Role Of Chromatin Modifications Research Articles

Nuclear Acly protects the liver from ischemia-reperfusion injury.

Hepatic ischemia-reperfusion (IR) injury is the most common complication that occurs in liver surgery and hemorrhagic shock. ATP citrate lyase (Acly) plays a pivotal role in chromatin modification via generating acetyl-CoA for histone acetylation to influence biological processes. We aim to examine the roles of Acly, which is highly expressed in hepatocytes, in liver IR injury. The functions of Acly in hepatic IR injury were examined in the mouse model with a hepatocyte-specific knockout of Acly . The Acly target genes were analyzed by CUT&RUN assay and RNA sequencing. The relationship between the susceptibility of the steatotic liver to IR and Acly was determined by the gain of function studies in mice. Hepatic deficiency of Acly exacerbated liver IR injury. IR induced Acly nuclear translocation in hepatocytes, which spatially fueled nuclear acetyl-CoA. This alteration was associated with enhanced acetylation of H3K9 and subsequent activation of the Foxa2 signaling pathway. Nuclear localization of Acly enabled Foxa2-mediated protective effects after hypoxia-reperfusion in cultured hepatocytes, while cytosolic Acly demonstrated no effect. The presence of steatosis disrupted Acly nuclear translocation. In the steatotic liver, restoration of Acly nuclear localization through overexpression of Rspondin-1 or Rspondin-3 ameliorated the IR-induced injury. Our results indicate that Acly regulates histone modification by means of nuclear AcCoA production in hepatic IR. Disruption of Acly nuclear translocation increases the vulnerability of the steatotic liver to IR. Nuclear Acly thus may serve as a potential therapeutic target for future interventions in hepatic IR injury, particularly in the context of steatosis.

The Role of Chromatin Modifications in the Evolution of Giant Plant Genomes.

Angiosperm genome sizes (GS) range ~2400-fold and comprise genes and their regulatory regions, repeats, semi-degraded repeats, and 'dark matter'. The latter represents repeats so degraded that they can no longer be recognised as repetitive. In exploring whether the histone modifications associated with chromatin packaging of these contrasting genomic components are conserved across the diversity of GS in angiosperms, we compared immunocytochemistry data for two species whose GS differ ~286-fold. We compared published data for Arabidopsis thaliana with a small genome (GS = 157 Mbp/1C) with newly generated data from Fritillaria imperialis, which has a giant genome (GS = 45,000 Mbp/1C). We compared the distributions of the following histone marks: H3K4me1, H3K4me2, H3K9me1, H3K9me2, H3K9me3, H3K27me1, H3K27me2, and H3K27me3. Assuming these histone marks are associated with the same genomic features across all species, irrespective of GS, our comparative analysis enables us to suggest that while H3K4me1 and H3K4me2 methylation identifies genic DNA, H3K9me3 and H3K27me3 marks are associated with 'dark matter', H3K9me1 and H3K27me1 mark highly homogeneous repeats, and H3K9me2 and H3K27me2 mark semi-degraded repeats. The results have implications for our understanding of epigenetic profiles, chromatin packaging and the divergence of genomes, and highlight contrasting organizations of the chromatin within the nucleus depending on GS itself.

Genome-Wide Identification and Characterization of Argonaute, Dicer-like and RNA-Dependent RNA Polymerase Gene Families and Their Expression Analyses in Fragaria spp.

In the growth and development of plants, some non-coding small RNAs (sRNAs) not only mediate RNA interference at the post-transcriptional level, but also play an important regulatory role in chromatin modification at the transcriptional level. In these processes, the protein factors Argonaute (AGO), Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) play very important roles in the synthesis of sRNAs respectively. Though they have been identified in many plants, the information about these gene families in strawberry was poorly understood. In this study, using a genome-wide analysis and a phylogenetic approach, 13 AGO, six DCL, and nine RDR genes were identified in diploid strawberry Fragaria vesca. We also identified 33 AGO, 18 DCL, and 28 RDR genes in octoploid strawberry Fragaria × ananassa, studied the expression patterns of these genes in various tissues and developmental stages of strawberry, and researched the response of these genes to some hormones, finding that almost all genes respond to the five hormone stresses. This study is the first report of a genome-wide analysis of AGO, DCL, and RDR gene families in Fragaria spp., in which we provide basic genomic information and expression patterns for these genes. Additionally, this study provides a basis for further research on the functions of these genes and some evidence for the evolution between diploid and octoploid strawberries.

ANKRD11 pathogenic variants and 16q24.3 microdeletions share an altered DNA methylation signature in patients with KBG syndrome.

Pathogenic variants in ANKRD11 or microdeletions at 16q24.3 are the cause of KBG syndrome (KBGS), a neurodevelopmental syndrome characterized by intellectual disability, dental and skeletal anomalies, and characteristic facies. The ANKRD11 gene encodes the ankyrin repeat-containing protein 11A transcriptional regulator, which is expressed in the brain and implicated in neural development. Syndromic conditions caused by pathogenic variants in epigenetic regulatory genes show unique patterns of DNA methylation (DNAm) in peripheral blood, termed DNAm signatures. Given ANKRD11's role in chromatin modification, we tested whether pathogenic ANKRD11 variants underlying KBGS are associated with a DNAm signature. We profiled whole-blood DNAm in 21 individuals with ANKRD11 variants, 2 individuals with microdeletions at 16q24.3 and 28 typically developing individuals, using Illumina's Infinium EPIC array. We identified 95 differentially methylated CpG sites that distinguished individuals with KBGS and pathogenic variants in ANKRD11 (n = 14) from typically developing controls (n = 28). This DNAm signaturewas then validated in an independent cohort of seven individuals with KBGS and pathogenic ANKRD11 variants. We generated a machine learning model from the KBGS DNAm signature and classified the DNAm profiles of four individuals with variants of uncertain significance (VUS) in ANKRD11. We identified an intermediate classification score for an inherited missense variant transmitted from a clinically unaffected mother to her affected child. In conclusion, we show that the DNAm profiles of two individuals with 16q24.3 microdeletions were indistinguishable from the DNAm profiles of individuals with pathogenic variants in ANKRD11, and we demonstrate the diagnostic utility of the new KBGS signature by classifying the DNAm profiles of individuals with VUS in ANKRD11.

Establishment of a CRISPR/Cas9-mediated GATAD2B homozygous knockout human embryonic stem cell line

As a subunit of the nucleosome remodeling and histone deacetylation (NuRD) complex, GATA zinc finger domain containing 2B (GATAD2B) plays a vital role in chromatin modification and transcriptional regulation. To further investigate the role of GATAD2B in cell fate determination of human ESCs, we generated two GATAD2B homozygous knockout human ESC lines by CRISPR/Cas9 technology. The cell line exhibits normal karyotype and typical stem cell morphology, following the high expression of pluripotent genes and differentiation potential in vitro. These cell lines will provide cell resources to investigate epigenetic regulation in extensive biological processes as menthioned above.

The Emerging Roles of Long Noncoding RNAs as Hallmarks of Lung Cancer.

Noncoding ribonucleic acids (ncRNAs) are closely associated with tumor initiation, growth, and progress in lung cancer. Long ncRNAs (lncRNAs), as one of the three subclasses of ncRNAs, play important roles in chromatin modification, transcription, and post-transcriptional processing. Various lncRNAs have recently been reported to be dysfunctional or dysregulated in cancers and have pro- or anti-tumor potential. Importantly, as a new class of cancer biomarkers, studies have demonstrated the plausibility of using certain subsets of lncRNAs as promising diagnostic, therapeutic, or prognostic strategies to manage cancers. This review focuses on lncRNAs associated with hallmarks of lung cancer, especially those discovered in the last five years. The expression levels of these lncRNAs in tumor samples are discussed, alongside their mechanisms of action, drug resistance, and potential as diagnostic and prognostic markers for lung cancer.

Is BRD7 associated with spermatogenesis impairment and male infertility in humans? A case-control study in a Han Chinese population

BackgroundBromodomain-containing protein 7 (BRD7), a member of the bromodomain-containing protein family, plays important roles in chromatin modification and transcriptional regulation. A recent model of Brd7-knockout mice presented azoospermia and male infertility, implying the potential role of BRD7 in spermatogenic failure in humans. This case-control study aimed to explore the association of the BRD7 gene with spermatogenic efficiency and the risk of spermatogenic defects in humans.ResultsA total of six heterozygous variants were detected in the coding and splicing regions of the BRD7 gene in patients with azoospermia. For each of four rare variants predicted to potentially damage BRD7 function, we further identified these four variants in oligozoospermia and normozoospermia as well. However, no difference in the allele and genotype frequencies of rare variants were observed between cases with spermatogenic failure and controls with normozoospermia; the sperm products of variant carriers were similar to those of noncarriers. Moreover, similar distribution of the alleles, genotypes and haplotypes of seven tag single nucleotide polymorphisms (tagSNPs) was observed between the cases with azoospermia and oligozoospermia and controls with normozoospermia; associations of tagSNP-distinguished BRD7 alleles with sperm products were not identified.ConclusionsThe lack of an association of BRD7-linked rare and common variants with spermatogenic failure implied a limited contribution of the BRD7 gene to spermatogenic efficiency and susceptibility to male infertility in humans.

PARP1-modulated chromatin remodeling is a new target for cancer treatment.

Cancer progression requires certain tumorigenic mutations in genes encoding for different cellular and nuclear proteins. Altered expressions of these mutated genes are mediated by post-translational modifications and chromatin remodeling. Chromatin remodeling is mainly regulated by the chromatin remodeling enzyme complexes and histone modifications. Upon DNA damage, Poly-(ADP-ribose) Polymerase1 (PARP1) plays a very important role in the induction of chromatin modifications and activation of DNA repair pathways to repair the DNA lesion. It has been targeted to develop different anti-cancer therapeutic interventions and PARP inhibitors have been approved by the U.S. Food and Drug Administration (FDA) for clinical use. But it has been found that the cancer cells often develop resistance to these PARP inhibitors and chromatin remodeling helps in enhancing this process. Hence, it may be beneficial to target PARP1-mediated chromatin remodeling, which may allow to reverse the drug resistance. In the current review, we have discussed the role of chromatin remodeling in DNA repair, how PARP1 regulates modifications of chromatin dynamics, and the role of chromatin modifications in cancer. It has also been discussed how the PARP1-mediated chromatin remodeling can be targeted by PARP inhibitors alone or in combination with other chemotherapeutic agents to establish novel anti-cancer therapeutics. We have also considered the use of PARG inhibitors that may enhance the action of PARP inhibitors to target different types of cancers.

R-Loops and Its Chro-Mates: The Strange Case of Dr. Jekyll and Mr. Hyde.

Since their discovery, R-loops have been associated with both physiological and pathological functions that are conserved across species. R-loops are a source of replication stress and genome instability, as seen in neurodegenerative disorders and cancer. In response, cells have evolved pathways to prevent R-loop accumulation as well as to resolve them. A growing body of evidence correlates R-loop accumulation with changes in the epigenetic landscape. However, the role of chromatin modification and remodeling in R-loops homeostasis remains unclear. This review covers various mechanisms precluding R-loop accumulation and highlights the role of chromatin modifiers and remodelers in facilitating timely R-loop resolution. We also discuss the enigmatic role of RNA:DNA hybrids in facilitating DNA repair, epigenetic landscape and the potential role of replication fork preservation pathways, active fork stability and stalled fork protection pathways, in avoiding replication-transcription conflicts. Finally, we discuss the potential role of several Chro-Mates (chromatin modifiers and remodelers) in the likely differentiation between persistent/detrimental R-loops and transient/benign R-loops that assist in various physiological processes relevant for therapeutic interventions.

'Enhancing' red cell fate through epigenetic mechanisms.

Transcription of erythroid-specific genes is regulated by the three-dimensional (3D) structure and composition of chromatin, which dynamically changes during erythroid differentiation. Chromatin organization and dynamics are regulated by several epigenetic mechanisms involving DNA (de-)methylation, posttranslational modifications (PTMs) of histones, chromatin-associated structural proteins, and higher-order structural changes and interactions. This review addresses examples of recent developments in several areas delineating the interface of chromatin regulation and erythroid-specific lineage transcription. We survey and discuss recent studies that focus on the erythroid chromatin landscape, erythroid enhancer-promotor interactions, super-enhancer functionality, the role of chromatin modifiers and epigenetic crosstalk, as well as the progress in mapping red blood cell (RBC) trait-associated genetic variants within cis-regulatory elements (CREs) identified in genome-wide association study (GWAS) efforts as a step toward determining their impact on erythroid-specific gene expression. As one of the best characterized and accessible cell differentiation systems, erythropoiesis has been at the forefront of studies aiming to conceptualize how chromatin dynamics regulate transcription. New emerging technologies that bring a significantly enhanced spatial and temporal resolution of chromatin structure, and allow investigation of small cell numbers, have advanced our understanding of chromatin dynamics during erythroid differentiation in vivo.

Platinum-Induced Ubiquitination of Phosphorylated H2AX by RING1A Is Mediated by Replication Protein A in Ovarian Cancer.

Platinum resistance is a common occurrence in high-grade serous ovarian cancer and a major cause of ovarian cancer deaths. Platinum agents form DNA cross-links, which activate nucleotide excision repair (NER), Fanconi anemia, and homologous recombination repair (HRR) pathways. Chromatin modifications occur in the vicinity of DNA damage and play an integral role in the DNA damage response (DDR). Chromatin modifiers, including polycomb repressive complex 1 (PRC1) members, and chromatin structure are frequently dysregulated in ovarian cancer and can potentially contribute to platinum resistance. However, the role of chromatin modifiers in the repair of platinum DNA damage in ovarian cancer is not well understood. We demonstrate that the PRC1 complex member RING1A mediates monoubiquitination of lysine 119 of phosphorylated H2AX (γH2AXub1) at sites of platinum DNA damage in ovarian cancer cells. After platinum treatment, our results reveal that NER and HRR both contribute to RING1A localization and γH2AX monoubiquitination. Importantly, replication protein A, involved in both NER and HRR, mediates RING1A localization to sites of damage. Furthermore, RING1A deficiency impairs the activation of the G2-M DNA damage checkpoint, reduces the ability of ovarian cancer cells to repair platinum DNA damage, and increases sensitivity to platinum. IMPLICATIONS: Elucidating the role of RING1A in the DDR to platinum agents will allow for the identification of therapeutic targets to improve the response of ovarian cancer to standard chemotherapy regimens.

The plasticity of DNA replication forks in response to clinically relevant genotoxic stress.

Complete and accurate DNA replication requires the progression of replication forks through DNA damage, actively transcribed regions, structured DNA and compact chromatin. Recent studies have revealed a remarkable plasticity of the replication process in dealing with these obstacles, which includes modulation of replication origin firing, of the architecture of replication forks, and of the functional organization of the replication machinery in response to replication stress. However, these specialized mechanisms also expose cells to potentially dangerous transactions while replicating DNA. In this Review, we discuss how replication forks are actively stalled, remodelled, processed, protected and restarted in response to specific types of stress. We also discuss adaptations of the replication machinery and the role of chromatin modifications during these transactions. Finally, we discuss interesting recent data on the relevance of replication fork plasticity to human health, covering its role in tumorigenesis, its crosstalk with innate immunity responses and its potential as an effective cancer therapy target.

Improvements on the quantitative analysis of Trypanosoma cruzi histone post translational modifications: Study of changes in epigenetic marks through the parasite's metacyclogenesis and life cycle

Trypanosome histone N-terminal sequences are very divergent from the other eukaryotes, although they are still decorated by post-translational modifications (PTMs). Here, we used a highly robust workflow to analyze histone PTMs in the parasite Trypanosoma cruzi using mass spectrometry-based (MS-based) data-independent acquisition (DIA). We adapted the workflow for the analysis of the parasite's histone sequences by modifying the software EpiProfile 2.0, improving peptide and PTM quantification accuracy. This workflow could now be applied to the study of 141 T. cruzi modified histone peptides, which we used to investigate the dynamics of histone PTMs along the metacyclogenesis and the life cycle of T. cruzi. Global levels of histone acetylation and methylation fluctuates along metacyclogenesis, however most critical differences were observed between parasite life forms. More than 66 histone PTM changes were detected. Strikingly, the histone PTM pattern of metacyclic trypomastigotes is more similar to epimastigotes than to cellular trypomastigotes. Finally, we highlighted changes at the H4 N-terminus and at H3K76 discussing their impact on the trypanosome biology. Altogether, we have optimized a workflow easily applicable to the analysis of histone PTMs in T. cruzi and generated a dataset that may shed lights on the role of chromatin modifications in this parasite. SignificanceTrypanosomes are unicellular parasites that have divergent histone sequences, no chromosome condensation and a peculiar genome/gene regulation. Genes are transcribed from divergent polycistronic regions and post-transcriptional gene regulation play major role on the establishment of transcripts and protein levels. In this regard, the fact that their histones are decorated with multiple PTMs raises interesting questions about their role. Besides, this digenetic organism must adapt to different environments changing its metabolism accordingly. As metabolism and epigenetics are closely related, the study of histone PTMs in trypanosomes may enlighten this strikingly, and not yet fully understood, interplay. From a biomedical perspective, the comprehensive study of molecular mechanisms associated to the metacyclogenesis process is essential to create better strategies for controlling Chagas disease.

Loss of H3K36 Methyltransferase SETD2 Impairs V(D)J Recombination during Lymphoid Development.

SummaryRepair of DNA double-stranded breaks (DSBs) during lymphocyte development is essential for V(D)J recombination and forms the basis of immunoglobulin variable region diversity. Understanding of this process in lymphogenesis has historically been centered on the study of RAG1/2 recombinases and a set of classical non-homologous end-joining factors. Much less has been reported regarding the role of chromatin modifications on this process. Here, we show a role for the non-redundant histone H3 lysine methyltransferase, Setd2, and its modification of lysine-36 trimethylation (H3K36me3), in the processing and joining of DNA ends during V(D)J recombination. Loss leads to mis-repair of Rag-induced DNA DSBs, especially when combined with loss of Atm kinase activity. Furthermore, loss reduces immune repertoire and a severe block in lymphogenesis as well as causes post-mitotic neuronal apoptosis. Together, these studies are suggestive of an important role of Setd2/H3K36me3 in these two mammalian developmental processes that are influenced by double-stranded break repair.

Role of chromatin modification and remodeling in stem cell regulation and meristem maintenance in Arabidopsis

In higher plants, pluripotent stem cells reside in the specialized microenvironment called stem cell niches (SCNs) harbored at the shoot apical meristem (SAM) and root apical meristem (RAM), which give rise to the aerial and underground parts of a plant, respectively. The model plant Arabidopsis thaliana (Arabidopsis) has been extensively studied to decipher the intricate regulatory mechanisms involving some key transcriptions factors and phytohormones that play pivotal roles in stem cell homeostasis, meristem maintenance, and organ formation. However, there is increasing evidence to show the epigenetic regulation of the chromatin architecture, gene expression exerting an influence on an innate balance between the self-renewal of stem cells, and differentiation of the progeny cells to a specific tissue type or organ. Post-translational histone modifications, ATP-dependent chromatin remodeling, and chromatin assembly/disassembly are some of the key features involved in the modulation of chromatin architecture. Here, we discuss the major epigenetic regulators and illustrate their roles in the regulation of stem cell activity, meristem maintenance, and related organ patterning in Arabidopsis.

Uncovering the mouse olfactory long non-coding transcriptome with a novel machine-learning model.

Very little is known about long non-coding RNAs (lncRNAs) in the mammalian olfactory sensory epithelia. Deciphering the non-coding transcriptome in olfaction is relevant because these RNAs have been shown to play a role in chromatin modification and nuclear architecture reorganization, processes that accompany olfactory differentiation and olfactory receptor gene choice, one of the most poorly understood gene regulatory processes in mammals. In this study, we used a combination of in silico and ex vivo approaches to uncover a comprehensive catalogue of olfactory lncRNAs and to investigate their expression in the mouse olfactory organs. Initially, we used a novel machine-learning lncRNA classifier to discover hundreds of annotated and unannotated lncRNAs, some of which were predicted to be preferentially expressed in the main olfactory epithelium and the vomeronasal organ, the most important olfactory structures in the mouse. Moreover, we used whole-tissue and single-cell RNA sequencing data to discover lncRNAs expressed in mature sensory neurons of the main epithelium. Candidate lncRNAs were further validated by in situ hybridization and RT-PCR, leading to the identification of lncRNAs found throughout the olfactory epithelia, as well as others exquisitely expressed in subsets of mature olfactory neurons or progenitor cells.

Stress - (self) eating: Epigenetic regulation of autophagy in response to psychological stress.

Autophagy is a constitutive and cytoprotective catabolic process. Aberrations in autophagy lead to a multitude of degenerative disorders, with neurodegeneration being one of the most widely studied autophagy-related disorders. While the field has largely been focusing on the cytosolic constituents and processes of autophagy, recent studies are increasingly appreciating the role of chromatin modifications and epigenetic regulation in autophagy maintenance. Autophagy has been implicated in the regulation of neurogenesis, and disruption of neurogenesis in response to psychological stress is a proximal risk factor for development of neuropsychiatric disorders such as major depressive disorder (MDD). In this review, we will discuss the regulation of autophagy in normal neurogenesis as well as during chronic psychological stress, focusing on the epigenetic control of autophagy in these contexts, and also highlight the lacunae in our understanding of this process. The systematic study of these regulatory mechanisms will provide a novel therapeutic strategy, based on the use epigenetic regulators of autophagy to enhance neurogenesis and potentially alleviate stress-related behavioral disorders.

Identification of genes functionally involved in the detrimental effects of mutant histone H3.3-K27M in Drosophila melanogaster.

Recurrent specific mutations in evolutionarily conserved histone 3 (H3) variants drive pediatric high-grade gliomas (HGGs), but little is known about their downstream effects. The aim of this study was to identify genes involved in the detrimental effects of mutant H3.3-K27M, the main genetic driver in lethal midline HGG, in a transgenic Drosophila model. Mutant and wild-type histone H3.3-expressing flies were generated using a φC31-based integration system. Genetic modifier screens were performed by crossing H3.3-K27M expressing driver strains and 194 fly lines expressing short hairpin RNA targeting genes selected based on their potential role in the detrimental effects of mutant H3. Expression of the human orthologues of genes with functional relevance in the fly model was validated in H3-K27M mutant HGG. Ubiquitous and midline glia-specific expression of H3.3-K27M but not wild-type H3.3 caused pupal lethality, morphological alterations, and decreased H3K27me3. Knockdown of 17 candidate genes shifted the lethal phenotype to later stages of development. These included histone modifying and chromatin remodeling genes as well as genes regulating cell differentiation and proliferation. Notably, several of these genes were overexpressed in mutant H3-K27M mutated HGG. Rapid screening, identification, and validation of relevant targets in "oncohistone" mediated pathogenesis have proven a challenge and a barrier to providing novel therapies. Our results provide further evidence on the role of chromatin modifiers in the genesis of H3.3-K27M. Notably, they validate Drosophila as a model system for rapid identification of relevant genes functionally involved in the detrimental effects of H3.3-K27M mutagenesis.

The Lysine Histone Methyltransferase SETD2 Is Required for Appropriate Immunoglobulin VDJ Recombination

Rearrangement of the immunoglobulin heavy chain locus (IgH) through non-homologous end-joining (NHEJ)-mediated VDJ recombination is a requisite step in normal lymphocyte development. This process is initiated through cleavage of DNA by RAG1/2 recombinases and repair by classical NHEJ pathway factors. Loss or defects of these factors can impair end-processing (DNA-PKcs, ARTEMIS) or end-ligation (KU70/80, XRCC4, XLF, LIGIV) of rearrangement and are drivers of disease, notably, severe combined immunodeficiency (SCID). In addition to these core NHEJ factors, other proteins, such as ATM kinase, have been shown to be critical for the stabilization of the post-cleavage VDJ intermediates to ensure proper signal joint formation. While epigenetic regulation of this process has been described through the maintenance of accessibility for transcription factors and RAG1/2, very little is known about the role of chromatin modifications on NHEJ-mediated VDJ recombination.Histone H3 lysine-36 tri-methylation (H3K36me3) is a histone modification associated with actively transcribed genes and has been shown to be critical for many biological processes, including transcription, mismatch repair and homologous recombination. Tri-methylation is catalyzed by the non-redundant histone methyltransferase, SETD2. Loss of function mutations in SETD2 or dominant negative onco-histone mutations in the H3K36 residue have been described in a broad array of tumors, including several hematopoietic malignancies. To study the role of Setd2 in hematopoiesis, we generated conditional knockout mice, where loss of Setd2 results in the ablation of the H3K36me3 mark. While heterozygous mice had no overt phenotype, mice with homozygous loss of Setd2(Setd2KO) displayed aberrant hematopoiesis, with a significant depletion of lymphoid primed progenitor population and B/T-cells in hematopoietic tissues. Setd2 loss early in hematopoiesis lead to a profound block of B-cell development at the proB cell stage and an early block in T-cell development, in a compartment where TCRβ rearrangements initiate. In Setd2KO mice, V to DJ rearrangements of the IgH locus were impaired, suggesting that the block at the proB stage of development was due to defective VDJ recombination. To assess this, we crossed our knockout mice with an IgHelMD4 mouse strain, and found that the block at the proB stage could be bypassed by the expression of a transgenic, fully rearranged IgH locus.We confirmed global loss of H3K36me3 in proB cells from our Setd2KO mice by ChIP-Seq and found that the ablation of this mark did not affect germline IgH locus accessibility (by ATAC-seq) or local chromatin architecture (H3K4me3 or H3K9ac) at a well-studied critical regulatory region near the Eμ enhancer. Loss of Setd2 and H3K36me3 only mildly effected sterile transcription of IgH genes and did not effect the overall cellular proliferative rate or the recruitment of NHEJ factors. Furthermore, loss of Setd2 did not cooperate to generate B-cell lymphomas in a p53-/- background. However, sequencing of VDJ recombination products from knockout mice indicated not only decreased clonality of products and variable gene usage but also resulted in alterations of complementarity defining region-3 (CDR3) hypervariable sequences. These results phenocopy defects or loss of NHEJ end-ligation factors and suggest that Setd2 and H3K36me3 could play a pivotal role in the end-ligation steps of NHEJ.Defects in end-ligation and resolution of VDJ intermediates have previously been shown to result in the formation of aberrant coding-signal hybrid joints. To see if this was occurring with loss of Setd2, we generated knockout murine proB v-Abl transformed lines where we could induce κ light chain rearrangement upon addition of an Abl kinase inhibitor. Remarkably, upon induction in Setd2KO v-Abl lines, we detected the formation of hybrid joints by both PCR and by the loss of a GFP-based coding-joint formation signal from a reporter expressed in these cells. We additionally could detect hybrid joint formation in the endogenous κ light chain locus of Setd2KO splenocytes, further indicating a role for Setd2/H3K36me3 in the end-ligation process. These studies demonstrate a novel and critical role for SETD2 and H3K36me3 in preventing aberrant, non-functional VDJ end joining of NHEJ-induced DNA breaks during lymphocyte development. DisclosuresNo relevant conflicts of interest to declare.

Abstract A170: Discovery of a potent and selective ATAD2 inhibitor with antiproliferative activity in breast cancer models

Abstract ATAD2 is a bromodomain (BD)-containing protein binding to chromatin at sites enriched in acetylated histones to modulate chromatin-related processes including transcription, DNA replication, and repair. ATAD2 is frequently amplified and/or overexpressed in breast cancer and is associated with aggressive disease, tumor metastasis, and poor outcome. Given the importance of the BD in targeting ATAD2 to relevant sites on the chromatin, we and others have hypothesized that the BD is essential for its mechanism of action and its targeting would have antiproliferative effects in cancer cells. The ATAD2 BD is one of the least tractable and to date reported inhibitors have been weak, nonselective, or exhibit poor cell permeability. Here we describe a potent, selective, and cell-permeable inhibitor of ATAD2 AZ4374 that modulates ATAD2 interaction with chromatin in cells and induces growth inhibition in breast cancer cell lines. AZ4374 has a potency (IC50) of 6.3 nM in the biochemical assay with >100-fold selectivity against other bromodomain-containing proteins. Chemoproteomic analysis of AZ4374 confirmed its selectivity. ATAD2 and BRD4 inhibition in cells was determined using displacement assays (NanoBRETTM) against the bromodomains. The IC50 for AZ4374 against ATAD2 was 80 nM and no inhibition of BRD4 was observed. The recently published GSK8814 was equipotent to AZ4374 in biochemical assays (IC50 ~6 nM) but less potent in the cell-based assay (IC50 ~600 nM). AZ4374 (10 µM) modulated the mRNA levels of CDK1, CDK2, and CCNE1 in EVSA-T cells (ATAD2 amplified). CRISPR/Cas9-based knockout of ATAD2 resulted in significant decrease in colony formation in breast cancer cell lines. Exposure of these cell lines to AZ4374 also reduced colony formation in a dose-dependent manner with IC50s ranging from 100 nM in SK-BR-3 to ~1µM in EVSA-T, so consistent with the potency observed in the cell-based assay. In addition, ATAD2 protacs caused a similar reduction of colony formation as AZ4374. Considering the role of chromatin modifiers in DNA damage response (DDR), we evaluated the impact of AZ4374 on the repair pathways HR and NHEJ using a CRISPR-reporter assay (traffic light reporter). A reduction in homology-directed repair was observed with 1 uM AZ4374, consistent with its biologic effect. Studies are ongoing to probe further the impact of ATAD2 inhibition on DNA repair. This is the first demonstration of highly potent and selective ATAD2 inhibition showing consistent cellular activity and biologic effect in cancer cell lines. Citation Format: Meghana M. Kulkarni, Jon Winter, David Whalley, ian Dale, Christopher Denz, Nin Guan, Deepa Bhavsar, Andrew Zhang, Timothy Rasmusson, Adrian Fretland, Sylvie M. Guichard. Discovery of a potent and selective ATAD2 inhibitor with antiproliferative activity in breast cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A170.