Polycomb Repressive Complex Research Articles

PRC2-AgeIndex as a universal biomarker of aging and rejuvenation

DNA methylation (DNAm) is one of the most reliable biomarkers of aging across mammalian tissues. While the age-dependent global loss of DNAm has been well characterized, DNAm gain is less characterized. Studies have demonstrated that CpGs which gain methylation with age are enriched in Polycomb Repressive Complex 2 (PRC2) targets. However, whole-genome examination of all PRC2 targets as well as determination of the pan-tissue or tissue-specific nature of these associations is lacking. Here, we show that low-methylated regions (LMRs) which are highly bound by PRC2 in embryonic stem cells (PRC2 LMRs) gain methylation with age in all examined somatic mitotic cells. We estimated that this epigenetic change represents around 90% of the age-dependent DNAm gain genome-wide. Therefore, we propose the “PRC2-AgeIndex,” defined as the average DNAm in PRC2 LMRs, as a universal biomarker of cellular aging in somatic cells which can distinguish the effect of different anti-aging interventions.

Nucleoporins cooperate with Polycomb silencers to promote transcriptional repression and repair at DNA double strand breaks.

DNA Double-strand breaks (DSBs) are harmful lesions and major sources of genomic instability. Studies have suggested that DSBs induce local transcriptional silencing that consequently promotes genomic stability. Several factors have been proposed to actively participate in this process, including ATM and Polycomb repressive complex 1 (PRC1). Here we found that disrupting PRC1 clustering disrupts DSB-induced gene silencing. Interactome analysis of PHC2, a PRC1 subunit that promotes the formation of the Polycomb body, found several nucleoporins that constitute the Nuclear Pore Complex (NPC). Similar to PHC2, depleting the nucleoporins also disrupted the DSB-induced gene silencing. We found that some of these nucleoporins, such as NUP107 and NUP43, which are members of the Y-complex of NPC, localize to DSB sites. These nucleoporin-enriched DSBs were distant from the nuclear periphery. The presence of nucleoporins and PHC2 at DSB regions were inter-dependent, suggesting that they act cooperatively in the DSB-induced gene silencing. We further found two structural components within NUP107 to be necessary for the transcriptional repression at DSBs: ATM/ATR-mediated phosphorylation at Serine37 residue within the N-terminal disordered tail, and the NUP133-binding surface at the C-terminus. These results provide a new functional interplay among nucleoporins, ATM and the Polycomb proteins in the DSB metabolism, and underscore their emerging roles in genome stability maintenance. *Hongseon Song, Yubin Bae, Sangin Kim, and Dante Deascanis contributed equally to this work.

Nucleocytoplasmic β-catenin expression contributes to neuroendocrine differentiation in muscle invasive bladder cancer.

Bladder cancers are heterogeneous in nature, showing diverse molecular profiles and histopathological characteristics, which pose challenges for diagnosis and treatment. However, understanding the molecular basis of such heterogeneity has remained elusive. This study aimed to elucidate the molecular landscape of neuroendocrine-like bladder tumors, focusing on the involvement of β-catenin localization. Analyzing the transcriptome data and benefiting from the molecular classification tool, we undertook an in-depth analysis of muscle-invasive bladder cancers to uncover the molecular characteristics of the neuroendocrine-like differentiation. The study explored the contribution of transcription factors and chromatin remodeling complexes to neuroendocrine differentiation in bladder cancer. The study revealed a significant correlation between β-catenin localization and neuroendocrine differentiation in muscle-invasive bladder tumors, highlighting the molecular complexity of neuroendocrine-like tumors. Enrichment of YY1 transcription factor, E2F family members, and Polycomb repressive complex components in β-catenin-positive tumors suggest their potential contribution to neuroendocrine phenotypes. Our findings contribute valuable insights into the molecular complexity of neuroendocrine-like bladder tumors. By identifying potential therapeutic targets and refining diagnostic strategies, this study advances our understanding of endocrinology in the context of bladder cancer. Further investigations into the functional implications of these molecular relationships are warranted to enhance our knowledge and guide future therapeutic interventions.

PRC1.6 localizes on chromatin with the human silencing hub (HUSH) complex for promoter-specific silencing.

An obligate step in the life cycle of HIV-1 and other retroviruses is the establishment of the provirus in target cell chromosomes. Transcriptional regulation of proviruses is complex, and understanding the mechanisms underlying this regulation has ramifications for fundamental biology, human health, and gene therapy implementation. The three core components of the Human Silencing Hub (HUSH) complex, TASOR, MPHOSPH8 (MPP8), and PPHLN1 (Periphilin 1), were identified in forward genetic screens for host genes that repress provirus expression. Subsequent loss-of-function screens revealed accessory proteins that collaborate with the HUSH complex to silence proviruses in particular contexts. To identify proteins associated with a HUSH complex-repressed provirus in human cells, we developed a technique, Provirus Proximal Proteomics, based on proximity labeling with C-BERST (dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging). Our screen exploited a lentiviral reporter that is silenced by the HUSH complex in a manner that is independent of the integration site in chromatin. Our data reveal that proviruses silenced by the HUSH complex are associated with DNA repair, mRNA processing, and transcriptional silencing proteins, including L3MBTL2, a member of the non-canonical polycomb repressive complex 1.6 (PRC1.6). A forward genetic screen confirmed that PRC1.6 components L3MBTL2 and MGA contribute to HUSH complex-mediated silencing. PRC1.6 was then shown to silence HUSH-sensitive proviruses in a promoter-specific manner. Genome wide profiling showed striking colocalization of the PRC1.6 and HUSH complexes on chromatin, primarily at sites of active promoters. Finally, PRC1.6 binding at a subset of genes that are silenced by the HUSH complex was dependent on the core HUSH complex component MPP8. These studies offer new tools with great potential for studying the transcriptional regulation of proviruses and reveal crosstalk between the HUSH complex and PRC1.6.

Polycomb function in early mouse development.

Epigenetic factors are crucial for ensuring proper chromatin dynamics during the initial stages of embryo development. Among these factors, the Polycomb group (PcG) of proteins plays a key role in establishing correct transcriptional programmes during mouse embryogenesis. PcG proteins are classified into two complexes: Polycomb repressive complex 1 (PRC1) and PRC2. Both complexes decorate histone proteins with distinct post-translational modifications (PTMs) that are predictive of a silent transcriptional chromatin state. In recent years, a critical adaptation of the classical techniques to analyse chromatin profiles and to study biochemical interactions at low-input resolution has allowed us to deeply explore PcG molecular mechanisms in the very early stages of mouse embryo development- from fertilisation to gastrulation, and from zygotic genome activation(ZGA) to specific lineages differentiation. These advancements provide a foundation for a deeper understanding of the fundamental role Polycomb complexes play in early development and have elucidated the mechanistic dynamics of PRC1 and PRC2. In this review, we discuss the functions and molecular mechanisms of both PRC1 and PRC2 during early mouse embryo development, integrating new studies with existing knowledge. Furthermore, we highlight the molecular functionality of Polycomb complexes from ZGA through gastrulation, with a particular focus on non-canonical imprinted and bivalent genes, and Hox cluster regulation.

PICKLE and HISTONE DEACETYLASE6 coordinately regulate genes and transposable elements in Arabidopsis.

Chromatin dynamics play essential roles in transcriptional regulation. The chromodomain helicase DNA-binding domain 3 chromatin remodeler PICKLE (PKL) and HISTONE DEACETYLASE6 (HDA6) are required for transcriptional gene silencing, but their coordinated function in gene repression requires further study. Through a genetic suppressor screen, we found that a point mutation at PKL could partially restore the developmental defects of a weak Polycomb repressive complex 1 (PRC1) mutant (ring1a-2 ring1b-3), in which RING1A expression is suppressed by a T-DNA insertion at the promoter. Compared to ring1a-2 ring1b-3, the expression of RING1A is increased, nucleosome occupancy is reduced, and the histone 3 lysine 9 acetylation (H3K9ac) level is increased at the RING1A locus in the pkl ring1a-2 ring1b-3 triple mutant. HDA6 interacts with PKL and represses RING1A expression similarly to PKL genetically and molecularly in the ring1a-2 ring1b-3 background. Furthermore, we show that PKL and HDA6 suppress the expression of a set of genes and transposable elements (TEs) by increasing nucleosome density and reducing H3K9ac. Genome-wide analysis indicated they possibly coordinately maintain DNA methylation as well. Our findings suggest that PKL and HDA6 function together to reduce H3K9ac and increase nucleosome occupancy, thereby facilitating gene/TE regulation in Arabidopsis (Arabidopsis thaliana).

Polycomb repressive complex 2 is critical for mouse cortical glutamatergic neuron development.

The Polycomb Repressive Complex 2 (PRC2) regulates corticogenesis, yet the consequences of mutations to this epigenetic modifier in the mature brain are poorly defined. Importantly, PRC2 core genes are haploinsufficient and causative of several human neurodevelopmental disorders. To address the role of PRC2 in mature cortical structure and function, we conditionally deleted the PRC2 gene Eed from the developing mouse dorsal telencephalon. Adult homozygotes displayed smaller forebrain structures. Single-nucleus transcriptomics revealed that glutamatergic neurons were particularly affected, exhibiting dysregulated gene expression profiles, accompanied by aberrations in neuronal morphology and connectivity. Remarkably, homozygous mice performed well on challenging cognitive tasks. In contrast, while heterozygous mice did not exhibit clear anatomical or behavioral differences, they displayed dysregulation of neuronal genes and altered neuronal morphology that was strikingly different from homozygous phenotypes. Collectively, these data reveal how alterations to PRC2 function shape the mature brain and reveal a dose-specific role for PRC2 in determining glutamatergic neuron identity.

Mono-methylation of lysine 27 at histone 3 confers lifelong susceptibility to stress

Histone post-translational modifications are critical for mediating persistent alterations in gene expression. By combining unbiased proteomics profiling and genome-wide approaches, we uncovered a role for mono-methylation of lysine 27 at histone H3 (H3K27me1) in the enduring effects of stress. Specifically, mice susceptible to early life stress (ELS) or chronic social defeat stress (CSDS) displayed increased H3K27me1 enrichment in the nucleus accumbens (NAc), a key brain-reward region. Stress-induced H3K27me1 accumulation occurred at genes that control neuronal excitability and was mediated by the VEFS domain of SUZ12, a core subunit of the polycomb repressive complex-2, which controls H3K27 methylation patterns. Viral VEFS expression changed the transcriptional profile of the NAc, led to social, emotional, and cognitive abnormalities, and altered excitability and synaptic transmission of NAc D1-medium spiny neurons. Together, we describe a novel function of H3K27me1 in the brain and demonstrate its role as a "chromatin scar" that mediates lifelong stress susceptibility.

PRDM16-DT: A Brain and Astrocyte-Specific lncRNA Implicated in Alzheimer's Disease.

Astrocytes provide crucial support for neurons, contributing to synaptogenesis, synaptic maintenance, and neurotransmitter recycling. Under pathological conditions, deregulation of astrocytes contributes to neurodegenerative diseases such as Alzheimer's disease (AD), highlighting the growing interest in targeting astrocyte function to address early phases of AD pathogenesis. While most research in this field has focused on protein-coding genes, non-coding RNAs, particularly long non-coding RNAs (lncRNAs), have emerged as significant regulatory molecules. In this study, we identified the lncRNA PRDM16-DT as highly enriched in the human brain, where it is almost exclusively expressed in astrocytes. PRDM16-DT and its murine homolog, Prdm16os, are downregulated in the brains of AD patients and in AD models. In line with this, knockdown of PRDM16-DT and Prdm16os revealed its critical role in maintaining astrocyte homeostasis and supporting neuronal function by regulating genes essential for glutamate uptake, lactate release, and neuronal spine density through interactions with the RE1-Silencing Transcription factor (Rest) and Polycomb Repressive Complex 2 (PRC2). Notably, CRISPR-mediated overexpression of Prdm16os mitigated functional deficits in astrocytes induced by stimuli linked to AD pathogenesis. These findings underscore the importance of PRDM16-DT in astrocyte function and its potential as a novel therapeutic target for neurodegenerative disorders characterized by astrocyte dysfunction.

Structural and mechanistic basis for nucleosomal H2AK119 deubiquitination by single-subunit deubiquitinase USP16.

Epigenetic regulators have a crucial effect on gene expression based on their manipulation of histone modifications. Histone H2AK119 monoubiquitination (H2AK119Ub), a well-established hallmark in transcription repression, is dynamically regulated by the opposing activities of Polycomb repressive complex 1 (PRC1) and nucleosome deubiquitinases including the primary human USP16 and Polycomb repressive deubiquitinase (PR-DUB) complex. Recently, the catalytic mechanism for the multi-subunit PR-DUB complex has been described, but how the single-subunit USP16 recognizes the H2AK119Ub nucleosome and cleaves the ubiquitin (Ub) remains unknown. Here we report the cryo-EM structure of USP16-H2AK119Ub nucleosome complex, which unveils a fundamentally distinct mode of H2AK119Ub deubiquitination compared to PR-DUB, encompassing the nucleosome recognition pattern independent of the H2A-H2B acidic patch and the conformational heterogeneity in the Ub motif and the histone H2A C-terminal tail. Our work highlights the mechanism diversity of H2AK119Ub deubiquitination and provides a structural framework for understanding the disease-causing mutations of USP16.

HGG-40. UNRAVELING THE IMMUNOLOGIC VULNERABILITIES OF DIFFUSE HEMISPHERIC GLIOMA, H3 G34-MUTANT

Abstract BACKGROUND Insights into the pathogenesis of diffuse hemispheric glioma, H3 G34-mutant (DHG-G34), an incurable high-grade glioma, have opened new therapeutic possibilities. The DHG-G34 oncohistone mutation impairs the epigenetic regulator SETD2, which normally orchestrates DNA-mismatch-repair and negatively regulates Polycomb Repressive Complex 2 (PRC2). In DHG-G34, a mismatch-repair-deficiency phenotype is predicted, but an anti-tumor immune response is notably absent, which we hypothesize is linked to the aberrant transcriptional repression by PRC2. Here, we investigate DHG-G34 immunosuppression mechanisms in a real-world multi-omics cohort and cell line model. METHODS Clinical, molecular, and immunologic characteristics of a DHG-G34 cohort, sequenced at Caris Life Sciences, are contrasted against diffuse midline glioma (DMG) and pediatric low-grade glioma (LGG). KNS-42 DHG-G34 cell line was exposed to valemetostat (a EZH1/2 inhibitor) or DMSO in-vitro. Flow cytometry and RNA-sequencing were utilized to determine differential immunologic characteristics. RESULTS 29 DHG-G34, 51 DMG, and 52 LGG were identified. DHG-G34 median OS was 15.4 mo., like DMG (HR=1.1, p=0.686). Common DHG-G34 alterations were TP53 (93.1%), ATRX (69.0%), PDGFRA (31.0%). Tumor mutational burden (TMB) was high, >=10mt/MB, in 10% of DHG-G34, with 0 DMG or LGG events (p<0.01, q>0.05), but innate and adaptive immune fractions and antigen-presenting genes were decreased (fold-change: 0.19–0.83, q<0.05). In KNS-42, PRC2 inhibition upregulated MHC class 1 (p<0.001). Of 402 missense variants, 43 were upregulated following EHZ2 inhibition, and 4 were downregulated (q<0.05). Among immunostimulatory genes, P2RX7, which engages T-cells, was downregulated in patient samples (q<0.001) and upregulated in EZH2 inhibited KNS-42 (q<0.001). CONCLUSIONS DHG-G34 has a propensity for high TMB but remains immunologically cold. Our pilot experiment suggests inhibiting PRC2’s unique role in DHG-G34 may sensitize DHG-G34 to an immune response by inducing MHC class 1 expression and transcriptional read-through of mutated transcripts. Understanding whether treatment induces TMB in DHG-G34 may affect timing of an immunotherapeutic intervention.

ATRT-07. PRC1/BMI1 ACTIVITY ALTERS CHROMATIN ACCESSIBILITY TO REGULATE DIFFERENTIATION IN ATYPICAL TERATOID RHABDOID TUMORS

Abstract BACKGROUND SMARCB1 is a member of the SWI/SNF chromatin remodeling complex that is responsible for determining cellular pluripotency and lineage commitment. Atypical Teratoid Rhabdoid Tumor (ATRT) is a highly aggressive pediatric brain tumor and characterized by SMARCB1 deletion. METHODS Genome wide RNAi screen, RNA-seq and ChIP-Seq analysis, colony formation assay, cell cycle, western blotting, immunoprecipitation, mass spectrometry analysis, mouse xenograft model, IVIS and MRI imaging. RESULTS We performed an RNAi-based functional genomic screen to identify key epigenetic regulators that co-operate with SMARCB1 loss and contribute to tumorigenesis in ATRT. 406 genes associated with epigenetic regulation in patient-derived ATRT cell lines were targeted. This screen identified BMI1, a component of the Polycomb Repressive Complex 1 (PRC1), as one of the top targets essential for ATRT cell growth. Using RNA-seq and ChIP-Seq analysis we demonstrated that BMI1 co-operates with SMARCB1 deletion to suppress transcription of pro-differentiation pathways and promote self-renewal of tumor stem cells. In SMARCB1 deficient cells BMI1 forms a partial PRC1 complex devoid of DNA binding components. We found that genetic inactivation of BMI1 suppresses ATRT cell clonogenicity and neurosphere formation, leads to S-phase arrest and induces ATRT cell differentiation in vitro. In vivo BMI1 inhibition induces apoptosis, leading to significantly increasing survival of treated mice. By Doxycycline-inducible SMARCB1 re-expression system we revealed activation of proapoptotic proteins p16 and p21. With immunoprecipitation for BMI1, followed by mass spectrometry analysis we demonstrated that re-expression of SMARCB1 activates two PRC1 chromatin localizing components CBX4 and CBX8. Moreover, SMARCB1 re-expression significantly prolongs mice survival and decreases tumor size as evaluated by IVIS imaging system and T2-MRI. CONCLUSION Thus, we demonstrated that SMARCB1 deletion results in reprograming of BMI1 chromatin occupancy away from lineage specification by altering the components of the PRC1 complex and that PRC1/BMI1 inhibition is a novel therapeutic approach in ATRT.

EPEN-11. CHARACTERIZING EPIGENETIC MODULATIONS AND EZHIP DOWNSTREAM TARGETS IN PFA EPENDYMOMA

Abstract BACKGROUND Ependymomas can arise from all compartments of the central nervous system and ten distinct molecular groups have been identified in children and adults. In children, the most aggressive group is posterior fossa group A ependymoma (PFA). In PFA ependymomas, the aberrant overexpression of EZHIP (EZH inhibitory protein) has been implicated as a relevant driver of the disease. EZHIP suppresses the installing and spreading of the repressive epigenetic mark H3K27me3 via disturbing PRC2 (polycomb repressive complex 2) activity through inhibiting EZH2 (enhancer of zeste 2), which causes overall low levels of H3K27me3 in PFA. METHODS To get a better understanding how EZHIP may drive the disease, how it affects the epigenetic landscape and what the downstream targets of EZHIP are, we performed DNA sequencing, RNA expression profiling and ChIP-seq experiments of H3K27me3, H3K27ac, EZH2, and EZHIP in PFA, PFB, and ST-ZFTA ependymomas (n = 3 tumor cases each). RESULTS After validating the high levels of EZHIP and global reduction of H3K27me3 in PFA, we characterized the epigenetic landscape and EZHIP chromatin binding characteristics in PFA compared to PFB and ST-ZFTA ependymoma. Transcriptome analysis showed that overall more genes are up-regulated in PFA compared with PFB and ZFTA, which might be caused by its relatively high accessibility with a global loss of H3K27me3. EZHIP knockdown experiments confirmed direct changes on H3K27me3, H3K27ac and gene expression. We also found that PLAG1 (pleomorphic adenoma gene 1) expression linearly correlated with EZHIP expression in PFA tumors. ChIP-seq data indeed showed the PLAG1 locus to be bound by EZHIP and marked with increased H3K27ac and decreased H3K27me3 signals in PFA. CONCLUSIONS We identified PLAG1 as a potential downstream target of EZHIP in PFA tumors. Currently, more experiments on PLAG1 are being performed to further explore its role as a potential vulnerability in PFA ependymoma.

MDB-08. MYC AND TGFΒ PROMOTE GROUP 3 MEDULLOBLASTOMA TUMOR RESISTANCE THROUGH DEREGULATION OF POLYCOMB TARGETS

Abstract Medulloblastoma (MB) is one of the most prevalent malignant brain tumors in children, with tremendous cognitive and neuroendocrine disability among survivors. Group 3 (G3) MBs have poor overall survival at <50%, higher frequency of metastasis, and no targeted therapies. Amplification of MYC and activation of TGFβ signaling are frequent in G3MB. Remarkably, some tumors have no reported mutations, suggesting roles for epigenetic mechanisms in driving disease. We have generated new humanized models for G3MB by transforming neuroepithelial stem cells (NESC) derived from human induced pluripotent stem cells with TGFβ effectors alone and/or in combination with MYC, prioritizing combinations observed in patients. Excitingly, both MYC and TGFβ effectors drove tumor formation in vivo with the combination of TGFβ effectors and MYC leading to more aggressive tumors. Moreover, NESC-derived tumors clustered with human G3/G4MB samples, indicating our models recapitulates human disease. We next found that NESCs expressing MYC with either TGFβR1 or TGFβ1 showed resistance to clinical TGFβR1 inhibitors, compared to cells driven by either TGFβR1 or TGFβ1 alone. To decipher mechanisms of resistance, we integrated CUT&RUN to probe for MYC genomic localization and relevant histone PTMs with RNA-seq analysis and discovered a subset of genes upregulated in MYC and TGFβ-driven lines that are targets of the histone demethylase KDM2B and Polycomb Repressive Complex 2 (PRC2) member SUZ12. Moreover, we found that MYC is bound to regions normally regulated by PRC interactors. We postulate that chromatin remodeling via MYC and recruitment of other MYC-interacting cofactors to PRC targets culminates in transcriptional changes that lead to aggressive disease. Work from others suggests that PRC-mediated chromatin regulation is critical in many adult and pediatric brain tumors, underscoring its significance in MB. Our studies provide important insight on identifying new therapeutic avenues with a focus on the epigenetic landscape for patients with MYC and TGFβ driven G3MB.

ETMR-19.BCOR/L1-ALTERATIONS REWIRE HISTONE REGULATION TO INDUCE ONCOGENIC PATHWAYS IN PEDIATRIC CENTRAL NERVOUS SYSTEM TUMORS

Abstract BACKGROUND Central nervous system (CNS) tumors with BCOR internal tandem duplication (ITD) or BCOR/L1 fusions are recently described tumor types with distinct molecular characteristics and poor clinical outcome. BCOR and its homologue BCORL1 are essential components of the non-canonical polycomb repressor complex (PRC) 1.1 thereby acting as epigenetic regulators exerting a profound impact on central cellular mechanisms. However, the influence of BCOR/L1-alterations on the functionality of PRC1.1 in pediatric CNS tumors remains understudied. METHODS We performed bulk as well as single-cell/nucleus RNA sequencing (scRNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) of CNS tumor tissue harboring BCOR-ITD or BCOR/L1 fusions. Additionally, BCOR/L1-altered, patient-derived cell models were used for downstream in vitro analyses. RESULTS ChIP-Seq analysis demonstrated binding of both KDM2B and mutant BCOR/L1 at PRC1.1 target genes whereas PRC1/2-mediated histone modifications H3K27me3 and H2AK119ub were absent. In addition, PRC1.1 target gene loci were characterized by high H3K27ac levels indicating increased transcriptional activity in BCOR/L1 mutant samples. Indeed, PRC1.1 targets such as FGFR1, GATA6, and ERBB3 were found to be upregulated in bulk transcriptome (n=37) analyses. Using scRNA-seq (n=10), we resolved a transcriptionally distinct cell population, specific for BCOR/L1-driven CNS tumors, which expressed high levels of PRC1.1 target genes. Besides PRC1.1 target genes, the targetable receptor tyrosine kinases PDGFR/FGFR(1/3), and IGF1R were found to be upregulated in pediatric BCOR/L1-altered CNS cancers. Accordingly, drug screens of patient-derived cell models revealed sensitivity towards small molecule inhibitors targeting the respective pathways. This translated well in anti-tumor effects of the FGFR/PDGFRA inhibitor nintedanib in BCORL1::NUTM2HP-fused patient-derived xenograft bearing mice. CONCLUSIONS Our data demonstrate for the first time that BCOR/L1-altered CNS tumors are driven by dysfunction of PRC1.1 histone modifications inducing profound transcriptomic changes ultimately activating oncogenic pathways. We further identified first feasible therapeutic targets including FGFR and PDGFR which may be leveraged for improved therapy of patients suffering from this aggressive tumor type.

ATRT-16. MAPPING THE DISORDERED METHYLOME IN ATRT

Abstract BACKGROUND Atypical Teratoid Rhabdoid Tumor (ATRT) is an aggressive pediatric brain tumor driven by inactivation of the chromatin regulator SMARCB1, with remarkably few other mutations. SMARCB1 loss leads to epigenetic dysregulation which plays a pivotal role in ATRT development and progression, and DNA methylation profiling has distinguished molecular subgroups of this disease. However, studies that provide a static view of the epigenome may not capture epigenetic variability that drives cellular plasticity and heterogeneity. Mapping epigenetic variability is crucial to understanding the phenotypic plasticity of this tumor and its ability to evade therapeutic interventions in the clinic. METHODS We performed whole genome bisulfite sequencing (WGBS) on 22 ATRT samples, 4 normal fetal brain controls, and 1 hindbrain neural stem cell control, and applied a novel analytic pipeline (InformME) to generate DNA methylation potential energy landscapes encapsulating epigenetic stochasticity. This characterizes mean methylation as well as methylation entropy. We identified key genes and regulatory regions with the most stochastic methylation signatures. RESULTS We find that ATRT exhibits a globally destabilized epigenome, with markedly elevated methylation entropy (a measure of variability/disorder). Methylation discordance mapped to key regulatory regions including bivalent promoters, quiescent chromatin regions, and enhancers, and specific genes, with an enrichment for targets of the Polycomb repressive complex 2 and regulators of pluripotency and differentiation. CONCLUSIONS The disordered methylome of ATRT could play a critical role in the tumor’s ability to evade therapies. Mapping the targets of epigenetic instability can identify targets for intervention. Moreover, strategies to constrain epigenetic variability may have therapeutic benefit by limiting the number of epigenetic states available to cancer cells.

Homeobox and Polycomb target gene methylation in human solid tumors

DNA methylation is an epigenetic mark that plays an important role in defining cancer phenotypes, with global hypomethylation and focal hypermethylation at CpG islands observed in tumors. These methylation marks can also be used to define tumor types and provide an avenue for biomarker identification. The homeobox gene class is one that has potential for this use, as well as other genes that are Polycomb Repressive Complex 2 targets. To begin to unravel this relationship, we performed a pan-cancer DNA methylation analysis using sixteen Illumina HM450k array datasets from TCGA, delving into cancer-specific qualities and commonalities between tumor types with a focus on homeobox genes. Our comparisons of tumor to normal samples suggest that homeobox genes commonly harbor significant hypermethylated differentially methylated regions. We identified two homeobox genes, HOXA3 and HOXD10, that are hypermethylated in all 16 cancer types. Furthermore, we identified several potential homeobox gene biomarkers from our analysis that are uniquely methylated in only one tumor type and that could be used as screening tools in the future. Overall, our study demonstrates unique patterns of DNA methylation in multiple tumor types and expands on the interplay between the homeobox gene class and oncogenesis.

EZH2-associated tumor malignancy: A prominent target for cancer treatment.

The discussion in this review centers around the significant relationships between EZH2 and the initiation, progression, metastasis, metabolism, drug resistance, and immune regulation of cancer. Polycomb group (PcG) proteins, which encompass two primary Polycomb repressor complexes (PRC1 and PRC2), have been categorized. PRC2 consists mainly of four subunits, namely EZH2, EED, SUZ12, and RbAp46/48. As the crucial catalytic component within the PRC2 complex, EZH2 plays a pivotal role in controlling a wide range of biological processes. Overexpression/mutations of EZH2 have been detected in a wide variety of tumors. Several mechanisms of EZH regulation have been identified, including regulation EZH2 mRNA by miRNAs, LncRNAs, accessibility to DNA via DNA-binding proteins, post-translational modifications, and transcriptional regulation. EZH2 signaling triggers cancer progression and may intervene with anti-tumor immunity; therefore it has charmed attention as an effective therapeutic target in cancer therapy. Numerous nucleic acid-based therapies have been used in the modification of EZH2. In addition to gene therapy approaches, pharmaceutical compounds can be used to target the EZH2 signaling pathway in the treatment of cancer. EZH2-associated tumor cells and immune cells enhance the effects of the immune response in a variety of human malignancies. The combination of epigenetic modifying agents, such as anti-EZH2 compounds with immunotherapy, could potentially be efficacious even in the context of immunosuppressive tumors. Summary, understanding the mechanisms underlying resistance to EZH2 inhibitors may facilitate the development of novel drugs to prevent or treat relapse in treated patients.

Stat3-mediated Atg7 expression enhances anti-tumor immunity in melanoma.

Epigenetic modifications to DNA and chromatin control oncogenic and tumor suppressive mechanisms in melanoma. EZH2, the catalytic component of the Polycomb repressive complex 2 (PRC2), which mediates methylation of lysine 27 on histone 3 (H3K27me3), can regulate both melanoma initiation and progression. We previously found that mutant Ezh2 Y641F interacts with the immune regulator Stat3 and together they affect anti-tumor immunity. However, given the numerous downstream targets and pathways affected by EZH2, many mechanisms that determine its oncogenic activity remain largely unexplored. Using genetically engineered mouse models we further investigated the role of pathways downstream of EZH2 in melanoma carcinogenesis and identified significant enrichment in several autophagy signatures, along with increased expression of autophagy regulators, such as Atg7. In this study, we investigated the effect of Atg7 on melanoma growth and tumor immunity within the context of an Ezh2 Y641F epigenetic state. We found that expression of Atg7 is largely dependent on Stat3 expression and that deletion of Atg7 slows down melanoma cell growth in vivo, but not in vitro. Atg7 deletion also results in increased CD8+ T cells and reduced myelosuppressive cell infiltration in the tumor microenvironment, suggesting a strong immune system contribution in the role of Atg7 in melanoma progression. These findings highlight the complex interplay between genetic mutations, epigenetic regulators, and autophagy in shaping tumor immunity in melanoma.

Brassica rapa CURLY LEAF is a major H3K27 methyltransferase regulating flowering time

Main conclusionIn Brassica rapa, the epigenetic modifier BraA.CLF orchestrates flowering by modulating H3K27me3 levels at the floral integrator genes FT, SOC1, and SEP3, thereby influencing their expression.CURLY LEAF (CLF) is the catalytic subunit of the plant Polycomb Repressive Complex 2 that mediates the trimethylation of histone H3 lysine 27 (H3K27me3), an epigenetic modification that leads to gene silencing. While the function of CURLY LEAF (CLF) has been extensively studied in Arabidopsis thaliana, its role in Brassica crops is barely known. In this study, we focused on the Brassica rapa homolog of CLF and found that the loss-of-function mutant braA.clf-1 exhibits an accelerated flowering together with pleiotropic phenotypic alterations compared to wild-type plants. In addition, we carried out transcriptomic and H3K27me3 genome-wide analyses to identify the genes regulated by BraA.CLF. Interestingly, we observed that several floral regulatory genes, including the B. rapa homologs of FT, SOC1 and SEP3, show reduced H3K27me3 levels and increased transcript levels compared to wild-type plants, suggesting that they are direct targets of BraA.CLF and key players in regulating flowering time in this crop. In addition, the results obtained will enhance our understanding of the epigenetic mechanisms regulating key developmental traits and will aid to increase crop yield by engineering new Brassica varieties with different flowering time requirements.