did not form detectable RNP complexes with TIP60 / H2A.Z (Fig.3D and Fig.3E), thus supporting the strong element of both primary and secondary structure recognition between TIP60 / H2A.Z and SPEAR.

[0131] To assess whether RNA may be important for the binding of remodeling complexes to chromatin (e.g., the ability of SPEAR to promote TIP60 binding to chromatin), TIP60 binding was compared to RNA (single-stranded RM9A) and DNA (double-stranded DM9A) oligonucleotides of the same primary sequence. REMSA / EMSA results demonstrated that TIP60 binding was stronger to RNA than to DNA (Figure 3E, lane 4 vs. lane 6). A similar weak complex formation was observed between TIP60 and RNA oligonucleotides (UR2 and UR3) and DNA oligonucleotide duplexes of the same primary sequence (DM2 and DM3, respectively) (shown in Figure 3E, lanes 13 and 20).

[0132] The experiments in this example demonstrate that binding between TIP60 and RNA is dependent on both primary and secondary structure when compared to TIP60 binding to DNA.

[0133] In conclusion, these experimental results suggest an interaction of SPEAR with the histone acetyltransferase TIP60 and histone H2A.Z, particularly its modified form acH2A.Z.

[0134] Example 4: SPEAR is involved in the acetylation and exchange of H2A.Z To directly examine whether SPEAR is required for acH2A.Z loading at the TSS of its corresponding gene, we pharmacologically inhibited transcription followed by chromatin immunoprecipitation (ChIP) with H2A.Z and acH2A.Z antibodies (see, e.g., Figure 4A). Two transcription inhibitors, actinomycin D (ActD; an RNA polymerase I, II, and III inhibitor) and 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DRB; an RNAPII inhibitor), were used at concentrations sufficient to block both RNA polymerase II and III. Cells were synchronized with a double thymidine block and released into S phase when ActD / DRB was added. The rationale for using ActD and DRB was to (i) globally assess the effect of SPEAR inhibition in a short time frame, (ii) test the effects resulting from two different pathways triggered by ActD and DRB, and (iii) take advantage of the reversibility of DRB treatment. Cells were treated with this inhibitor for 2 hours, during which time the overall levels of H2A.Z and TIP60 proteins were unaffected (Figure 10A), while the global expression levels of both the coding genes and SPEAR were significantly decreased (Figure 10B and Figure 10C). After 2 hours of drug treatment, cells were crosslinked and subjected to ChIP-Seq analysis. Figure 4B demonstrated that loci where SPEAR expression was silenced showed reduced enrichment of acH2A.Z, indicating that SPEAR is involved in the precise placement of this epigenetic mark. We then investigated the relative changes in unmodified H2A.Z and acH2A.Z footprints near the TSS resulting from DRB- and ActD-induced inhibition of SPEAR. The enrichment of H2A.Z and acH2A.Z ChIP-Seq signals around 38,512 TSSs (defined in the hg38 UCSC standard gene table) following drug treatment was then compared to their occupancy in mock-treated (DMSO) cells.In these experiments, we observed a dramatic decrease in acH2A.Z enrichment at TSSs in samples treated with either DRB or ActD, in contrast to only minor changes in the occupancy levels of unmodified H2A.Z (Fig. 4C; see Methods below for details). DRB-treated cells showed at least a two-fold decrease in acH2A.Z occupancy at 3,394 TSSs when compared to untreated cells, whereas only 398 TSSs showed a similar trend in total H2A.Z signal. ActD-treated cells also followed this pattern, with 6,283 TSSs showing a more than two-fold decrease in the levels of acH2A.Z compared to untreated samples. In contrast, 650 TSSs exhibited a comparable decrease in total H2A.Z occupancy. A 10-fold greater reduction in acH2A.Z was observed in treated samples compared to H2A.Z, suggesting that SPEAR is involved in maintaining normal levels of H2A.Z acetylation at TSSs, i.e., generating the active epigenetic mark acH2H2A.Z. Figure 4D (top and middle panels) illustrates an example of a combined snapshot of two individual loci, c-MYC and PU.1, in which SPEAR, both negatively affected by ActD and DRB (shown in Figure 10C), demonstrates a reduction in the intensity of H2A.Z and acH2A.Z peaks, i.e., a more pronounced reduction in acH2A.Z levels (full snapshots shown in Figures 10D and 10E). The ratio of peak intensity of drug-treated H2A.Z to control (DMSO) for c-MYC and PU.1 loci, respectively, is [DMSO / DRB / ActD]. c‐MYC =1 / 0.76 / 0.67, and [DMSO / DRB / ActD] PU.1 = 1 / 0.87 / 0.82; the ratio of acH2A.Z peak intensities of drug-treated to control (DMSO) is [DMSO / DRB / AcD] c‐MYC =1 / 0.62 / 0.36, and [DMSO / DRB / ActD] PU.1=1.0 / 0.25 / 0.13. These results were confirmed by quantitative ChIP-PCR for the c-MYC locus (Fig. 4E). In contrast, loci that escaped the downregulatory effects of drugs on SPEAR exhibited acH2A.Z intensity ratios that were consistent with changes in the levels of SPEAR. For example, the MYB locus, whose SPEAR was positively affected by ActD and DRB (Fig. 10C), exhibits an increase in the intensity of the acH2A.Z peak, while the intensity of the unmodified H2A.Z peak does not change significantly (Fig. 4D, lower panel; and Fig. 10D and Fig. 10E). In MYB, the ratio of drug-treated acH2A.Z peak intensity to control (DMSO) was [DMSO / DRB / ActD] MYB = 0.62 / 0.89 / 1.0, which corresponds to an increase in the SPEAR level (FIG. 10C). Thus, unexpectedly, at a locus where SPEAR expression is positively affected by ActD / DRB, the experiments in this example illustrate how increasing SPEAR levels is accompanied by a corresponding increase in the intensity of the acH2A.Z peak.

[0135] To confirm the ChIP-Seq results and follow the loading of H2A.Z and acH2A.Z, we performed nascent chromatin immunoprecipitation (nasChIP-PCR) with antibodies against H2A.Z, acH2A.Z, and TIP60, because nasChIP-PCR can be used to follow the loading of histones. Cells were synchronized with a double thymidine block, released to S phase, and treated with DRB for 2 h. Edu DNA analog was added to the medium to allow collection of nascent chromatin DNA (Figure 4A). During inhibition of transcription with DRB, the c-MYC locus showed reduced enrichment of H2A.Z, acH2A.Z, and TIP60 (Figure 4F, three left panels). Because there was an even greater degree of loss of acetylated acH2A.Z at the TSS of the c-MYC locus, the results of these experiments provide an important link between the suppression of c-MYC SPEAR expression and reduced levels of replacement histone H2A.Z. Similar results were obtained for another locus, PU.1 (Figure 4F, right panel).

[0136] Taken together, the results of ChIP-Seq and nasChIP-PCR experiments underscore the role of transcription in the loading of the substituted histone H2A.Z. The robust reduction in acetylated levels of H2A.Z compared to the reduction in the unmodified form at the gene's TSS, as well as the loss of TIP60 at the same sites, suggest a role for transcription in mediating H2A.Z acetylation.

[0137] In the reverse experiment, we tested RNAi-mediated downregulation of specific SPEARs, followed by ChIP-Seq and ChIP-qPCR analysis, to see whether RNAi-mediated downregulation resulted in reduced H2A.Z and acH2A.Z levels at the TSS of the target locus, as well as reduced expression of the corresponding gene. Comparison of the two panels in Figure 5A demonstrates that a reduction of c-MYC SPEAR by approx. 75% reduces c-MYC mRNA expression by a similar magnitude (approx. 70%). In contrast, the unaffected control PU.1 SPEAR is accompanied by virtually no change in PU.1 mRNA levels. Figure 5B shows snapshots of H2A.Z and acH2A.Z levels at the target and control loci, c-MYC and PU.1, respectively (full snapshots are shown in Figure 10F and Figure 10G). Knockdown of c-MYC SPEAR was associated with a significant reduction in acH2A.Z levels at the TSS of the c-MYC gene, when compared to the TSS of the non-targeted control PU.1 gene. Results from RNAi / ChIP experiments demonstrate that expression of c-MYC SPEAR correlates with the level of H2A.Z acetylation at the TSS of c-MYC. These experimental results demonstrate a causal relationship between SPEAR expression and loading of activating histone marks, resulting in transcriptional activation of adjacent genes. Figure 5C shows validation of ChIP-Seq analysis of acH2A.Z using quantitative ChIP-PCR for the c-MYC locus.

[0138] Overall, downregulation of SPEAR using independent pharmacological and RNAi-mediated methods substantially reduced the epigenetic mark acH2A.Z, implying that loss of the acetylated form of H2A.Z from chromatin in the vicinity of active TSSs reduces its native activity and, consequently, expression of the respective genes (Figure 5A, right panel).

[0139] Example 5: SPEAR controls the expression of respective mRNAs via recruitment / loading of TIP60 / acH2AZ To demonstrate that SPEAR-mediated regulation of the corresponding mRNA is carried out through the TIP60 / acH2AZ pathway, in this example, we tested the effect of TIP60 inhibition on the c-MYC locus. Two TIP60 / HAT inhibitors, TH1834 and MG-149, were used in HL60 cells to reduce the overall level of H2A.Z acetylation (Figure 11A). This resulted in a significant downregulation of the mRNA, in contrast to little change in the level of SPEAR transcript (Figure 6A). To investigate the possibility of direct regulation of c-MYC expression by TIP60 and acH2A.Z interacting with SPEAR, we took advantage of the reversibility of DRB transcription inhibitors to monitor the regulatory effect of nascent SPEAR on the level of nascent mRNA expression in the presence or absence of the two TIP60 inhibitors. The rationale for this approach was to analyze only those transcripts that reappeared after being released from the transcriptional block during HAT inhibition. The short treatment time should rule out any secondary effects of HAT inhibition.

[0140] Cells were synchronized with a double thymidine block and released into S phase, at which time DMSO / DRB was added. After 2 hours (during which H2A.Z levels remained unaffected, but SPEAR and mRNA levels were reduced, see FIG. 10A), cells were washed and then incubated for another 2 hours with or without TIP60 inhibitors. This interval is sufficient for the levels of both c-MYC mRNA and its SPEAR to recover in the absence of TIP60 inhibitors (results shown in FIG. 11B). The medium was supplemented with EU RNA and EdU DNA analogs to allow the collection of nascent RNA and newly formed chromatin that escaped drug-induced inhibition of transcription or acetylation. After 2 h in the presence of TIP60 inhibitors (200 μM MG-149 and 500 μM TH1834), during which acH2A.Z levels decreased (see Figure 11A), cells were crosslinked and subjected to nasChIP and nascent RNA expression analysis (nas-qRT-PCR) (Figure 6B). Collected RNA was biotinylated by click chemistry, isolated with streptavidin beads, and analyzed by nas-qRT-PCR (see Methods below; Figure 8A; lower panel for details). Results in Figure 6C show that when TIP60 activity is inhibited, SPEAR expression no longer correlates with mRNA, i.e., restoring SPEAR levels cannot rescue c-MYC mRNA expression to the level defined by the reversed DRB. These data suggest that the function of SPEAR in controlling c-MYC mRNA expression is disrupted by inhibition of TIP60 HAT activity.

[0141] To further confirm that SPEAR directly achieves its c-MYC regulatory function through the TIP60 / acH2A.Z pathway, we used NASChIP-qPCR to assess the differences in chromatin occupancy of H2A.Z, acH2A.Z, and TIP60 after inhibition of transcription ("DRB"), reversal of inhibition ("DRB REV"), and reversal of inhibition in the presence of two TIP60 inhibitors. In particular, it was important to check whether the reduction in c-MYC expression after treating cells with DRB and TIP60 inhibitors correlated with the decreased enrichment of TIP60, acH2A.Z, or H2A.Z. Nascent DNA was isolated from immunoprecipitated chromatin with antibodies against H2A.Z or acH2A.Z or TIP60, biotinylated by click chemistry, then isolated with streptavidin beads (see Methods below for details) and finally analyzed by qPCR with amplicons corresponding to maximum enrichment within the c-MYC locus (Figure 4E). By analyzing nascent RNA and chromatin, we investigated only the immediate changes in c-MYC expression and occupancy of TIP60, acH2A.Z and H2A.Z within the locus imposed by inhibition of transcription and / or acetylation. The middle and right panels of Figure 6D demonstrate that the c-MYC locus in which SPEAR expression is silenced shows a decrease in enrichment of both TIP60 and acH2A.Z, and that reversing the inhibition of transcription fully restores enrichment of both TIP60 and acH2A.Z. This indicates that SPEAR is involved in the recruitment of TIP60 and the precise placement of epigenetic acetylation marks. In the presence of two HAT inhibitors, reversal of transcriptional inhibition could only restore the enrichment levels of unmodified H2A.Z and TIP60 (Fig. 6D, left and right panels), but not acH2A.Z (middle panel). In contrast, no significant changes were observed in the distribution of unmodified H2A.Z (Fig. 6D; left panel). Moreover, no enrichment of H2A.Z, acH2A.Z, or TIP60 was detected within gene desert loci (Fig. S11C).Considering that the global levels of H2A.Z did not change locally (near the TSS of the c-MYC locus; Fig. 6D; left panel) but also throughout the experiment (Fig. 11A), these results establish a leading role for the TIP60 / acH2A.Z pathway in the c-MYC SPEAR-mediated regulation of the corresponding gene c-MYC. Similar results were obtained for the PU.1 locus (Fig. 11D and Fig. 11E). These findings using nasChIP are consistent with the general trends obtained globally by ChIP-Seq (Fig. 4C).

[0142] Taken together, these experiments and the results presented in the Examples demonstrate that the TIP60 / acH2A.Z pathway is a general mechanism for SPEAR-mediated regulation of their corresponding adjacent coding genes.

[0143] Example 6: Controlling the direction of replication using SPEAR In this example, we show that SPEAR controls the direction of replication. As shown in this example, by changing the direction of replication, we can reverse the expansion of trinucleotide repeats. This has implications for the treatment of triplet disease ("TRD") caused by trinucleotide repeat expansion. Trinucleotide repeat expansion is a mutation that increases the copy number of three nucleotide repeats to a level that leads to instability in gene expression. In this example, we show that SPEAR reverses the expansion of trinucleotide repeats, thereby demonstrating that SPEAR can treat triplet disease. Figure 12A (without wishing to be bound by theory), Figure 12B, Figure 12C, and Figure 12D are images showing how induction of SPEAR-like transcription affects the size of trinucleotide repeats. These experiments suggest that one or more SPEARs can be overexpressed to control the direction of replication, and that one or more SPEAR inhibitors can eradicate the cause of triplet disease ("TRD"; e.g., Huntington's disease (HD), spinocerebellar ataxia, movement disorders, autism) by reversing the expansion of trinucleotide repeats ("TNRs," which include CAG, CTG, CGG, and GAA) that occur during replication and repair.

[0144] method mammalian cell culture The human leukemia cell line HL-60 was obtained from ATCC and grown in glutamine-containing medium in the absence of antibiotics at 37°C in a humidified atmosphere with 5% CO2.

[0145] Antibodies used: Rabbit polyclonal against human histone H2A.Z - ChIP Grade (1:2,000; Abcam ab4174), sheep polyclonal against human H2A.Z (K4+K7+K11) (1:2,000; Abcam ab18262), rabbit polyclonal against human TIP60 antibody.

[0146] RNA isolation Total RNA isolation was performed and all RNA samples used in this study were treated with DNase I (10 U DNase I per 3 μg total RNA; 1 h at 37°C; in the presence of RNase inhibitors). After DNase I treatment, RNA samples were extracted with acid phenol (pH 4.3) to eliminate any remaining traces of DNA. cDNA synthesis was performed with Random Primers (Invitrogen) or gene-specific primers using Transcriptor Reverse Transcriptase (Roche Applied Science) according to the manufacturer's recommendations. cDNA was purified with the High Pure PCR Product Purification Kit (Roche Applied Science).

[0147] qRT‐PCR Sybr Green reactions were performed using iQ Sybr Green supermix (Biorad, Hercules, CA) with the following parameters: 95°C (10 min); 40 cycles of 95°C (15 s) and 60°C (1 min) 72°C (1 min). TaqMan analysis was performed using Hotstart Probe One-step qRT-PCR master mix (USB) with the following conditions: 50°C (10 min), 95°C (2 min), then 40 cycles of 95°C (15 s) and 60°C (60 s).

[0148] Primers used for TaqMan real-time PCR: Human MYC mRNA:ABI Cat.# Hs00153408_m1 Human MYB mRNA:ABI Cat.# Hs00920556_m1, Human IRF2BP2 mRNA:ABI Cat.# Hs00766250_g1, Human RRM2 mRNA:ABI Cat.# Hs00357247_g, Human CTCF mRNA:ABI Cat.# Hs00902008_m1, rRNA:ABI Cat.# 4310893E, Human 5S rRNA:ABI Cat.# Hs02385257_g1.

[0149] Primers used for strand-specific real-time RT PCR (Sybr): Reverse Transcriptase primer for c-MYC SPEAR: 5'-AAC CGC ATC CTT GTC CTG TGA GTA-3' (SEQ ID NO: 1); PCR primers: Forward: 5'-ACA GGC AGA CAC ATC TCA GGG CTA-3' (SEQ ID NO: 2); Reverse: 5'-ATA GGG AGG AAT GAT AGA GGC ATA-3' (SEQ ID NO: 3); and Reverse Transcriptase primer for PU.1 SPEAR: 5'-GGC TTT TGC TCT AAC CCA AC-3' (SEQ ID NO: 4); PCR primers: Forward: 5'-ACT ATG CTG AAG ACC CTA CAC-3' (SEQ ID NO: 5); Reverse: 5'-GCT CTA ACC CAA CAA ATG CC-3' (SEQ ID NO: 6).

[0150] Capture of nascent RNA / DNA was performed using the Click-iT Nascent RNA Capture Kit (ThermoFisher) according to the manufacturer's instructions with minor modifications. Briefly, 1. Labeling of cells with EU / EdU. Stock solutions of 200 mM EU or 30 mM EdU were added to cells to give final concentrations of 0.5 mM or 30 μM, respectively. 2. Incubation. Cells were incubated for 1 or 2 h. 3. Isolation of RNA / DNA. Cells were harvested and RNA / DNA was isolated and dissolved in 14 μL of H2O. 4. Biotinylation of RNA / DNA by click reaction. Click-iT reaction cocktail (50 μL per reaction) was prepared according to the manufacturer's instructions: a mixture containing 1× Click-iT EU buffer; 2 mM CuSO4; 1 mM biotin azide; 13.25 μL of isolated RNA; 10 mM Click-iT reaction buffer additive 1; 12 mM Click-iT reaction buffer additive 2 was prepared. After adding each component, the reaction cocktail was mixed gently by vortexing. Addition of Click-iT reaction buffer additive 1 stock initiates the click reaction between EU-RNA / EdU-DNA and biotin azide. Click-iT® reaction buffer additive 2 is then added and incubated for 30 minutes with gentle vortexing. 5. RNA / DNA precipitation. 1 μL of ULTRAPURE™ Glycogen, 55 μL of 7M ammonium acetate, and 750 μL of chilled 100% ethanol were added to the click reaction and incubated at -70°C for at least 30 minutes. After centrifugation, the pellet was dissolved in 125 μL of H2O. 6. Binding of biotinylated RNA / DNA with Dynabeads® MYONE™ Streptavidin T1 magnetic beads. The RNA / DNA binding reaction mixture contained 125 μL of 2× Click-iT RNA binding buffer, 2 μL of ribonuclease inhibitor or 2 μL of water in the case of DNA, and 125 μL of isolated biotinylated RNA / DNA. The RNA binding reaction mixture was heated at 68-70°C for 5 minutes, and 50 μL of the bead suspension was added to the heated RNA binding reaction mixture.The tubes containing the RNA / DNA binding reactions were incubated at room temperature for 30 minutes with gentle vortexing to avoid settling of the beads. The beads were immobilized using a magnet and washed five times with 500 μL of Click-iT® Reaction Wash Buffer 1 and five times with 500 μL of Click-iT® Reaction Wash Buffer 2. Finally, the beads were resuspended in 50 μL of Click-iT Reaction Wash Buffer 2 and the captured RNA was immediately processed for cDNA synthesis. The captured DNA was released into 50 μL of boiling water and used for qPCR analysis.

[0151] Primer extension and 5' / 3'RACE cDNA was synthesized from the HL-60 cell line as described above and run on urea-PAGE. 5' / 3'RACE was performed using the Exact START™ Eukaryotic mRNA 5'-&3'-RACE Kit according to the manufacturer's instructions.

[0152] Double thymidine block (early S-phase block) was performed as described. Briefly, HL-60 cells were grown overnight to 70–80% confluence, washed twice with 1× PBS, and cultured for 18 h in DMEM (10% FCS) + 2.5 mM thymidine (first block). Thymidine was washed out with 1× PBS, and cells were grown in DMEM (10% FCS). After 8 h, cells were cultured for 18 h in the presence of thymidine (second block) and then released as described. Synchronization was monitored by flow cytometric analysis of propidium iodide-stained cells using an LSRII flow cytometer (BD Biosciences) at the Harvard Stem Cell Institute / Beth Israel Deaconess Center Flow Cytometry Facility.

[0153] DRB and actinomycin D treatments were performed as described. Briefly, after release from double thymidine block, HL-60 cells were treated with 100 μM 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) (Sigma Aldrich) or 0.8 μM actinomycin D (Sigma Aldrich) as indicated for each time point.

[0154] Downregulation of c-MYC SPEAR 27 siRNAs targeting c-MYC SPEAR were designed and synthesized by siTOOLs Biotech as c-MYC SPEAR siPool (siSPEAR). The sequences are shown in Table 1 below. The siMyc was dissolved in nuclease-free water and used to transfect HL-60 cells. Transfection was performed using Amaxa Cell Line Nucleoeffector Kit V, Program T-019 (Nucleoeffector II Device) according to the manufacturer's instructions. Briefly, 2 × 10 6Cells / reaction were cultured in RPMI medium + 10% FBS. Cells were harvested by centrifugation. 1 μL of siSPEAR and negative control siPool (siControl) were mixed with 4 μL of nuclease-free water per reaction (siPool solution). Cells were resuspended in 100 μL of room temperature Nucleofector Solution per reaction and combined with the siPool solution. The final concentration of siSPEAR and siControl was 100 nM. Cell / siSPEAR and cell / siControl suspensions were transferred into certified cuvettes and passed in triplicate through a Nucleofector Program T-019 (Nucleofector II Device). 500 μL of pre-incubated culture medium was immediately added to the cuvette and the samples were gently transferred into wells of a 6-well plate containing 500 μL of pre-incubated culture medium. Samples were cultured for 24 hours and then electroporation was repeated. Samples were cultured for another 24 hours. Viable cells were harvested with Ficoll-Paque PLUS medium (GE Healthcare, #17144003) and RNA / chromatin was extracted.

[0155] The short hairpin RNA sequences are shown in Table 1.

[0156] Ribonucleoprotein (RNP) fractionation The same number of viable cells (approximately 2 million) counted after Ficoll gradient purification was used for each isolation. 1–5. 2 × 10 6Nuclei were isolated from cells and, briefly, equal amounts of viable cells were washed with ice-cold PBS supplemented with 5 mM vanadyl complex, 1 mM PMSF and resuspended in ice-cold lysis buffer. Ice-cold lysis buffer was as follows: 1x buffer A (10 mM HEPES-NaOH pH 7.6; 25 mM KCl; 0.15 mM spermine; 0.5 mM spermidine; 1 mM EDTA; 2 mM sodium butyrate); 1.25 M sucrose; 10% glycerol; 5 mg / mL BSA; 0.5% NP-40; freshly supplemented, protease inhibitors (2 mM leupeptin, added as ×400; 2 mM pepstatin, added as ×400; 100 mM benzamidine, added as ×400; protease inhibitor cocktail (Roche Applied Science, catalog no. 1836153), 1 tablet / 375 μL HO, added as ×100; 100 mM PMSF, added as ×100), 2 mM vanadyl complex (New England Biolabs), and 20 units / mL RNase inhibitor (RNAguard; Amersham Biosciences). Samples were incubated at 0°C for approximately 10 minutes and passed through a Dounce homogenizer several times. Pelleted nuclei were resuspended in 0.5 ml lysis buffer, diluted with 2.25 mL dilution buffer (2.13 mL "cushion" buffer + 0.12 mL 0.1 g / mL BSA), freshly supplemented with protease inhibitors, layered on top of 2 mL "cushion" (200 mL "cushion" buffer, consisting of 15 mL ddH2O; 15 mL 20x buffer A; 30 mL glycerol; 240 mL 2.5 M sucrose; freshly supplemented protease inhibitors) in one SW 55Ti tube and centrifuged at 24,400 rpm for 60 minutes at 4°C. Washed with PBS / 1 mM PMSF. 7. The nuclei were then resuspended in 1.8 ml of cytoskeleton buffer (CSK-50: 10 mM Pipes, pH 6.8; 300 mM sucrose, 50 mM NaCl; 3 mM MgCl2; 1 mM EGTA; 5 mM vanadyl complex; 1 mM PMSF). 8. 20 μl (200 U) of DNase I was added and incubated at 37° C. for 30 minutes and cooled on ice.9. After addition of 200 μl of 2.5 M (NH4)2SO4 (250 mM final), nuclei were incubated for 40 min at 4°C on a rocking platform and spun down at 2000 g for 5 min at 4°C. 10. The supernatant fraction was discarded. 11. The RNP-containing pellet was washed twice with ice-cold PBS and resuspended in 2 ml of RIP buffer #4 (50 mM HEPES-KOH, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na deoxycholate, 0.5% N-lauroyl sarcosine, 1× protease inhibitors). 12. After solubilization by sonication, the pellet was cooled on ice and spun down at 5,000 g for 5 min at 4°C. 13. The supernatant-RNP fractions (approximately 2 ml each) were collected. 14. 1.2 μl of 20% SDS; 0.8 μl of 1 M DTT solution was added to 40 μl of RNP fraction and boiled for at least 1 min. 15. Samples were desalted on a pre-equilibrated G25 column (with 100 mM Tris-HCl, pH 7.4; 10 mM DTT; 0.6% SDS) and subjected to SDS-PAGE / Western blotting analysis.

[0157] Western blotting analysis Approximately 0.2×10 for each sample 6 Total cell lysates from 100 cells were separated on 12% SDS-PAGE gels and transferred to nitrocellulose membranes. Immunoblots were stained overnight at 4°C with the following primary antibodies: H2A.Z (1:2,000; Abcam ab4174), H2A.Z acetyl(K4+K7+K11) (1:2,000; Abcam ab18262) or TIP60 (1:1000; a gift from Bruno Amati). All secondary horseradish peroxidase (HRP)-conjugated antibodies were diluted 1:5,000 and incubated with TBST / 5% BSA for 1 h at room temperature. Immunoreactive proteins were detected using the Pierce® ECL system (Thermo Scientific #32106).

[0158] Tandem mass spectrometry (LC-MS / MS) Whole cell extracts were resolved on SDS-PAGE gels, which were stained with Coomassie Brilliant Blue to excite protein bands of interest. Gel pieces were reduced with 55 mM DTT, alkylated with 10 mM iodoacetamide (Sigma-Aldrich), and digested with TPCK-modified trypsin / LysC mix (Promega) overnight at pH = 8.3. Digestion was stopped with 1% TFA, and peptides were dried down to 10 μL in a SpeedVac. Peptide mixtures were analyzed by positive ion mode LC-MS / MS using a high-resolution hybrid QExactive HF Orbitrap Mass Spectrometer (Thermo Fisher Scientific) by HCD with data-dependent analysis (DDA). Peptides were delivered and separated using an EASY-nLC nanoflow HPLC (Thermo Fisher Scientific) using a self-packed 15 cm long × 75 μm inner diameter C18 frit microcapillary column at 300 nL / min. Solvent gradient conditions were as follows: 3%–38% buffer B (100% acetonitrile) in buffer A (0.9% acetonitrile / 0.1% formic acid / 99.0% water) for 90 min. Raw files were processed with MaxQuant version 1.5.2.8 with pre-set standard settings and a multiplicity of 1. Carbamidomethylation was set as a fixed modification, and methionine oxidation and protein N-acetylation were considered as variable modifications. Search results were filtered with a false discovery rate of 0.01. Only reverse hits and site specifics, as well as potential contaminants, were removed. MS data will be deposited in the ProteomeXChange Consortium via PRIDE upon manuscript acceptance.

[0159] Nuclear chromatin (ChIP) and RNA immunoprecipitation (nRIP) ChIP was performed as follows: cells were crosslinked with 1% formaldehyde for 10 min at room temperature. 6Pellets of cells were used for immunoprecipitation, lysed on ice for 10 min, and chromatin was sheared using a Branson 250 digital sonicator. Each ChIP was performed with 4 μg of antibody and incubated overnight at 4°C. A 50 / 50 slurry of Protein A and Protein G Dynabeads was used to capture enriched chromatin, which was then washed before reverse cross-linking and proteinase K digestion at 65°C. AMPure XP beads were used to clean up and isolate ChIP DNA for subsequent library construction. The following antibodies were used for ChIP: H2A.Z (Abcam ab4174, lot GR3176820-1), acH2A.Z (Abcam ab18262, lot GR306397-1), TIP60 antibody; and IgG (Abcam ab171870). Fold enrichment was calculated using the following equation: (-ΔΔCt(ChIP / 非免疫血清)) The primer sets used for ChIP are listed in Table 2. nRIP was performed and crosslinked nuclei were collected as follows: 1. 60 × 10 6HL-60 cells were crosslinked with 1% formaldehyde (formaldehyde solution, freshly made: 50 mM HEPES-KOH; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 11% formaldehyde) for 10 min at room temperature. Crosslinking was stopped by adding 1 / 10 volume of 2.66 M glycine and keeping for 5 min at room temperature and 3 min on ice. 3. Cell pellets were washed twice with ice-cold PBS (freshly supplemented with 1 mM PMSF). 4. The cell pellet was resuspended in the following cell lysis buffer (volume = 4 mL): 1x buffer (10 mM Tris pH 7.4; 10 mM NaCl; 0.5% NP-40, freshly supplemented with protease inhibitors (protease inhibitor cocktail: Roche Applied Science, catalogue no. 1836153, 1 tablet / 375 μL H2O, added as ×100), 0.1 mM PMSF, and 0.2 mM vanadyl complex (NEB). 5. The cells were incubated at 0°C for 10-15 min and homogenized in a Dounce (10 times in inner tube A and 40 times in inner tube B). 6. Nuclei were collected by centrifugation at 2,000 rpm for 10 min at 4°C. 7. The nuclei were resuspended in 3 ml of 1x resuspension buffer (50 mM ESB) supplemented with 0.1 mM PMSF and 0.2 mM vanadyl complex. HEPES-NaOH, pH 7.4; 10 mM MgCl2). 8. DNase I treatment (250 U / ml) was performed for 30 min at 37°C and the reaction was stopped by adding EDTA (final concentration 20 mM). 9. Resuspended nuclei were sonicated once for 20 s (one pulse every 3 s) at 30% power (Branson Digital Sonifer, Danbury, CT). Immunoprecipitation was performed as follows: 1. Before pre-clearing, samples were adjusted to the following: 1% Triton X-100; 0.1% sodium deoxycholate; 0.01% SDS; 140 mM NaCl; protease inhibitors; 0.2 mM vanadyl complex; 0.1 mM PMSF. 2. Pre-clearing step: Approximately 50 μl of magnetic beads (Protein A or G Magnetic Beads; #S1425S or #S1430S NEB) was added to the sample and incubation was carried out on a rocking platform for 1 hour at 4° C. 3. The beads were removed in a magnetic field.4. The samples were then split into 5 aliquots: (i) antibody of interest: (i) H2A.Z antibody (ab4174); (ii) acH2A.Z antibody (ab18262); (iii) TIP60 antibody; (iv) pre-immune serum: IgG (ab171870); (v) no antibody, no serum (input). 5. 5 μg of antibody or pre-immune serum was added to each aliquot and incubation was performed overnight at 4° C. on a rocking platform. Input was stored at −20° C. after adding SDS to a final concentration of 2%. Day II. 6. 200 μl of Protein A coated superparamagnetic beads (enough to bind 8 μg IgG) was added to the sample and incubated on a rocking platform at 4° C. for 1 hour. 7. The beads were washed six times in the immunoprecipitation buffer (150 mM NaCl; 10 mM Tris-HCl, pH 7.4; 1 mM EDTA; 1 mM EGTA pH 8.0; 1% Triton X-100; 0.5% NP-40, freshly supplemented, 0.2 mM vanadyl complex and 0.2 mM PMSF) in a magnetic field. 8. Proteinase K treatment to release DNA / RNA into solution and reverse crosslinks was performed overnight at 56°C using 500 μg / ml proteinase K in 200 μl of: 100 mM Tris-HCl, pH 7.4; 0.5% SDS for immunoprecipitation samples and in parallel for inputs. 9. Day III. The beads were removed in a magnetic field. 10. Phenol (pH 4.3) extraction was performed after the addition of NaCl (0.2 M final concentration). 11. Ethanol precipitation (in the presence of glycogen); 3 hours at -20°C. 12. Pellets were dissolved in 180 μl H2O, heated to 75°C for 3 minutes and immediately chilled on ice. 13. Samples were treated with DNase I (250 U / ml) in ×1 buffer #2 (NEB) in the presence of 300 U / ml RNase inhibitor at 37°C for 30 minutes. 14. Phenol (pH 4.3) extraction and EtOH precipitation were repeated. 15. RNA pellets were dissolved in 50 μl H2O.

[0160] RNA electrophoresis gel mobility shift assay (REMSA) RNA oligonucleotides (15 pmol) were added to the 32End-labeling was performed with [P]ATP (Perkin Elmer) and T4 polynucleotide kinase (New England Biolabs). Reactions were incubated at 37°C for 1 h and then passed through a G-25 spin column (GE Healthcare) according to the manufacturer's instructions to remove unincorporated radioactivity. Labeled samples were gel purified on a 10% polyacrylamide gel. Binding reactions were performed in a volume of 10 μL in the following buffer: 5 mM Tris pH 7.4, 5 mM MgCl2, 1 mM DTT, 3% v / v glycerol, 100 mM NaCl. Five micrograms of full-length purified H2A.Z (Abcam) and TIP60 (SignalChem) proteins were diluted with 1.1 nM 32 The probe was incubated with P-labeled ssRNA. All reactions were assembled on ice and then incubated at room temperature for 30 min. Samples were loaded onto a 6% native polyacrylamide gel (0.5× TBE) at 140 V for 3 h at 4 °C. Various concentrations (1 μM to 10 mM) of H2A.Z and K7-acetylated H2A.Z peptides (AnaSpec) were incubated with a constant amount of probe in a final volume of 10 μL of 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.4 mM EDTA, and 40 U / ml RNasin, 1 mM DTT, 50% glycerol. Then, 1 μl of glutaraldehyde (final concentration 0.2%) was added into the mixture and incubated at room temperature for 15 min. Samples were loaded onto a 10% native polyacrylamide gel (0.5× TBE) at 170 V for 3 h at 4 °C. All gels were fixed with methanol and acetic acid, then dried and exposed to X-ray film. The RNA oligonucleotides are listed in Table 3.

[0161] Quantification and statistical analysis ChIP-sequencing analysis ChIP libraries were constructed and paired-end sequenced on a NextSeq500 platform with a read length of 36 nucleotides. The resulting alignment files were trimmed to 150 bp and processed with trim_galore, Cutadapt, and FastQC for adapter trimming and sequence quality control. CHIP-Seq reads were aligned to the hg38 human reference genome in STAR using the following settings: "--outFilterMultimapNmax 1", "--outFilterMatchNminOverLread 0.8", "--alignIntronMax 1", "--alignEndsType EndToEnd", and the remaining options were set to default. acH2AZ bam files were converted to bigWig using deepTools to remove duplicated reads, normalize by library size, and convert values ​​to "counts per million". Duplicate reads were removed from the H2AZ libraries using MarkDuplicates and then analyzed using the DPOS algorithm from the DANPOS2 software. The resulting smoothed and quantile-normalized wig files were converted to bigWig files using the wigToBigWig tool from UCSC. DeepTools was used to quantify and plot heat maps of ChIP-Seq signal around all transcription start sites of genes annotated in the UCSC known norm table. Metaplots of the regions around transcription start sites were generated in R. Scatter plots were generated in R using the function smoothScatter, using as input the arcsine transformed values ​​of the area under the ChIP-Seq signal around the TSS extracted from the matrix used to generate the heatmap. Scatter plots comparing enrichment around the TSS were generated by calculating the area under the signal in bigWig using ma and then transforming the signal. For acH2AZ, the mapped reads of ChIP-Seq and IgG control were processed in HOMER to assess the statistical significance of the ChIP-Seq peaks.Peak size was set to 500 bp, adjacent peaks within a 1000 bp region were stitched together, and ChIP signal versus IgG control peaks were analyzed using a Poisson p -value threshold of 1·10. -3 and a Poisson tag threshold of 32 was used to filter on a region of size 1000 bp with a peak fold change of 4 between ChIP signal versus IgG control.

[0162] Comparison of enrichment of H2AZ and acH2AZ ChIP-Seq signals around (+ / -2kb) the gene TSS locus Enrichment was calculated as the area under the curve generated by the ChIP-Seq signal around the TSS for all genes in the genome, then transformed using an arcsine function. The resulting values ​​were used to compare the ChIP-Seq occupancy of the DMSO sample to the DRB and ACTD samples. This comparison is shown as a scatter plot with a color gradient, in which each TSS is a dot and areas of high concentration of the dot are shown in red, whereas blue means no concentration.

[0163] RIP sequencing analysis Immunoprecipitated RNA was processed for sequencing and ribosomal RNA was depleted from the RNA using the Ribo-Zero™ Magnetic Gold Kit (Cat# MRZG126 Epicentre). Double-stranded cDNA libraries were constructed using the ScriptSeq™ v2 RNA-Seq Library Preparation Kit (Cat# SSV21106 Epicentre). Libraries were subjected to final size selection on a 3% agarose gel. Fragments of 250–500 bp were excised and recovered using the Qiaquy Gel Extraction Kit (QIAGEN). Paired-end reads generated by the sequencer (Illumina HiSeq‐2000) were trimmed to 150 bp and further processed with trim_galore, Cutadapt, and FastQC for adapter trimming and sequence quality control, and the preprocessed sequencing files were mapped to the GRCh38 human reference genome (release 88) using STAR with an overhang length of 150 bp for the fragments used to build the splice junction database. The mapped reads of the RIP and IgG control were processed with HOMER (PMID: 20513432) to assess the statistical significance of the RIP peaks. In this process, individual peaks spanning 100 bp long bins were stitched together into variable length regions, and the RIP signal versus IgG control peaks were matched against a Poisson p‐value threshold of 1·10 -3 and filtered on regions of size 1000 bp with a peak fold change of 4 between RIP signal versus IgG control, using a Poisson tag threshold of 32. Statistically significant peaks were annotated based on their distance to nearby coding regions (corresponding to 10403 loci that overlap with at least one significant RIPseq peak) using HOMER.

[0164] RNA sequencing RNA was extracted with TRI Reagent® (MRC). RNA samples were treated with DNase I (10 U DNase I (Roche) per 3 μg total RNA for 1 h at 37°C in the presence of RNase A inhibitor. Ribo-Zero™ Magnetic Gold Kit (Cat# MRZG126 Epicentre) was used to deplete ribosomal RNA from RNA. Double-stranded cDNA libraries were constructed using ScriptSeq™ v2 RNA-Seq Library Preparation Kit (Cat# SSV21106 Epicentre). Libraries were subjected to final size selection on a 3% agarose gel. Fragments of 250–500 bp were excised and recovered using the Qiaquy Gel Extraction Kit (QIAGEN). Libraries were run on a Hi-Seq-2000 Sequences were generated on Illumina. Paired-end reads were preprocessed with trim_galore, Cutadapt, and FastQC for adapter trimming and sequence quality control, and further aligned to the GRCh38 human reference genome (release 88) using STAR with an overhang length of 150 bp for fragments used to build the splice junction database. Alignment files were analyzed with RSEM to estimate gene expression levels. To confirm transcription of significant RIP-Seq peaks, peak intervals were overlapped with the RIP-Seq assembly using the bedtools intersect Bed utility, and NGSPLOT was used to generate SPEAR and late gene expression overlaps in regions of transcription start sites and coding regions. All statistical analyses and associated plots were generated using ad-hoc R scripts (r-project.org on the World Wide Web (www)).

[0165] Motif discovery The RNA binding motif was identified according to the following steps: 1) Filtering for SPEAR expression, 2) Prediction of 5' and 3' SPEAR boundaries from RNA-Seq 3) Search for common motifs in selected SPEARs

[0166] In total, 13891 predicted promoter loci in HL60 (Broad ChromHMM) were subjected to coverage calculation. SPEARs were further selected according to their expression and the top quartile subset (75th percentile) was selected for further analysis. Prior to motif discovery, 5' and 3' SPEAR boundaries were inferred from RNA-Seq. Specifically, coverage tracks were scanned and 5' and 3' SPEAR boundaries were identified as localized drops in coverage levels via Friedman's SuperSmoother method (R, supsmu). A total of 1942 SPEARs were scanned for common motifs using findGenomeMotif in RNA mode (Homer suite: homer.ucsd.edu / homer / ngs on the world wide web) with the options "-len 10,20,30" and using human promoters as background (except those scanned for motifs). Motifs were classified as significant differences (p<=e -10 ) and fold enrichment (observed vs. expected ≥ 200), and three enriched motifs (RM3, 5, and 9, respectively) were identified.

[0167] The presence of motifs found in overlapping peaks between acH2A.Z and TIP60 RIP-Seq was assessed by running findGenomeMotif with the -mknown option using non-overlapping peaks as background. The same analysis was performed for SINEs, LINEs, and rRNA transcripts. Repeat elements were obtained from UCSC (RepeatMasker) and transcripts expressed at ≥ 2 fpkm were selected for motif scanning. rRNA genomic regions were obtained from UCSC Table Browser (Table:rmsk, repClass:rRNA).

[0168] Statistical analysis: All statistical analyses were performed using the R suite (on the World Wide Web (www), r-project.org / ). Statistical comparisons of distributions corresponding to the cumulative read count signals of ChIP-Seq, RIP-Seq, and RNA-Seq were performed using the Mann-Whitney-Wilcoxon signed rank test with confidence intervals (p<0.05).

[0169] Data Availability The data are available from the Gene Omnibus database under the accession ID number: GSE117663 (World Wide Web (www), ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE117663 Token input: azatzwcwlzcndez.

[0170] Files in a database submission: SM3305784 ChIPseq HL‐60 siMyc H2A.Z GSM3305785 ChIPseq HL‐60 siControl H2A.Z GSM3305786 ChIPseq HL‐60 siMyc acH2A.Z GSM3305787 ChIPseq HL‐60 siControl acH2A.Z GSM3305788 ChIPseq HL‐60 DMSO‐treated H2A.Z GSM3305789 ChIPseq HL‐60 DRB‐treated H2A.Z GSM3305790 ChIPseq HL‐60 ActD‐treated H2A.Z GSM3305791 ChIPseq HL‐60 DMSO‐treated acH2A.Z GSM3305792 ChIPseq HL‐60 DRB‐treated acH2A.Z GSM3305793 ChIPseq HL‐60 ActD‐treated acH2A.Z GSM3305794 ChIPseq HL‐60 DMSO‐treated IgG GSM3305795 ChIPseq HL‐60 DRB‐treated IgG GSM3305796 ChIPseq HL‐60 ActD‐treated IgG GSM3305797 ChIPseq HL‐60 DMSO‐treated input GSM3305798 ChIPseq HL‐60 DRB‐treated input GSM3305799 ChIPseq HL‐60 ActD‐treated input GSM3305800 RIPseq HL‐60 H2A.Z GSM3305801 RIPseq HL‐60a acH2A.Z GSM3305802 RIPseq HL‐60 Tip60 GSM3305803 RIPseq HL‐60 IgG rabbit GSM3305804 RIPseq HL‐60 IgG sheep GSM3305805 RIPseq HL‐60 IgG goat GSM3305806 RNAseq HL‐60 GSM3305807 nasRNAseq HL‐60 GSM3305808 nasRNAseq HL‐60 DMSO‐treated GSM3305809 nasRNAseq HL‐60 DRB‐treated GSM3305810 nasRNAseq HL‐60 ActD‐treated GSM3305784 ChIPseq HL‐60 siMyc H2A.Z GSM3305785 ChIPseq HL‐60 siControl H2A.Z GSM3305786 ChIPseq HL‐60 siMyc acH2A.Z GSM3305787 ChIPseq HL‐60 siControl acH2A.Z GSM3305788 ChIPseq HL‐60 DMSO‐treated H2A.Z GSM3305789 ChIPseq HL‐60 DRB‐treated H2A.Z GSM3305790 ChIPseq HL‐60 ActD‐treated H2A.Z GSM3305791 ChIPseq HL‐60 DMSO‐treated acH2A.Z GSM3305792 ChIPseq HL‐60 DRB‐treated acH2A.Z GSM3305793 ChIPseq HL‐60 ActD‐treated acH2A.Z GSM3305794 ChIPseq HL‐60 DMSO‐treated IgG GSM3305795 ChIPseq HL‐60 DRB‐treated IgG GSM3305796 ChIPseq HL‐60 ActD‐treated IgG GSM3305797 ChIPseq HL‐60 DMSO‐treated input GSM3305798 ChIPseq HL‐60 DRB‐treated input GSM3305799 ChIPseq HL‐60 ActD‐treated input GSM3305800 RIPseq HL‐60 H2A.Z GSM3305801 RIPseq HL‐60a acH2A.Z GSM3305802 RIPseq HL‐60 Tip60 GSM3305803 RIPseq HL‐60 IgG rabbit GSM3305804 RIPseq HL‐60 IgG sheep GSM3305805 RIPseq HL‐60 IgG goat GSM3305806 RNAseq HL‐60 GSM3305807 nasRNAseq HL‐60 GSM3305808 nasRNAseq HL‐60 DMSO‐treated GSM3305809 nasRNAseq HL‐60 DRB‐treated GSM3305810 nasRNAseq HL‐60 ActD‐treated

[0171] [Table 2-1] [Table 2-2]

[0172] [Table 3]

[0173] [Table 4]

[0174] All of the features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only one example of a generic series of equivalent or similar features.

[0175] From the above description, those skilled in the art can easily ascertain the essential features of the present disclosure, and can make various changes and modifications to the present disclosure to suit various applications and situations without departing from the spirit and scope thereof. Accordingly, other embodiments are within the scope of the appended claims.

Claims

1. A pharmaceutical composition for treating cancer in a subject in need thereof comprising cells derived from a subject that have been contacted with one or more inhibitors of S-phase early RNA (SPEAR), or an effective amount of one or more inhibitors of SPEAR, wherein said treatment comprises: (i) administering to said subject an effective amount of said one or more inhibitors of SPEAR; or (ii) administering an effective amount of cells derived from said subject. Including, The pharmaceutical composition, wherein the cancer is characterized by epigenetic dysregulation.

2. A pharmaceutical composition for treating a genetic disease or disorder associated with epigenetic dysregulation in a subject in need thereof, comprising a cell derived from a subject that has been contacted with one or more inhibitors of SPEAR, or an effective amount of one or more inhibitors of SPEAR, wherein said treatment comprises: (i) administering to said subject an effective amount of said one or more inhibitors of SPEAR; or (ii) administering an effective amount of cells derived from the subject.

3. The pharmaceutical composition according to claim 1 or 2, wherein said inhibitor causes modulation of the expression level of one or more genes controlled by said SPEAR.

4. The pharmaceutical composition according to claim 3 , wherein said modulation of the expression level of one or more genes regulated by SPEAR is upregulation of said genes.

5. The pharmaceutical composition according to claim 3, wherein said modulation of the expression level of one or more genes regulated by SPEAR is downregulation of said genes.

6. The pharmaceutical composition of claim 5, wherein the modulation of the expression level of one or more genes controlled by SPEAR is a restoration of the level of the one or more genes compared to an untreated state.

7. The pharmaceutical composition according to claim 6 , wherein the gene is a myc gene.

8. The pharmaceutical composition according to claim 1 or 2, wherein one or more SPEARs are overexpressed to generate an artificial origin of replication complex.

9. The pharmaceutical composition according to claim 1 or 2, wherein one or more SPEARs are overexpressed to control the direction of replication.

10. The pharmaceutical composition of claim 1 or 2, wherein the epigenetic dysregulation is dysregulation of one or more epigenetic marks.

11. The pharmaceutical composition of claim 1 or 2, wherein the subject is suffering from a cancer associated with epigenetic dysregulation.

12. The cancer may be basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, cancer of the peritoneum, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatoma, intraepithelial neoplasia, kidney or renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (lips, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, cancer), testicular cancer, thyroid cancer, uterine or endometrial cancer, cancer of the urinary system, vulvar cancer, Hodgkin and non-Hodgkin lymphomas, and lymphomas including B-cell lymphomas (low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, bulky mass disease NHL, 3. The pharmaceutical composition of claim 1 or 2, wherein the patient is one or more of the following: mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic myeloblastic ... other carcinomas and sarcomas, and post-transplant lymphoproliferative disorders (PTLDs), as well as phacomatosis, abnormal blood vessel proliferation associated with edema, and Meigs' syndrome.

13. The pharmaceutical composition of claim 1 or 2, wherein the SPEAR is a non-coding RNA.

14. The pharmaceutical composition of claim 1 or 2, wherein the inhibitor is a nucleic acid.

15. The pharmaceutical composition of claim 1 or 2, wherein the inhibitor modulates the expression and / or activity of SPEAR.

16. A pharmaceutical composition described in claim 1 or 2, wherein the use includes administering one or more epigenetic drugs or contacting the cells with one or more epigenetic drugs.

17. 17. The pharmaceutical composition of claim 16, wherein the epigenetic drug is a DNA methyltransferase inhibitor optionally selected from azacytidine, escitabine, zebularine, panobinostat, belinostat, dacinostat, xinostat, tefinostat, acediinaline, entinostat, mocetinostat, chidamide, butyric acid, pibanex, phenylbutyric acid, and valproic acid.

18. 1. A method of making an epigenetic modulating agent, comprising: (a) identifying an epigenetic modulating agent by: (i) determining whether the agent binds to or interacts with one or more SPEARs; (ii) classifying the agent as epigenetically modulating based on its ability to bind to or interact with one or more SPEARs; and (b) formulating the agent for use in a therapy selected from the treatment or prevention of cancer associated with epigenetic dysregulation or a genetic disease or disorder associated with epigenetic dysregulation.

19. 20. The method of claim 18, wherein the agent reduces or substantially eliminates the activity of the epigenetic mark associated with SPEAR.

20. 20. The method of any one of claims 18 or 19, wherein the agent reduces or substantially eliminates the formation and / or recycling of epigenetic marks.

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