Polycomb Repressive Complex Research Articles

Phosphorylation-mediated disassembly of C-terminal binding protein 2 tetramer impedes epigenetic silencing of pluripotency in mouse embryonic stem cells.

Cells need to overcome both intrinsic and extrinsic threats. Although pluripotency is associated with damage responses, how stem cells respond to DNA damage remains controversial. Here, we elucidate that DNA damage activates Chk2, leading to the phosphorylation of serine 164 on C-terminal binding protein 2 (Ctbp2). The phosphorylation of Ctbp2 induces the disruption of Ctbp2 tetramer, weakening interactions with zinc finger proteins, leading to the dissociation of phosphorylated Ctbp2 from chromatin. This transition to a monomeric state results in the separation of histone deacetylase 1 from Ctbp2, consequently slowing the rate of H3K27 deacetylation. In contrast to the nucleosome remodeling and deacetylase complex, phosphorylated Ctbp2 increased binding affinity to polycomb repressive complex (PRC)2, interacting through the N-terminal domain of Suz12. Through this domain, Ctbp2 competes with Jarid2, inhibiting the function of PRC2. Thus, the phosphorylation of Ctbp2 under stress conditions represents a precise mechanism aimed at preserving stemness traits by inhibiting permanent transcriptional shutdown.

Increased translation driven by non-canonical EZH2 creates a synthetic vulnerability in enzalutamide-resistant prostate cancer

Overcoming resistance to therapy is a major challenge in castration-resistant prostate cancer (CRPC). Lineage plasticity towards a neuroendocrine phenotype enables CRPC to adapt and survive targeted therapies. However, the molecular mechanisms of epigenetic reprogramming during this process are still poorly understood. Here we show that the protein kinase PKCλ/ι-mediated phosphorylation of enhancer of zeste homolog 2 (EZH2) regulates its proteasomal degradation and maintains EZH2 as part of the canonical polycomb repressive complex (PRC2). Loss of PKCλ/ι promotes a switch during enzalutamide treatment to a non-canonical EZH2 cistrome that triggers the transcriptional activation of the translational machinery to induce a transforming growth factor β (TGFβ) resistance program. The increased reliance on protein synthesis creates a synthetic vulnerability in PKCλ/ι-deficient CRPC.

Polycomb group protein Mel18 inhibits hematopoietic stem cell self-renewal through repressing the transcription of self-renewal and proliferation genes.

Polycomb group (PcG) proteins play important roles in hematopoietic stem cell (HSC) self-renewal. Mel18 and Bmi1 are homologs of the PCGF subunit within the Polycomb repressive complex 1 (PRC1). Bmi1 (PCGF4) enhances HSC self-renewal and promotes terminal differentiation. However, the role of Mel18 (PCGF2) in hematopoiesis is not fully understood and how Mel18 regulates gene transcription in HSCs remains elusive. We found that acute deletion of Mel18 in the hematopoietic compartment significantly increased the frequency of functional HSCs in the bone marrow. Furthermore, we demonstrate that Mel18 inhibits HSC self-renewal and proliferation. RNA-seq studies revealed that HSC self-renewal and proliferation gene signatures are enriched in Mel18-/- hematopoietic stem and progenitors (HSPCs) compared to Mel18+/+ HSPCs. Notably, ATAC-seq revealed increased chromatin accessibility at genes important for HSC self-renewal, whereas CUT&RUN showed decreased enrichment of H2AK119ub1 at genes important for proliferation, leading to increased expression of both Hoxb4 and Cdk4 in Mel18-/- HSPCs. Thus, we demonstrate that Mel18 inhibits hematopoietic stem cell self-renewal through repressing the transcription of genes important for HSC self-renewal and proliferation.

PTEN depletion reduces H3K27me3 levels to promote epithelial-to-mesenchymal transition in epithelial colorectal cancer cells.

Epithelial-to-mesenchymal (EMT) transition is one of the best-known examples of tumor cell plasticity. EMT enhances cancer cell metastasis, which is the main cause of colorectal cancer (CRC)-related mortality. Therefore, understanding underlying molecular mechanisms contributing to the EMT process is crucial to finding druggable targets and more effective therapeutic approaches in CRC. In this study, we demonstrated that phosphatase and tensin homolog (PTEN) knockdown (KD) induces EMT in epithelial CRC, likely through the activation of AKT. PTEN KD modulated chromatin accessibility and reprogrammed gene transcription to mediate EMT in epithelial CRC cells. Active AKT can phosphorylate enhancer of zeste homolog 2 (EZH2) on serine 21, which switches EZH2 from a transcriptional repressor to an activator. Interestingly, PTEN KD reduced the global levels of trimethylation of histone 3 at lysine 27(H3K27me3) in an EZH2-phosphorylation-dependent manner. Additionally, EZH2 phosphorylation at serine 21 reduced the interaction of EZH2 with another polycomb repressive complex 2 (PRC2) component, suppressor of zeste 12 (SUZ12), suggesting that the reduced H3K27me3 levels in PTEN KD cells were due to a disruption of the PRC2 complex. Overall, we demonstrated that PTEN KD modulates changes in gene expression to induce the EMT process in epithelial CRC cells by phosphorylating EZH2 and activates transcription factors such as activator protein 1 (AP1).

RADIP technology comprehensively identifies H3K27me3-associated RNA-chromatin interactions.

Many RNAs associate with chromatin, either directly or indirectly. Several technologies for mapping regions where RNAs interact across the genome have been developed to investigate the function of these RNAs. Obtaining information on the proteins involved in these RNA-chromatin interactions is critical for further analysis. Here, we developed RADIP [RNA and DNA interacting complexes ligated and sequenced (RADICL-seq) with immunoprecipitation], a novel technology that combines RADICL-seq technology with chromatin immunoprecipitation to characterize RNA-chromatin interactions mediated by individual proteins. Building upon the foundational principles of RADICL-seq, RADIP extends its advantages by increasing genomic coverage and unique mapping rate efficiency compared to existing methods. To demonstrate its effectiveness, we applied an anti-H3K27me3 antibody to the RADIP technology and generated libraries from mouse embryonic stem cells (mESCs). We identified a multitude of RNAs, including RNAs from protein-coding genes and non-coding RNAs, that are associated with chromatin via H3K27me3 and that likely facilitate the spread of Polycomb repressive complexes over broad regions of the mammalian genome, thereby affecting gene expression, chromatin structures and pluripotency of mESCs. Our study demonstrates the applicability of RADIP to investigations of the functions of chromatin-associated RNAs.

Abstract A030: Polycomb repressive complex 2 (PRC2) mediates the epigenetic regulation of cancer-associated fibroblasts in esophageal cancer

Abstract Cancer-associated fibroblasts (CAFs) are a heterogenous cell population that can promote esophageal cancer progression through multiple molecular mechanisms including increased cancer cell proliferation and migration, immunosuppression, and therapeutic resistance. Despite the divergence of CAFs from normal fibroblasts with regards to phenotype and functions, CAFs rarely exhibit widespread genetic alterations. Instead, it is proposed that CAFs are regulated at the epigenetic level. Our objective is to delineate this epigenetic regulation of CAFs in esophageal cancer. Ultimately, we aim to identify the epigenetic regulatory complexes that mediate CAF heterogeneity and pro-tumorigenic functions. To this end, we completed RNA-seq and ATAC-seq on fibroblasts isolated from esophageal squamous cell carcinoma (ESCC), esophageal adenocarcinoma (EAC) and non-cancerous esophageal tissue. Enrichment analysis identified the polycomb repressive complex 2 (PRC2) as a potential regulator of ESCC and EAC CAFs. PRC2 catalyzes the methylation (mono-, di- or tri-) of lysine 27 on histone 3 (H3K27me1/2/3) resulting in transcriptional repression. Inhibition of two key proteins of PRC2 (EZH2 and SUZ12) through either pharmacological or genetic modulation, altered expression of multiple CAF phenotype related proteins and inhibited CAF functions including CAF proliferation. Ongoing studies, using co-cultures of fibroblasts and tumor cells, seek to identify if inhibition of PRC2 can prevent the initial transition of normal esophageal fibroblasts into CAFs. Importantly, we are conducting in vivo studies to determine if selective PRC2 inhibition in CAFs can inhibit esophageal tumor progression with specific focus on primary tumor growth and metastatic spread. In summary, initial studies have identify that PRC2 complex mediates CAF phenotype and functions, and we aim to establish the larger impact of these CAF specific effects on esophageal cancer. Citation Format: Karen J. Dunbar, Raul Navaridas Fernandez de Bobadil, Katherine M. Cunningham, Emily Esquea, Gizem Efe, Anil K. Rustgi. Polycomb repressive complex 2 (PRC2) mediates the epigenetic regulation of cancer-associated fibroblasts in esophageal cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A030.

Maternally expressed OsFERTILIZATION INDEPENDENT ENDOSPERM1 regulates seed dormancy and aleurone development in rice.

Seed dormancy, an essential trait for plant adaptation, is determined by the embryo itself and the surrounding tissues. Here, we found that rice (Oryza sativa) FERTILIZATION INDEPENDENT ENDOSPERM1 (OsFIE1) regulates endosperm-imposed dormancy and the dorsal aleurone thickness in a manner dependent on the parent of origin. Maternally expressed OsFIE1 suppresses gibberellin (GA) biosynthesis in the endosperm by depositing trimethylation of lysine 27 on histone H3 (H3K27me3) marks on GA biosynthesis-related genes, thus inhibiting germination and aleurone differentiation. Knockout of rice GA 20-oxidase1 (OsGA20ox1) alleviated the phenotypic defects in osfie1. The aleurone-positive determinant Crinkly 4 (OsCR4) is another target of the OsFIE1-containing Polycomb repressive complex 2 (PRC2). We found that OsFIE1 plays an important role in genomic imprinting in the endosperm of germinating seeds, particularly for paternally expressed genes associated with H3K27me3. The increased aleurone thickness of osfie1 substantially improved grain nutritional quality, indicating that the osfie1 gene may be utilized for breeding nutrient-enriched rice. The findings provide insights into the essential roles of PRC2-mediated H3K27me3 methylation in the acquisition of seed dormancy and endosperm cell differentiation in rice.

Read-write mechanisms of H2A ubiquitination by Polycomb repressive complex 1.

Epigenetic inheritance of silent chromatin domains is fundamental to cellular memory during embryogenesis, but it must overcome the dilution of repressive histone modifications during DNA replication1. One such modification, histone H2A lysine 119 monoubiquitination (H2AK119Ub), needs to be re-established by the Polycomb repressive complex 1 (PRC1) E3 ligase to restore the silent Polycomb domain2,3. However, the exact mechanism behind this restoration remains unknown. Here, combining cryo-electron microscopy (cryo-EM) and functional approaches, we characterize the read-write mechanism of the non-canonical PRC1-containing RYBP (ncPRC1RYBP). This mechanism, which functions as a positive-feedback loop in epigenetic regulation4,5, emphasizes the pivotal role of ncPRC1RYBP in restoring H2AK119Ub. We observe an asymmetrical binding of ncPRC1RYBP to H2AK119Ub nucleosomes, guided in part by the N-terminal zinc-finger domain of RYBP binding to residual H2AK119Ub on nascent chromatin. This recognition positions the RING domains of RING1B and BMI1 on the unmodified nucleosome side, enabling recruitment of the E2 enzyme to ubiquitinate H2AK119 within the same nucleosome (intra-nucleosome read-write) or across nucleosomes (inter-nucleosome read-write). Collectively, our findings provide key structural and mechanistic insights into the dynamic interplay of epigenetic regulation, highlighting the significance of ncPRC1RYBP in H2AK119Ub restoration to sustain repressive chromatin domains.

HCV infection activates the proteasome via PA28γ acetylation and heptamerization to facilitate the degradation of RNF2, a catalytic component of polycomb repressive complex 1.

We previously reported that hepatitis C virus (HCV) infection or HCV core protein expression induces HOX gene expression by impairing histone H2A monoubiquitination via a proteasome-dependent reduction in the level of RNF2, a key catalytic component of polycomb repressive complex 1 (H. Kasai, K. Mochizuki, T. Tanaka, A. Yamashita, et al., J Virol 95:e01784-20, 2021, https://doi.org/10.1128/jvi.01784-20). In this study, we aimed to investigate the mechanism by which HCV infection accelerates RNF2 degradation. Yeast two-hybrid screening and an immunoprecipitation assay revealed that RNF2 is a PA28γ-binding protein. The proteasome activator PA28γ destabilized the RNF2 protein in a proteasome-dependent manner, since RNF2 degradation was impaired by PA28γ knockout or MG132 treatment. HCV infection or core protein expression reduced the levels of RNF2 and histone H2A K119 monoubiquitination and induced the expression of HOX genes in the presence of PA28γ, while PA28γ knockout reversed these changes. Treatment with a lysine acetyltransferase inhibitor inhibited the acetylation of PA28γ at K195 and the degradation of the RNF2 protein, while treatment with a lysine deacetylase inhibitor accelerated these events in a PA28γ-dependent manner. RNF2 protein degradation was increased by expression of the acetylation mimetic PA28γ mutant but not by expression of the acetylation-defective mutant or the proteasome activation-defective mutant. Furthermore, HCV infection or core protein expression facilitated the interaction between PA28γ and the lysine acetyltransferase CBP/p300 and then accelerated PA28γ acetylation and heptazmerization to promote RNF2 degradation. These data suggest that HCV infection accelerates the acetylation-dependent heptamerization of PA28γ to increase the proteasomal targeting of RNF2.IMPORTANCEHCV is a causative agent of HCV-related liver diseases, including hepatic steatosis, cirrhosis, and hepatocellular carcinoma. PA28γ, which, in heptameric form, activates the 20S core proteasome for the degradation of PA28γ-binding proteins, is responsible for HCV-related liver diseases. HCV core protein expression or HCV infection accelerates RNF2 degradation, leading to the induction of HOX gene expression via a decrease in the level of H2Aub on HOX gene promoters. However, the mechanism of RNF2 degradation in HCV-infected cells has not been clarified. The data presented in this study suggest that PA28γ acetylation and heptamerization are promoted by HCV infection or by core protein expression to activate the proteasome for the degradation of RNF2 and are responsible for HCV propagation. This study provides novel insights valuable for the development of therapies targeting both HCV propagation and HCV-related diseases.

EPCO-04. H3F3A K27M MUTATION DRIVES A REPRESSIVE TRANSCRIPTOME VIA REDUCED SERINE 31 PHOSPHORYLATION BY MODULATING CHROMATIN ACCESSIBILITY, INDEPENDENT OF H3K27ME3 IN DIFFUSE MIDLINE GLIOMA

Abstract Heterozygous H3.3 K27M mutations are central to the pathogenesis of diffuse midline gliomas (DMG), causing a global reduction in H3K27 trimethylation (H3K27me3) and subsequent epigenetic dysregulation. H3.3 contains a unique Serine 31 (S31) phosphorylation site crucial for mitotic checkpoint regulation. In DMG cells, reduced mitotic S31 phosphorylation correlates with chromosomal instability and aneuploidy. Using a mouse model, we demonstrate that both H3.3K27M and H3.3S31A mutations promote high-grade glioma development, whereas wild-type H3.3 does not. Importantly, S31A, which maintains normal K27me, suggests that S31 phosphorylation loss alone is oncogenic. The H3.3K27M mutation disrupts epigenetic regulation by inhibiting the Polycomb Repressive Complex 2 (PRC2), leading to decreased H3K27me3 and unintended gene induction. Concurrently, K27M exerts a compensatory repressive effect on the transcriptome, mediated by reduced S31 phosphorylation, independent of PRC2 activity. Using CRISPR-engineered isogenic DMG cells and analyzing the transcriptome and chromatin accessibility with RNA-seq and ATAC-seq, we found that the K27M mutation produces a balanced gene expression profile, while PRC2 loss primarily leads to gene induction. Notably, cells with S31A mutations also maintain this balanced profile, linking the PRC2-independent effect of K27M to reduced S31 phosphorylation. Conversely, cells with wild-type K27 but S31A mutation show a repressive genomic state, evidenced by an up/down gene ratio of 0.75, owing to functional PRC2. We further show that the genes uniquely dysregulated by K27M or S31A, independent of PRC2, overlap significantly, with dysregulated genes displaying altered chromatin accessibility. This suggests that the effect of K27M and S31A mutations on gene regulation is primarily due to changes in chromatin state. This complex interplay points to targeting K27M-associated pathways, particularly S31 phosphorylation, as a promising therapeutic avenue in DMG, a malignancy with fewer effective treatments and dismal prognosis.

CSIG-10. DEFINING THE ROLE OF EZHIP ON CHROMOSOMAL INSTABILITY IN EPENDYMOMA

Abstract Diffuse Midline Gliomas (DMGs) are highly aggressive pediatric cancers with no effective treatments. Over 80% of DMGs harbor histone mutations, particularly H3.1 K27M, H3.3 K27M, and H3.3 G34R/V. These mutations impair the function of the Polycomb Repressive Complex 2 (PRC2), leading to reduced levels of H3K27me3. Overexpression of the EZHIP has been identified in ependymomas and DMGs. EZHIP overexpression is marked by widespread H3K27me3 loss and is mutually exclusive with histone mutations. EZHIP overexpression mimics the effects of the H3K27M mutation by reducing H3K27me3 levels. It is believed to inhibit the catalytic activity of EZH2 by binding to its SET domain, similar to the K27M mutant. Expression of EZHIP is tightly regulated and typically absent in most cell types. The transcriptional regulation of EZHIP is poorly understood; however, promoter hypermethylation is likely involved in regulating its expression. We investigate EZHIP’s role in DMG tumorigenesis, focusing on its regulation, impact on histone modifications, and chromosomal stability. Initial analyses have revealed transcription factor binding sites within the EZHIP promoter, including EGR2, SOX2, and ICP4, indicating that various transcription factors and viral infections might directly affect EZHIP expression. Utilizing RNA-Seq, we have identified both shared and unique transcriptomic alterations in DMG cells related to EZHIP expression and H3.3 K27M mutations. To study the role of EZHIP in tumorigenesis, we developed a mouse model expressing EZHIP-HA p2A PDFGB in RCAS-Nestin TVA mice. EZHIP expression induces high-grade tumors and significantly reduces survival. Our ongoing research suggests that EZHIP functions similarly to H3.3 K27M mutations in driving tumor growth, not only by reducing K27me3 but also through chromosomal missegregation. We aim to elucidate the full extent of EZHIP’s role in epigenetic control, histone post-translational modifications, chromosomal accessibility, and instability. Our findings will provide critical insights into the mechanisms underlying DMG tumorigenesis and potential therapeutic targets.

PWOs repress gene transcription by regulating chromatin structures in Arabidopsis.

PWWP-DOMAIN INTERACTOR OF POLYCOMBS (PWO) family proteins play a vital role in regulating plant development. However, the molecular mechanisms of how PWOs regulate chromatin structure is elusive. Our data show that the PWO1 binding sites are enriched with positive modifications but exclusive with H3K27me3. Moreover, PWO1 binds to the H3K27me3-enriched compartment domain (H3K27me3-CD) boundary regions, and functions to maintain the boundary strength. Meanwhile, we found that PWOs and Polycomb repressive complex 2 (PRC2) function parallelly in maintaining H3K27me3-CDs' structure. Loss of either PWOs or PRC2 leads to H3K27me3-CD strength reduction, B to A compartment switching as well as the H3K27me3-CD relocating away from the nuclear periphery. Additionally, PWOs and lamin-like proteins collaborate to regulate multiple chromatin structures to repress gene transcription within H3K27me3-CDs. We conclude that PWOs maintain H3K27me3-CDs' repressive state and regulate their spatial position in the nucleus.

Hypermethylation of CDKN2A CpG island drives resistance to PRC2 inhibitors in SWI/SNF loss-of-function tumors.

Polycomb repressive complex 2 (PRC2) catalyzes the writing of the tri-methylated histone H3 at Lys27 (H3K27me3) epigenetic marker and suppresses the expression of genes, including tumor suppressors. The function of the complex can be partially antagonized by the SWI/SNF chromatin-remodeling complex. Previous studies have suggested that PRC2 is important for the proliferation of tumors with SWI/SNF loss-of-function mutations. In the present study, we have developed an EED-directed allosteric inhibitor of PRC2 termed BR0063, which exhibits anti-proliferative properties in a subset of solid tumor cell lines harboring mutations of the SWI/SNF subunits, SMARCA4 or ARID1A. Tumor cells sensitive to BR0063 exhibited several distinct phenotypes, including cell senescence, which was mediated by the up-regulation of CDKN2A/p16. Further experiments revealed that the expression of p16 was suppressed in the BR0063-resistant cells via DNA hypermethylation in the CpG island (CGI) promoter region, rather than via PRC2 occupancy. The expression of TET1, which is required for DNA demethylation, was found to be inversely correlated with p16 CGI methylation, and this may serve as a biomarker for the prediction of resistance to PRC2 inhibitors in SWI/SNF LOF tumors.

An EED/PRC2-H19 Loop Regulates Cerebellar Development.

EED (embryonic ectoderm development) is a core subunit of the polycomb repressive complex 2 (PRC2), which senses the trimethylation of histone H3 lysine 27 (H3K27). However, its biological function in cerebellar development remains unknown. Here, we show that EED deletion from neural stem cells (NSCs) or cerebellar granule cell progenitors (GCPs) leads to reduced GCPs proliferation, cell death, cerebellar hypoplasia, and motor deficits in mice. Joint profiling of transcripts and ChIP-seq analysis in cerebellar granule cells reveals that EED regulates bunches of genes involved in cerebellar development. EED ablation exhibits overactivation of a developmental repressor long non-coding RNA H19. Importantly, an obvious H3K27ac enrichment is found at Ctcf, a trans-activator of H19, and H3K27me3 enrichment at the H19 imprinting control region (ICR), suggesting that EED regulates H19 in an H3K27me3-dependent manner. Intriguingly, H19 deletion reduces EED expression and the reprogramming of EED-mediated H3K27me3 profiles, resulting in increased proliferation, differentiation, and decreased apoptosis of GCPs. Finally, molecular and genetic evidence provides that increased H19 expression is responsible for cerebellar hypoplasia and motor defects in EED mutant mice. Thus, this study demonstrates that EED, H19 forms a negative feedback loop, which plays a crucial role in cerebellar morphogenesis and controls cerebellar development.

Proteome Profiling of Cucurbita pepo Phyllosphere After Infection by Podosphaera xanthii and Application of Reynoutria sachalinensis Extract

Podosphaera xanthii is the main causal agent of powdery mildew (PM) disease for Cucurbita pepo. Disease control is attained principally by applications of chemical fungicides, along with parallel use of tolerant crop varieties and alternate application of elicitors to control development of disease resistance. To get insight into C. pepo molecular responses to P. xanthii infection and elicitor treatment we studied the proteomic profile differences at the phyllosphere of a zucchini cultivar susceptible to PM, at the onset of P. xanthii (PX) infection and after application of Reynoutria sachalinensis (RS) plant extract, respectively, using a nano-LC-HRMS/MS, Q-Exactive-Orbitrap approach. Analysis of peptide sequences regarding four treatment groups (Control; PX; RS; and RSPX (PX-infected priorly treated with RS)) resulted in 2070 CuGenDB annotations. Three comparisons (treatments vs. Control) encompassed most of the Differentially Expressed Proteins (DEPs). In these three comparisons, KEGG and Gene Ontology functional analyses highlighted unique differentially enriched pathways—some of which included highly expressed proteins—in PX-related (proteasome, pentose phosphate pathway, and carbon fixation), RS-related (biosynthesis of secondary metabolites, flavonoids, and starch and sucrose metabolism), and RSPX-related (pyruvate metabolism and polycomb repressive complex) comparisons, respectively, suggesting distinct mechanisms of early plant responses modulated by PX and RS. Furthermore, in four out of six comparisons the thiamine metabolism pathway was found to be enriched, suggesting a pivotal role in PX-induced responses.

Epigenetic and Immune Profile Characteristics in Sinonasal Undifferentiated Carcinoma.

Sinonasal undifferentiated carcinoma (SNUC) is a rare and highly aggressive malignancy originating in the nasal cavity and paranasal sinuses. Its pathogenesis and immune characteristics remain poorly understood. This study investigates the molecular aspects of SNUC, focusing on tumorigenesis and immunity. For this purpose, spatial transcriptome analysis was employed to compare the gene expression profiles of SNUC tumor cells with those of normal epithelial cells, as well as to compare tumor-infiltrating immune cells with immune cells from normal, tumor-free tissue areas. For validation, next-generation sequencing tests and clinical sample studies were conducted. Spatial transcriptome analysis revealed notable upregulation of EZH2 and the histone family gene such as H3C2 (H3-clustered histone 2) in SNUC tumor cells. Additionally, gene set enrichment analysis identified significant activations in the histone deacetylase (HDAC) signaling pathway, histone acetyltransferase (HAT) pathway, polycomb repressive complex 2 (PRC2), and DNA methylation pathways. A notable decrease was observed in downregulated genes and pathways, including the mucin family of protein genes, the keratin protein gene, and the mucin glycosylation pathway. Next-generation sequencing did not reveal specific genetic mutations within these pathways, although mutations such as IDH2 R172S were noted. Clinical SNUC tissues confirmed increased immunoexpression of EZH2 and PRC2 markers. Analysis of tumor immunity revealed a characteristic immune cell signature, with a notable predominance of naïve B cells, macrophages, CD8 memory T cells, and Tregs in the SNUC microenvironment, alongside the increased expression of LAG3 in tumor-infiltrating immune cells. Our study suggests epigenetic mechanisms, particularly via EZH2, play a crucial role in SNUC carcinogenesis. Furthermore, distinctive immune cell profiles in SNUC point to potential immune-related characteristics of this malignancy.

H3K27 dimethylation dynamics reveal stepwise establishment of facultative heterochromatin in early mouse embryos.

Facultative heterochromatin is formed by Polycomb repressive complex 2 (PRC2)-deposited H3K27 trimethylation (H3K27me3) and PRC1-deposited H2AK119 mono-ubiquitylation (H2AK119ub1). How it is newly established after fertilization remains unclear. To delineate the establishment kinetics, here we profiled the temporal dynamics of H3K27 dimethylation (H3K27me2), which represents the de novo PRC2 catalysis, in mouse preimplantation embryos. H3K27me2 is newly deposited at CpG islands (CGIs), the paternal X chromosome (Xp) and putative enhancers during the eight-cell-to-morula transition, all of which follow H2AK119ub1 deposition. We found that JARID2, a PRC2.2-specific accessory protein possessing an H2AK119ub1-binding ability, colocalizes with SUZ12 at CGIs and Xp in morula embryos. Upon JARID2 depletion, SUZ12 chromatin binding and H3K27me2 deposition were attenuated and H3K27 acetylation at putative enhancers was increased in morulae and subsequently H3K27me3 failed to be deposited in blastocysts. These data reveal that facultative heterochromatin is established by PRC2.2-driven stepwise H3K27 methylation along pre-deposited H2AK119ub1 during early embryogenesis.

DNA methylation shapes the Polycomb landscape during the exit from naive pluripotency.

In mammals, 5-methylcytosine (5mC) and Polycomb repressive complex 2 (PRC2)-deposited histone 3 lysine 27 trimethylation (H3K27me3) are generally mutually exclusive at CpG-rich regions. As mouse embryonic stem cells exit the naive pluripotent state, there is massive gain of 5mC concomitantly with restriction of broad H3K27me3 to 5mC-free, CpG-rich regions. To formally assess how 5mC shapes the H3K27me3 landscape, we profiled the epigenome of naive and differentiated cells in the presence and absence of the DNA methylation machinery. Surprisingly, we found that 5mC accumulation is not required to restrict most H3K27me3 domains. Instead, this 5mC-independent H3K27me3 restriction is mediated by aberrant expression of the PRC2 antagonist Ezhip (encoding EZH inhibitory protein). At the subset of regions where 5mC appears to genuinely supplant H3K27me3, we identified 163 candidate genes that appeared to require 5mC deposition and/or H3K27me3 depletion for their activation in differentiated cells. Using site-directed epigenome editing to directly modulate 5mC levels, we demonstrated that 5mC deposition is sufficient to antagonize H3K27me3 deposition and confer gene activation at individual candidates. Altogether, we systematically measured the antagonistic interplay between 5mC and H3K27me3 in a system that recapitulates early embryonic dynamics. Our results suggest that H3K27me3 restraint depends on 5mC, both directly and indirectly. Our study also implies a noncanonical role of 5mC in gene activation, which may be important not only for normal development but also for cancer progression, as oncogenic cells frequently exhibit dynamic replacement of 5mC for H3K27me3 and vice versa.

GTSF1 is required for transposon silencing in the unicellular eukaryote Paramecium tetraurelia.

The PIWI-interacting RNA (piRNA) pathway is crucial for transposon repression and the maintenance of genomic integrity. Gametocyte-specific factor 1 (GTSF1), a PIWI-associated protein indispensable for transposon repression, has been recently shown to potentiate the catalytic activity of PIWI in many metazoans. Whether the requirement of GTSF1 extends to PIWI proteins beyond metazoans is unknown. In this study, we identified a homolog of GTSF1 in the unicellular eukaryote Paramecium tetraurelia (PtGtsf1) and found that its role as a PIWI-cofactor is conserved. PtGtsf1 interacts with PIWI (Ptiwi09) and Polycomb Repressive Complex 2 and is essential for PIWI-dependent DNA elimination of transposons during sexual development. PtGtsf1 is crucial for the degradation of PIWI-bound small RNAs that recognize the organism's own genomic sequences. Without PtGtsf1, self-matching small RNAs are not degraded and results in an accumulation of H3K9me3 and H3K27me3, which may disturb transposon recognition. Our results demonstrate that the PIWI-GTSF1 interaction also exists in unicellular eukaryotes with a role in transposon silencing.

EMBRYONIC FLOWER 1 regulates male reproduction by repressing the jasmonate pathway downstream transcription factor MYB26.

The evolutionarily conserved Polycomb repressive complexes (PRC) mediate genome-wide transcriptional silencing and regulate a plethora of development, as well as environmental responses in multicellular organisms. The PRC2-catalyzed trimethylation of lysine 27 on histone H3 (H3K27me3) is recognized by reader-effector modules of PRC1 to implement gene repression. Here, we report that the Arabidopsis (Arabidopsis thaliana) H3K27me3 effector EMBRYONIC FLOWER 1 (EMF1) interacts with and constrains the R2R3 DNA binding transcription factor MYB26 by a eudicot-conserved motif in the stamen. MYB26 activates the transcription of two NAC domain genes, NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1) and NST2, whose encoded proteins mediate anther secondary cell thickening in jasmonate (JA)-regulated stamen maturation. In this process, the transcriptional activity of MYB26 is negatively modulated by the JAZ-PRC repressive complex to precisely regulate the expression of NST1 and NST2. Disruption of EMF1 repression stimulates MYB26, leading to the excessive transcription of the two NAC genes and male sterility. Our results reveal a novel mechanism in polycomb-mediated gene silencing and illustrate that the plant Polycomb complex regulates stamen development by preventing the hypersensitivity of JA responses in male reproduction.