Presence Of H3K27me3 Research Articles

Spatial 3D genome organization controls the activity of bivalent chromatin during human neurogenesis.

The nuclear genome is spatially organized into a three-dimensional (3D) architecture by physical association of large chromosomal domains with subnuclear compartments including the nuclear lamina at the radial periphery and nuclear speckles within the nucleoplasm1-5. However, how spatial genome architecture regulates human brain development has been overlooked owing to technical limitations. Here, we generate high-resolution maps of genomic interactions with the lamina and speckles in cells of the neurogenic lineage isolated from midgestational human cortex, uncovering an intimate association between subnuclear genome compartmentalization, chromatin state and transcription. During cortical neurogenesis, spatial genome organization is extensively remodeled, relocating hundreds of neuronal genes from the lamina to speckles including key neurodevelopmental genes bivalent for H3K27me3 and H3K4me3. At the lamina, bivalent genes have exceptionally low expression, and relocation to speckles enhances resolution of bivalent chromatin to H3K4me3 and increases transcription >7-fold. We further demonstrate that proximity to the nuclear periphery - not the presence of H3K27me3 - is the dominant factor in maintaining the lowly expressed, poised state of bivalent genes embedded in the lamina. In addition to uncovering a critical role of subnuclear genome compartmentalization in neurogenic transcriptional regulation, our results establish a new paradigm in which knowing the spatial location of a gene is necessary to understanding its epigenomic regulation.

ASH1L guards cis-regulatory elements against cyclobutane pyrimidine dimer induction.

The histone methyltransferase ASH1L, first discovered for its role in transcription, has been shown to accelerate the removal of ultraviolet (UV) light-induced cyclobutane pyrimidine dimers (CPDs) by nucleotide excision repair. Previous reports demonstrated that CPD excision is most efficient at transcriptional regulatory elements, including enhancers, relative to other genomic sites. Therefore, we analyzed DNA damage maps in ASH1L-proficient and ASH1L-deficient cells to understand how ASH1L controls enhancer stability. This comparison showed that ASH1L protects enhancer sequences against the induction of CPDs besides stimulating repair activity. ASH1L reduces CPD formation at C-containing but not at TT dinucleotides, and no protection occurs against pyrimidine-(6,4)-pyrimidone photoproducts or cisplatin crosslinks. The diminished CPD induction extends to gene promoters but excludes retrotransposons. This guardian role against CPDs in regulatory elements is associated with the presence of H3K4me3 and H3K27ac histone marks, which are known to interact with the PHD and BRD motifs of ASH1L, respectively. Molecular dynamics simulations identified a DNA-binding AT hook of ASH1L that alters the distance and dihedral angle between neighboring C nucleotides to disfavor dimerization. The loss of this protection results in a higher frequency of C->T transitions at enhancers of skin cancers carrying ASH1L mutations compared to ASH1L-intact counterparts.

Epigenetic signals that direct cell type-specific interferon beta response in mouse cells.

The antiviral response induced by type I interferon (IFN) via the JAK-STAT signaling cascade activates hundreds of IFN-stimulated genes (ISGs) across human and mouse tissues but varies between cell types. However, the links between the underlying epigenetic features and the ISG profile are not well understood. We mapped ISGs, binding sites of the STAT1 and STAT2 transcription factors, chromatin accessibility, and histone H3 lysine modification by acetylation (ac) and mono-/tri-methylation (me1, me3) in mouse embryonic stem cells and fibroblasts before and after IFNβ treatment. A large fraction of ISGs and STAT-binding sites was cell type specific with promoter binding of a STAT1/2 complex being a key driver of ISGs. Furthermore, STAT1/2 binding to putative enhancers induced ISGs as inferred from a chromatin co-accessibility analysis. STAT1/2 binding was dependent on the chromatin context and positively correlated with preexisting H3K4me1 and H3K27ac marks in an open chromatin state, whereas the presence of H3K27me3 had an inhibitory effect. Thus, chromatin features present before stimulation represent an additional regulatory layer for the cell type-specific antiviral response.

Presence of H3K4me3 on Paternally Expressed Genes of the Paternal Genome From Sperm to Implantation.

Genomic imprinting, parent-of-origin-specific gene expression, is controlled by differential epigenetic status of the parental chromosomes. While DNA methylation and suppressive histone modifications established during gametogenesis suppress imprinted genes on the inactive allele, how and when the expressed allele gains its active status is not clear. In this study, we asked whether the active histone-3 lysine-4 trimethylation (H3K4me3) marks remain at paternally expressed genes (PEGs) in sperm and embryos before and after fertilization using published data. Here we show that mouse sperm had the active H3K4me3 at more than half of known PEGs, and these genes were present even after fertilization. Using reciprocal cross data, we identified 13 new transient PEGs during zygotic genome activation. Next, we confirmed that the 12 out of the 13 new transient PEGs were associated with the paternal H3K4me3 in sperm. Nine out of the 12 genes were associated with the paternal H3K4me3 in zygotes. Our results show that paternal H3K4me3 marks escape inactivation during the histone-to-protamine transition that occurs during sperm maturation and are present in embryos from early zygotic stages up to implantation.

Principles of 3D compartmentalization of the human genome.

SUMMARYChromatin is organized in the nucleus via CTCF loops and compartmental domains. Here, we compare different cell types to identify distinct paradigms of compartmental domain formation in human tissues. We identify and quantify compartmental forces correlated with histone modifications characteristic of transcriptional activity and previously underappreciated roles for distinct compartmental domains correlated with the presence of H3K27me3 and H3K9me3, respectively. We present a computer simulation model capable of predicting compartmental organization based on the biochemical characteristics of independent chromatin features. Using this model, we show that the underlying forces responsible for compartmental domain formation in human cells are conserved and that the diverse compartmentalization patterns seen across cell types are due to differences in chromatin features. We extend these findings to Drosophila to suggest that the same principles are at work beyond humans. These results offer mechanistic insights into the fundamental forces driving the 3D organization of the genome.

Epigenetic Mechanisms Are Involved in the Oncogenic Properties of ZNF518B in Colorectal Cancer.

Simple SummaryThe ZNF518B gene, which is up-regulated in colorectal cancer, plays a role in metastasis, but neither the mechanisms involved in this process nor the role of the different isoforms of the gene are known. Here we show that the ratio of these isoforms is related to the relapsing of the disease, and that the protein ZNF518B interacts with enzymes able to introduce epigenetic changes, which may affect the activity of many genes. We also report a list of genes affected in common by ZNF518B and by two of those related enzymes, namely, G9A and EZH2. An in-depth analysis of five of those genes revealed that ZNF518B is involved in the recruitment of the enzymes and in the deposition of the corresponding epigenetic marks. The results highlight the relevance of epigenetic changes in cancer development, and open the possibility of developing therapeutic approaches, as the introduction of epigenetic modifications is reversible.The ZNF518B gene, which is up-regulated in colorectal cancer, plays a role in cell dissemination and metastasis. It encodes a zinc-finger protein, which interacts with histone methyltransferases G9A and EZH2. The expression of the two major mRNA isoforms 1 (coding for the full protein) and 2 was quantified by RT-qPCR in a cohort of 66 patients. The effects of silencing ZNF518B on the transcriptome of DLD1 and HCT116 cells were analysed by Clariom-S assays and validated by RT-qPCR. The recruitment of methyltransferases and the presence of H3K27me3 were studied by chromatin immunoprecipitation (ChIP). The ratio (isoform 2)/(isoform 1) negatively correlated with the relapsing of disease. The study of the transcriptome of DLD1 and HCT116 cells revealed that many genes affected by silencing ZNF518B are related to cancer. After crossing these results with the list of genes affected by silencing the histone methyltransferases (retrieved in silico), five genes were selected. ChIP analysis revealed that the recruitment of EZH2 is ZNF518B-dependent in KAT2B, RGS4 and EFNA5; the level of H3K27me3 changes in accordance. G9A also binds RGS4 and PADI3 in a ZNF518B-dependent manner. The results highlight the importance of epigenetics in cancer and open a novel therapeutic possibility, as inhibition of histone methyltransferases may reverse the disease-linked histone marks.

The RNA-binding protein SERBP1 functions as a novel oncogenic factor in glioblastoma by bridging cancer metabolism and epigenetic regulation

BackgroundRNA-binding proteins (RBPs) function as master regulators of gene expression. Alterations in RBP expression and function are often observed in cancer and influence critical pathways implicated in tumor initiation and growth. Identification and characterization of oncogenic RBPs and their regulatory networks provide new opportunities for targeted therapy.ResultsWe identify the RNA-binding protein SERBP1 as a novel regulator of glioblastoma (GBM) development. High SERBP1 expression is prevalent in GBMs and correlates with poor patient survival and poor response to chemo- and radiotherapy. SERBP1 knockdown causes delay in tumor growth and impacts cancer-relevant phenotypes in GBM and glioma stem cell lines. RNAcompete identifies a GC-rich region as SERBP1-binding motif; subsequent genomic and functional analyses establish SERBP1 regulation role in metabolic routes preferentially used by cancer cells. An important consequence of these functions is SERBP1 impact on methionine production. SERBP1 knockdown decreases methionine levels causing a subsequent reduction in histone methylation as shown for H3K27me3 and upregulation of genes associated with neurogenesis, neuronal differentiation, and function. Further analysis demonstrates that several of these genes are downregulated in GBM, potentially through epigenetic silencing as indicated by the presence of H3K27me3 sites.ConclusionsSERBP1 is the first example of an RNA-binding protein functioning as a central regulator of cancer metabolism and indirect modulator of epigenetic regulation in GBM. By bridging these two processes, SERBP1 enhances glioma stem cell phenotypes and contributes to GBM poorly differentiated state.

Canonical Wnt/β-catenin activity and differential epigenetic marks direct sexually dimorphic regulation of Irx3 and Irx5 in developing mouse gonads.

Members of the Iroquois B (IrxB) homeodomain cluster genes, specifically Irx3 and Irx5, are crucial for heart, limb and bone development. Recently, we reported their importance for oocyte and follicle survival within the developing ovary. Irx3 and Irx5 expression begins after sex determination in the ovary but remains absent in the fetal testis. Mutually antagonistic molecular signals ensure ovary versus testis differentiation with canonical Wnt/β-catenin signals paramount for promoting the ovary pathway. Notably, few direct downstream targets have been identified. We report that Wnt/β-catenin signaling directly stimulates Irx3 and Irx5 transcription in the developing ovary. Using in silico analysis of ATAC- and ChIP-Seq databases in conjunction with mouse gonad explant transfection assays, we identified TCF/LEF-binding sequences within two distal enhancers of the IrxB locus that promote β-catenin-responsive ovary expression. Meanwhile, Irx3 and Irx5 transcription is suppressed within the developing testis by the presence of H3K27me3 on these same sites. Thus, we resolved sexually dimorphic regulation of Irx3 and Irx5 via epigenetic and β-catenin transcriptional control where their ovarian presence promotes oocyte and follicle survival vital for future ovarian health.

Interactions between core histone marks and DNA methyltransferases predict DNA methylation patterns observed in human cells and tissues

ABSTRACTDNA methylation and histone modifications are two major epigenetic marks in mammalian cells. Previous studies have revealed that these two mechanisms interact although a quantitative model of these is still lacking in mammalian cells. Here we sought to develop such a model by systematically evaluating the quantitative relationship between DNA methylation and the core histone modification marks in human epigenomes. This model reflects the interactions of ADD and PWWP domains of DNA methyltransferase (DNMTs) with histone 3 lysine tails. Our analysis integrated 35 whole genome bisulphite sequencing data sets (about 800 million CpG sites), 35 chromatin states and 175 ChIP-Seq histone modification profiles across 35 human cell types. The logistic regression model we built shows that more than half of the variance across DNA methylomes can be explained by the five-core histone modification across varied types of human cell and tissue samples. Importantly, we find that H3K4me3 has a dramatic effect in DNA methylation patterning, highlighting the essential interaction between ADD domain of DNMTs and histone 3 lysine 4 in human. Moreover, our model suggests DNA methylation is generally inhibited by the presence of H3K4me3, H3K4me1 and H3K27me3, while increased levels are found in regions that are marked by H3K9me3 and H3K36me3. In summary, our results provide a comprehensive evaluation of the crosstalk between DNA methylation and histone modification in a variety of human cell types, and shows that DNA methylation patterns can be largely explained by interactions between histone 3 lysine tails and specific domains of DNA methyltransferases.

DNA sequence features in the establishing of H3K27ac.

The presence of H3K27me3 has been demonstrated to correlate with the CpG content. In this work, we tested whether H3K27ac has similar sequence preferences. We performed a translocation of DNA sequences with various properties into a beta-globin locus to control for the local chromatin environment. Our results suggest that in contrast to H3K27me3, H3K27ac gain is unlikely affected by the CpG content of the underlying DNA sequence, while extremely high GC-content might contribute to the gain of the H3K27ac.

The RAG-2 Inhibitory Domain Gates Accessibility of the V(D)J Recombinase to Chromatin.

Accessibility of antigen receptor loci to RAG is correlated with the presence of H3K4me3, which binds to a plant homeodomain (PHD) in the RAG-2 subunit and promotes V(D)J recombination. A point mutation in the PHD, W453A, eliminates binding of H3K4me3 and impairs recombination. The debilitating effect of the W453A mutation is ameliorated by second-site mutations that locate an inhibitory domain in the interval from residues 352 through 405 of RAG-2. Disruption of the inhibitory domain stimulates V(D)J recombination within extrachromosomal substrates and at endogenous antigen receptor loci. Association of RAG-1 and RAG-2 with chromatin at the IgH locus in B cell progenitors is dependent on recognition of H3K4me3 by the PHD. Strikingly, disruption of the inhibitory domain permits association of RAG with the IgH locus in the absence of H3K4me3 binding. Thus, the inhibitory domain acts as a gate that prohibits RAG from accessing the IgH locus unless RAG-2 is engaged by H3K4me3.

DNA sequence features in the establishing of H3K27ac

The presence of H3K27me3 has been demonstrated to correlate with the CpG content. In this work, we tested whether H3K27ac has similar sequence preferences. We performed a translocation of DNA sequences with various properties into a beta-globin locus to control for the local chromatin environment. Our results suggest that in contrast to H3K27me3, H3K27ac gain is unlikely affected by the CpG content of the underlying DNA sequence, while extremely high GC-content might contribute to the gain of the H3K27ac.

DNA sequence features in the establishing of H3K27ac.

The presence of H3K27me3 has been demonstrated to correlate with the CpG content. In this work, we tested whether H3K27ac has similar sequence preferences. We performed a translocation of DNA sequences with various properties into a beta globin locus to control for the local chromatin environment. We demonstrate that H3K27ac is not linked to CpG content of the sequence, while extremely high GC-content may contribute to the establishment of this mark.

Abstract 3047: PRC2-mediated silencing of circRNA CDR1as drives miR-7-independent melanoma metastasis

Abstract Circular RNAs are a novel class of non-coding RNAs with functions that remain poorly characterized in normal and pathological conditions. CDR1as is a non-canonical circRNA observed to act as a sponge for miR-7 in brain tissues. Analysis of RNA-seq data of melanocytes and melanoma cell lines and short-term cultures revealed loss of CDR1as expression as a hallmark of melanoma cells. We confirmed silencing of CDR1as in melanoma cells and tissues by RT-qPCR using divergent primers. Clinically, we observed CDR1as loss associated with metastatic progression and poor patient outcomes in a cohort of fresh-frozen melanoma tissue samples. Depletion of CDR1as in melanoma cell lines enhanced invasion in vitro and lung metastasis in vivo, demonstrating functional significance of CDR1as silencing. Surprisingly, CDR1as depletion had no clear effect on miR-7 activity in melanoma cells, and miR-7 inhibition was insufficient to rescue CDR1as silencing-induced invasion. Moreover, GSEA analyses of proteomic profiling of melanoma cells depleted of CDR1as revealed reductions of proteins involved in oxidative phosphorylation (OXPHOS) and mitochondrial function, suggesting CDR1as loss may alter metabolism of melanoma cells. Mining of CLIP-Seq data sets and subsequent RIP-PCR revealed direct interactions of CDR1as with the IGF2BP family of proteins and TARDBP, each of which are involved in regulation of mitochondrial function. These data suggest that CDR1as could regulate the subcellular localization and/or function of proteins involved in OXPHOS and mitochondrial respiration. To understand the upstream mechanism driving CDR1as loss in melanoma, we sought to examine the primary transcript from which CDR1as arises. Interestingly, examination of the CDR1as locus revealed an upstream long non-coding RNA, LINC00632, as a plausible primary transcript of CDR1as. CDR1as and LINC00632 expression is strikingly correlated across human tissues and in melanoma cell lines (r>.65). Moreover, depletion of LINC00632 using GapmeRs reduced expression of CDR1as, suggesting they arise from the same precursor transcript. Intriguingly, from data mining of ChIP-Seq datasets, we observed that the PRC2-dependent repressive chromatin mark H3K27me3, is abundant in the genomic locus of LINC00632 in melanoma cells. We validated the presence of H3K27me3 at LINC00632 by ChIP-PCR in melanoma cells lacking CDR1as expression. Strikingly, treatment of such melanoma cell lines with the EZH2 inhibitor, GSK126, induced robust re-expression of both CDR1as and LINC00632, and removal of H3K27me3 at LINC00632. Our data document that CDR1as is epigenetically silenced in melanoma, and its loss promotes melanoma invasion and metastasis. Moreover, CDR1as loss could contribute to metabolic adaptations during metastasis through misregulation of RNA binding proteins. Citation Format: Doug Hanniford, Rana Moubarak, Jochen Imig, Alejandro Ulloa, Beatriz Sanchez Sendra, Alcida Karz, Iman Osman, Ioannis Aifantis, Eva Hernando. PRC2-mediated silencing of circRNA CDR1as drives miR-7-independent melanoma metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3047. doi:10.1158/1538-7445.AM2017-3047

O-Linked N-Acetylglucosamine (O-GlcNAc) Expression Levels Epigenetically Regulate Colon Cancer Tumorigenesis by Affecting the Cancer Stem Cell Compartment via Modulating Expression of Transcriptional Factor MYBL1

To study the regulation of colorectal adenocarcinoma progression by O-GlcNAc, we have focused on the O-GlcNAc-mediated epigenetic regulation of human colon cancer stem cells (CCSC). Xenograft tumors from colon tumor cells with O-linked N-acetylglucosamine transferase (OGT) knockdown grew significantly slower than those formed from control cells, indicating a reduced proliferation of tumor cells due to inhibition of OGT expression. Significant reduction of the CCSC population was observed in the tumor cells after OGT knockdown, whereas tumor cells treated with the O-GlcNAcase inhibitor showed an increased CCSC population, indicating that O-GlcNAc levels regulated the CCSC compartment. When grown in suspension, tumor cells with OGT knockdown showed a reduced ability to form tumorspheres, indicating a reduced self-renewal of CCSC due to reduced levels of O-GlcNAc. ChIP-sequencing experiments using an anti-O-GlcNAc antibody revealed significant chromatin enrichment of O-GlcNAc-modified proteins at the promoter of the transcription factor MYBL1, which was also characterized by the presence of H3K27me3. RNA-sequencing analysis showed an increased expression of MYBL1 in tumor cells with OGT knockdown. Forced overexpression of MYBL1 led to a reduced population of CCSC and tumor growth in vivo, similar to the effects of OGT silencing. Moreover, two CpG islands near the transcription start site of MYBL1 were identified, and O-GlcNAc levels regulated their methylation status. These results strongly argue that O-GlcNAc epigenetically regulates MYBL1, functioning similarly to H3K27me3. The aberrant CCSC compartment observed after modulating O-GlcNAc levels is therefore likely to result, at least in part, from the epigenetic regulation of MYBL1 expression by O-GlcNAc, thereby significantly affecting tumor progression.

Abstract 4026: Epigenetic changes in T cells in response to immune checkpoint blockade

Abstract Immune checkpoint blockade therapy has led to clinical success and have established immunotherapy as one of the primary modality of cancer treatment. It leads to a durable response in around 20% of the patients (responders). Success of immune checkpoint blockade therapy relies on the increase of effector T cells and decrease of regulatory T cells in the tumor microenvironment. However, the underlying mechanism that govern the ratio of effector and regulatory T cells between responder and non-responder to checkpoint blockade is not understood. Understanding the process could reveal novel mechanisms to boost efficacy of immune checkpoint blockade. Differentiation of naïve T cells into effector and regulatory phenotype requires lineage-specifying transcription factors and epigenetic modifications that allow appropriate repression or activation of gene transcription. One key epigenetic modification for T cell differentiation is trimethylation of lysine 27 on histone H3 (H3K27me3). Loss of H3K27me3 results in increased Th-1 plasticity whereas presence of H3K27me3 on Foxp3 locus is required to maintain the function of T-regulatory cells. We hypothesize that immune checkpoint blockade changes the epigenetic landscape in tumor infiltrating T cells that results in an effector phenotype and epigenetic modification of H3K27me3 could promote an anti-tumor immune microenvironment in tumor bearing mice.We showed that in-vivo inhibition of H3K27me3 in combination with anti-CTLA4 results in reduction of tumor size and also reduction in regulatory T cells in a B16-F10 melanoma mice model compared to anti-CTLA4 treatment alone, suggesting H3K27me3 inhibition could increase the efficacy of checkpoint blockade therapy. Future studies will aim to elucidate epigenetic changes in H3K27me3 and define an epigenetic signature as a result of immune checkpoint therapy by ChIP-sequencing. Data generated in this project will greatly enhance our understanding of epigenetic regulation of T cell differentiation is response to immune checkpoint blockade and aid identifying key changes that could be further modified to overcome resistance to immune checkpoint blockade in non-responders. Citation Format: Sangeeta Goswami, Jianfeng Chen, Hao Zhao, Xuejun Zhang, Padmanee Sharma. Epigenetic changes in T cells in response to immune checkpoint blockade. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4026.

DNA methylation and histone modifications regulate SOX11 expression in lymphoid and solid cancer cells.

BackgroundThe neural transcription factor SOX11 is present at specific stages during embryo development with a very restricted expression in adult tissue, indicating precise regulation of transcription. SOX11 is strongly up-regulated in some malignancies and have a functional role in tumorgenesis. With the aim to explore differences in epigenetic regulation of SOX11 expression in normal versus neoplastic cells, we investigated methylation and histone modifications related to the SOX11 promoter and the possibility to induce re-expression using histone deacetylase (HDAC) or EZH2 inhibitors.MethodsThe epigenetic regulation of SOX11 was investigated in distinct non-malignant cell populations (n = 7) and neoplastic cell-lines (n = 42) of different cellular origins. DNA methylation was assessed using bisulfite sequencing, methylation-specific melting curve analysis, MethyLight and pyrosequencing. The presence of H3K27me3 was assessed using ChIP-qPCR. The HDAC inhibitors Vorinostat and trichostatin A were used to induce SOX11 in cell lines with no endogenous expression.ResultsThe SOX11 promoter shows a low degree of methylation and strong enrichment of H3K27me3 in non-malignant differentiated cells, independent of cellular origin. Cancers of the B-cell lineage are strongly marked by de novo methylation at the SOX11 promoter in SOX11 non-expressing cells, while solid cancer entities display a more varying degree of SOX11 promoter methylation. The silencing mark H3K27me3 was generally present at the SOX11 promoter in non-expressing cells, and an increased enrichment was observed in cancer cells with a low degree of SOX11 methylation compared to cells with dense methylation. Finally, we demonstrate that the HDAC inhibitors (vorinostat and trichostatin A) induce SOX11 expression in cancer cells with low levels of SOX11 methylation.ConclusionsWe show that SOX11 is strongly marked by repressive histone marks in non-malignant cells. In contrast, SOX11 regulation in neoplastic tissues is more complex involving both DNA methylation and histone modifications. The possibility to re-express SOX11 in non-methylated tissue is of clinical relevance, and was successfully achieved in cell lines with low levels of SOX11 methylation. In breast cancer patients, methylation of the SOX11 promoter was shown to correlate with estrogen receptor status, suggesting that SOX11 may be functionally re-expressed during treatment with HDAC inhibitors in specific patient subgroups.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1208-y) contains supplementary material, which is available to authorized users.

Epigenetic Regulation of Cell Cycle-Promoting Genes By Ikaros and HDAC1 in Acute Lymphoblastic Leukemia

B-cell acute lymphoblastic leukemia (B-ALL) is the most common childhood leukemia. Expression profiling has identified IKZF1 (Ikaros) as a major tumor suppressor in B-ALL and established reduced Ikaros function as a poor prognostic marker for this disease. Ikaros regulates expression of its target genes via chromatin remodeling. In vivo, Ikaros can form a complex with histone deacetylases HDAC1 and/or HDAC2 as well as the NuRD chromatin remodeling complex. The mechanisms by which Ikaros exerts its tumor suppressor function and regulates gene expression in B-ALL are unknown. Here we report the use of chromatin immunoprecipitation coupled with next generation sequencing (ChIP-SEQ) to identify genes that are regulated by Ikaros in vivo and to determine the role of Ikaros in chromatin remodeling in B-ALL.Results reveal that Ikaros binds to the promoter regions of a large number of genes that are critical for cell cycle progression. These include CDC2, CDC16, CDC25A, ANAPC1, and ANAPC7. Overexpression of Ikaros in leukemia cells resulted in transcriptional repression of Ikaros target genes. Results from luciferase reporter assays performed using the respective promoters of Ikaros target genes support a role for Ikaros as a transcriptional repressor of these genes. Downregulation of Ikaros by siRNA resulted in increased expression of Ikaros target genes that control cell cycle progression. These results suggest that Ikaros functions as a negative regulator of cell cycle progression by repressing transcription of cell cycle-promoting genes. Next, we studied how Ikaros binding affects the epigenetic signature at promoters of Ikaros target genes. Global epigenetic mapping showed that Ikaros binding at the promoter region of cell cycle-promoting genes is associated with the formation of one of two types of repressive epigenetic marks – either H3K27me3 or H3K9me3. While these epigenetic marks were mutually exclusive, they were both associated with the loss of H3K9 acetylation and transcriptional repression. Serial qChIP assays spanning promoters of the Ikaros target genes revealed that the presence of H3K27me3 is associated with Ikaros and HDAC1 binding, while the H3K9me3 modification is associated with Ikaros binding and the absence of HDAC1. ChIP-SEQ analysis of HDAC1 global genomic binding demonstrated that over 80% of H3K27me3 modifications at promoter regions are associated with HDAC1 binding at surrounding sites. The treatment of leukemia cells with the histone deacetylase inhibitor – trichostatin (TSA) resulted in a severe reduction of global levels of H3K27me3, as evidenced by Wesern blot. These data suggest that HDAC1 activity in leukemia is essential for the formation of repressive chromatin that is characterized by the presence of H3K27me3.Our data suggest that Ikaros binding at the promoters of its target genes can result in the formation of repressive chromatin by two distinct mechanisms: 1) direct Ikaros binding resulting in increased H3K9me3 or 2) Ikaros recruitment of HDAC1 with increased H3K27me3 modifications. These data suggest distinct mechanisms for the regulation of chromatin remodeling and target gene expression by Ikaros alone, and Ikaros in complex with HDAC1. In conclusion, the presented data suggest that HDAC1 has a key role in regulating cell cycle progression and proliferation in B-ALL. Our results identify novel, Ikaros-mediated mechanisms of epigenetic regulation that contribute to tumor suppression in leukemia. Supported by National Institutes of Health R01 HL095120, and the Four Diamonds Fund Endowment. DisclosuresNo relevant conflicts of interest to declare.

Polycomb repressive complex 2 and H3K27me3 cooperate with H3K9 methylation to maintain heterochromatin protein 1α at chromatin.

Methylation of histone H3 on lysine 9 or 27 is crucial for heterochromatin formation. Previously considered hallmarks of, respectively, constitutive and facultative heterochromatin, recent evidence has accumulated in favor of coexistence of these two marks and their cooperation in gene silencing maintenance. H3K9me2/3 ensures anchorage at chromatin of heterochromatin protein 1α (HP1α), a main component of heterochromatin. HP1α chromoshadow domain, involved in dimerization and interaction with partners, has additional but still unclear roles in HP1α recruitment to chromatin. Because of previously suggested links between polycomb repressive complex 2 (PRC2), which catalyzes H3K27 methylation, and HP1α, we tested whether PRC2 may regulate HP1α abundance at chromatin. We found that the EZH2 and SUZ12 subunits of PRC2 are required for HP1α stability, as knockdown of either protein led to HP1α degradation. Similar results were obtained upon overexpression of H3K27me2/3 demethylases. We further showed that binding of HP1α/β/γ to H3K9me3 peptides is greatly increased in the presence of H3K27me3, and this is dependent on PRC2. These data fit with recent proteomic studies identifying PRC2 as an indirect H3K9me3 binder in mouse tissues and suggest the existence of a cooperative mechanism of HP1α anchorage at chromatin involving H3 methylation on both K9 and K27 residues.

THU0115 Epigenetic Repression of the Long Noncoding Rna Hotair Regulates NF-KB Signalling and the Expression of Matrix Metalloproteases in Synovial Fibroblasts

BackgroundLong noncoding RNAs (lncRNA, >200nt) influence gene expression through their interaction with transcription factors and/or epigenetic enzymes. We have shown that the histone methyltransferase EZH2 is upregulated in rheumatoid arthritis...