A repressive H3K36me2 reader mediates Polycomb silencing.

In animals, evolutionarily conserved Polycomb repressive complex 2 (PRC2) catalyzes histone H3 lysine 27 trimethylation (H3K27me3) and PRC1 functions in recruitment and transcriptional repression. However, the mechanisms underlying H3K27me3-mediated stable transcriptional silencing are largely unknown, as PRC1 subunits are poorly characterized in fungi. Here, we report that in the filamentous fungus Magnaporthe oryzae, the N-terminal chromodomain and C-terminal MRG domain of Eaf3 play key roles in facultative heterochromatin formation and transcriptional silencing. Eaf3 physically interacts with Ash1, Eed, and Sin3, encoding an H3K36 methyltransferase, the core subunit of PRC2, and a histone deacetylation co-suppressor, respectively. Eaf3 co-localizes with a set of repressive Ash1-H3K36me2 and H3K27me3 loci and mediates their transcriptional silencing. Furthermore, Eaf3 acts as a histone reader for the repressive H3K36me2 and H3K27me3 marks. Eaf3-occupied regions are associated with increased nucleosome occupancy, contributing to transcriptional silencing in M. oryzae. Together, these findings reveal that Eaf3 is a repressive H3K36me2 reader and plays a vital role in Polycomb gene silencing and the formation of facultative heterochromatin in fungi.

EZH2 represses mesenchymal genes and upholds the epithelial state of breast carcinoma cells.

Emerging studies support that the polycomb repressive complex 2 (PRC2) regulates phenotypic changes of carcinoma cells by modulating their shifts among metastable states within the epithelial and mesenchymal spectrum. This new role of PRC2 in cancer has been recently proposed to stem from the ability of its catalytic subunit EZH2 to bind and modulate the transcription of mesenchymal genes during epithelial-mesenchymal transition (EMT) in lung cancer cells. Here, we asked whether this mechanism is conserved in other types of carcinomas. By combining TGF-ß-mediated reversible induction of epithelial to mesenchymal transition and inhibition of EZH2 methyltransferase activity, we demonstrate that EZH2 represses a large set of mesenchymal genes and favours the residence of breast cancer cells towards the more epithelial spectrum during EMT. In agreement, analysis of human patient samples supports that EZH2 is required to efficiently repress mesenchymal genes in breast cancer tumours. Our results indicate that PRC2 operates through similar mechanisms in breast and lung cancer cells. We propose that PRC2-mediated direct transcriptional modulation of the mesenchymal gene expression programme is a conserved molecular mechanism underlying cell dissemination across human carcinomas.

Leading medulloblastoma to a differentiation end.

Effective and less toxic therapies for medulloblastoma have proved to be highly elusive. In this issue of Cancer Cell, Yang et al. show that thyroid hormone treatment leads to the activation of neurogenic differentiation factor 1 (NeuroD1) and differentiation of medulloblastoma cells through reversing EZH2-mediated transcriptional repression of NeuroD1.

MTF2 facilitates the advancement of osteosarcoma through mediating EZH2/SFRP1/Wnt signaling.

BACKGROUND: Osteosarcoma is a soft tissue neoplasm with elevated recurrence risk and highly metastatic potential. Metal response element binding transcriptional factor 2 (MTF2) has been revealed to exert multiple activities in human tissues. The present research was conducted to explore the functions and related response mechanism of MTF2 in osteosarcoma which have not been introduced yet. METHODS: Bioinformatics tools identified the differential MTF2 expression in osteosarcoma tissues. MTF2 expression in osteosarcoma cells was examined with Western blot. Cell Counting Kit-8 (CCK-8) assay, 5-Ethynyl-2'-deoxyuridine (EDU) staining, wound healing as well as transwell assays measured cell proliferation, migration and invasion, respectively. Flow cytometry assay detected the cellular apoptotic level. Western blot also measured the expressions of proteins associated with epithelial mesenchymal transition (EMT), apoptosis and enhancer of zeste homolog 2 (EZH2)/secreted frizzled-related protein 1 (SFRP1)/Wnt signaling. Co-immunoprecipitation (Co-IP) assay confirmed MTF2-EZH2 interaction. RESULTS: MTF2 expression was increased in osteosarcoma tissues and cells. MTF2 interference effectively inhibited the proliferation, migration and invasion of osteosarcoma cells and promoted the cellular apoptotic rate. MTF2 directly bound to EZH2 and MTF2 silence reduced EZH2 expression, activated SFRP1 expression and blocked Wnt signaling in osteosarcoma cells. EZH2 upregulation or SFRP1 antagonist WAY-316606 partly counteracted the impacts of MTF2 down-regulation on the SFRP1/Wnt signaling and the biological phenotypes of osteosarcoma cells. CONCLUSIONS: MTF2 might down-regulate SFRP1 to activate Wnt signaling and drive the progression of osteosarcoma via interaction with EZH2 protein.

Structural basis for the H2AK119ub1-specific DNMT3A-nucleosome interaction.

Isoform 1 of DNA methyltransferase DNMT3A (DNMT3A1) specifically recognizes nucleosome monoubiquitylated at histone H2A lysine-119 (H2AK119ub1) for establishment of DNA methylation. Mis-regulation of this process may cause aberrant DNA methylation and pathogenesis. However, the molecular basis underlying DNMT3A1-nucleosome interaction remains elusive. Here we report the cryo-EM structure of DNMT3A1's ubiquitin-dependent recruitment (UDR) fragment complexed with H2AK119ub1-modified nucleosome. DNMT3A1 UDR occupies an extensive nucleosome surface, involving the H2A-H2B acidic patch, a surface groove formed by H2A and H3, nucleosomal DNA, and H2AK119ub1. The DNMT3A1 UDR's interaction with H2AK119ub1 affects the functionality of DNMT3A1 in cells in a context-dependent manner. Our structural and biochemical analysis also reveals competition between DNMT3A1 and JARID2, a cofactor of polycomb repression complex 2 (PRC2), for nucleosome binding, suggesting the interplay between different epigenetic pathways. Together, this study reports a molecular basis for H2AK119ub1-dependent DNMT3A1-nucleosome association, with important implications in DNMT3A1-mediated DNA methylation in development.

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.

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.

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.

TRPS1 expression in MPNST is correlated with PRC2 inactivation and loss of H3K27me3.

Initially described as a highly specific immunohistochemical marker for carcinomas of mammary origin, trichorhinophalangeal syndrome type 1 (TRPS1) has subsequently been detected in a variety of other non-mammary tumors. In this study, we examined the immunohistochemical expression of TRPS1 in 114 peripheral nerve sheath tumors, including 43 malignant peripheral nerve sheath tumors (MPNSTs), 58 schwannomas, including 9 cellular neurofibromas, and 13 neurofibromas, including 1 atypical neurofibroma. Notably, TRPS1 was expressed in 49% of MPNSTs and was absent in all schwannomas and neurofibromas. All MPNSTs showed TRPS1 labeling in >50% of nuclei, with 95% of cases demonstrating diffuse labeling. Most cases (67%) showed weak TRPS1 immunoreactivity, while a smaller subset showed moderate (24%) or strong (9%) intensity staining. Analysis of publicly available gene expression datasets revealed higher levels of TRPS1 mRNA in MPNSTs with PRC2 inactivation. In keeping with this finding, TRPS1 expression was more commonly observed in MPNSTs with loss of H3K27me3, suggesting a potential relationship between TRPS1 and the PRC2 complex. This study further broadens the spectrum of TRPS1-expressing tumors to include MPNST.

JARID2, a novel regulatory factor, promotes cell proliferation, migration, and invasion in oral squamous cell carcinoma.

BACKGROUND: Accurate regulation of gene expression is crucial for normal development and function of cells. The prognostic significance and potential carcinogenic mechanisms of the related gene JARID2 in OSCC are not yet clear, but existing research has indicated a significant association between the two. METHODS AND MATERIALS: The relationship between the expression of the JARID2 gene in tumor samples of OSCC patients and clinical pathological factors was analyzed using immunohistochemistry experiments and RT-qPCR analysis. Based on the clinical pathological data of patients, bioinformatics analysis was conducted using public databases to determine the function of JARID2 in OSCC. Knockdown OSCC cell lines were constructed, and the impact of JARID2 on the biological behavior of OSCC cell lines was assessed through CCK-8, wound healing assay, and transwell analysis. RESULTS: Immunohistochemistry experiments confirmed the correlation between JARID2 and the prognosis of OSCC patients, while RT-qPCR experiments demonstrated its expression levels in tissue and cells. CKK-8 experiments, wound healing assays, and Transwell experiments indicated that knocking down JARID2 had a negative impact on the proliferation, invasion, and migration of OSCC cells. Bioinformatics analysis results showed that the expression of JARID2 in OSCC is closely associated with patient gene co-expression, gene function enrichment, immune infiltration, and drug sensitivity. CONCLUSION: Our study indicates that JARID2 is a novel prognostic biomarker and potential therapeutic target for OSCC.

The Cell Cycle: a Key to Unlock EZH2-targeted Therapy Resistance.

SUMMARY: In this issue, a study by Kazansky and colleagues explored resistance mechanisms after EZH2 inhibition in malignant rhabdoid tumors (MRT) and epithelioid sarcomas (ES). The study identified genetic alterations in EZH2 itself, along with alterations that converge on RB1-E2F-mediated cell-cycle control, and demonstrated that inhibition of cell-cycle kinases, such as Aurora Kinase B (AURKB) could bypass EZH2 inhibitor resistance to enhance treatment efficacy. See related article by Kazansky et al., p. 965 (6).

A novel interplay between PRC2 and miR-3189 regulates epithelial-mesenchymal transition (EMT) via modulating COL6A2 in glioblastoma.

Recent studies have shed light on disrupted collagen signaling in Gliomas, yet the regulatory landscape remains largely unexplored. This study enquired into the role of polycomb repressive complex-2 (PRC2)-mediated H3K27me3 modification, a key epigenetic factor in glioma. Using in-house data, we identified miRNAs downregulated in glioblastoma (GBM) with the potential to regulate Collagen VI family genes. Notably, miR-3189 emerged as a prime PRC2 target. Its expression was significantly downregulated in Indian GBM patients as well as other glioma cohorts. Mechanistic insights, involving Luciferase assays, mutagenesis, and Western blot analysis, confirmed direct targeting of Collagen VI member COL6A2 by miR-3189-3p. Functional assays demonstrated that miR-3189-3p restrained GBM malignancy by inhibiting proliferation, migration, and epithelial-mesenchymal transition (EMT). Conversely, COL6A2 overexpressed in GBM patients, countered miR-3189, and promoted the malignant phenotype. Gene set enrichment analysis highlighted EMT enrichment in GBM patients with elevated COL6A2 expression, carrying prognostic implications. This study uncovers intricate interactions between two epigenetic regulators-H3K27me3 and miR-3189-working synergistically to modulate Collagen VI gene; thus, influencing the malignancy of GBM. Targeting this H3K27me3|miR-3189-3p|COL6A2 axis presents a potential therapeutic avenue against GBM.

Two MADS-box proteins, AGL9 and AGL15, recruit the FIS-PRC2 complex to trigger the phase transition from endosperm proliferation to embryo development in Arabidopsis.

Spatiotemporal regulation of gene expression by polycomb repressive complex 2 (PRC2) is critical for animal and plant development. The Arabidopsis fertilization independent seed (FIS)-PRC2 complex functions specifically during plant reproduction from gametogenesis to seed development. After a double fertilization event, triploid endosperm proliferates early, followed by the growth of a diploid embryo, which replaces the endosperm in Arabidopsis and many dicots. Key genes critical for endosperm proliferation such as IKU2 and MINI3 are activated after fertilization. Here we report that two MADS-box AGAMOUS-LIKE (AGL) proteins associate with the key endosperm proliferation loci and recruit the FIS-PRC2 repressive complex at 4-5 days after pollination (DAP). Interestingly, AGL9 and AGL15 only accumulate toward the end of endosperm proliferation at 4-5 DAP and promote the deposition of H3K27me3 marks at key endosperm proliferation loci. Disruption of AGL9 and AGL15 or overexpression of AGL9 or AGL15 significantly influence endosperm proliferation and cellularization. Genome-wide analysis with cleavage Under Targets and tagmentation (CUT&Tag) sequencing and RNA sequencing revealed the landscape of endosperm H3K27me3 marks and gene expression profiles in Col-0 and agl9 agl15. CUT&Tag qPCR also demonstrated the occupancy of the two MADS-box proteins and FIS-PRC2 on a few representative target loci. Our studies suggest that MADS-box proteins could potentially recruit PRC2 to regulate many other developmental processes in plants or even in fungi and animals.

Cancer-associated Histone H3 N-terminal arginine mutations disrupt PRC2 activity and impair differentiation.

Dysregulated epigenetic states are a hallmark of cancer and often arise from genetic alterations in epigenetic regulators. This includes missense mutations in histones, which, together with associated DNA, form nucleosome core particles. However, the oncogenic mechanisms of most histone mutations are unknown. Here, we demonstrate that cancer-associated histone mutations at arginines in the histone H3 N-terminal tail disrupt repressive chromatin domains, alter gene regulation, and dysregulate differentiation. We find that histone H3R2C and R26C mutants reduce transcriptionally repressive H3K27me3. While H3K27me3 depletion in cells expressing these mutants is exclusively observed on the minor fraction of histone tails harboring the mutations, the same mutants recurrently disrupt broad H3K27me3 domains in the chromatin context, including near developmentally regulated promoters. H3K27me3 loss leads to de-repression of differentiation pathways, with concordant effects between H3R2 and H3R26 mutants despite different proximity to the PRC2 substrate, H3K27. Functionally, H3R26C-expressing mesenchymal progenitor cells and murine embryonic stem cell-derived teratomas demonstrate impaired differentiation. Collectively, these data show that cancer-associated H3 N-terminal arginine mutations reduce PRC2 activity and disrupt chromatin-dependent developmental functions, a cancer-relevant phenotype.

IκBα controls dormancy in hematopoietic stem cells via retinoic acid during embryonic development.

Recent findings suggest that Hematopoietic Stem Cells (HSC) and progenitors arise simultaneously and independently of each other already in the embryonic aorta-gonad mesonephros region, but it is still unknown how their different features are established. Here, we uncover IκBα (Nfkbia, the inhibitor of NF-κB) as a critical regulator of HSC proliferation throughout development. IκBα balances retinoic acid signaling levels together with the epigenetic silencer, PRC2, specifically in HSCs. Loss of IκBα decreases proliferation of HSC and induces a dormancy related gene expression signature instead. Also, IκBα deficient HSCs respond with superior activation to in vitro culture and in serial transplantation. At the molecular level, chromatin regions harboring binding motifs for retinoic acid signaling are hypo-methylated for the PRC2 dependent H3K27me3 mark in IκBα deficient HSCs. Overall, we show that the proliferation index in the developing HSCs is regulated by a IκBα-PRC2 axis, which controls retinoic acid signaling.

Proximal termination generates a transcriptional state that determines the rate of establishment of Polycomb silencing.

The mechanisms and timescales controlling de novo establishment of chromatin-mediated transcriptional silencing by Polycomb repressive complex 2 (PRC2) are unclear. Here, we investigate PRC2 silencing at Arabidopsis FLOWERING LOCUS C (FLC), known to involve co-transcriptional RNA processing, histone demethylation activity, and PRC2 function, but so far not mechanistically connected. We develop and test a computational model describing proximal polyadenylation/termination mediated by the RNA-binding protein FCA that induces H3K4me1 removal by the histone demethylase FLD. H3K4me1 removal feeds back to reduce RNA polymerase II (RNA Pol II) processivity and thus enhance early termination, thereby repressing productive transcription. The model predicts that this transcription-coupled repression controls the level of transcriptional antagonism to PRC2 action. Thus, the effectiveness of this repression dictates the timescale for establishment of PRC2/H3K27me3 silencing. We experimentally validate these mechanistic model predictions, revealing that co-transcriptional processing sets the level of productive transcription at the locus, which then determines the rate of the ON-to-OFF switch to PRC2 silencing.

G-quadruplex folding in Xist RNA antagonizes PRC2 activity for stepwise regulation of X chromosome inactivation.

How Polycomb repressive complex 2 (PRC2) is regulated by RNA remains an unsolved problem. Although PRC2 binds G-tracts with the potential to form RNA G-quadruplexes (rG4s), whether rG4s fold extensively in vivo and whether PRC2 binds folded or unfolded rG4 are unknown. Using the X-inactivation model in mouse embryonic stem cells, here we identify multiple folded rG4s in Xist RNA and demonstrate that PRC2 preferentially binds folded rG4s. High-affinity rG4 binding inhibits PRC2's histone methyltransferase activity, and stabilizing rG4 in vivo antagonizes H3 at lysine 27 (H3K27me3) enrichment on the inactive X chromosome. Surprisingly, mutagenizing the rG4 does not affect PRC2 recruitment but promotes its release and catalytic activation on chromatin. H3K27me3 marks are misplaced, however, and gene silencing is compromised. Xist-PRC2 complexes become entrapped in the S1 chromosome compartment, precluding the required translocation into the S2 compartment. Thus, Xist rG4 folding controls PRC2 activity, H3K27me3 enrichment, and the stepwise regulation of chromosome-wide gene silencing.

H3K27me3 timely dictates uterine epithelial transcriptome remodeling and thus transformation essential for normal embryo implantation.

Uterine luminal epithelia (LE), the first layer contacting with the blastocyst, acquire receptivity for normal embryo implantation. Besides the well-accepted transcriptional regulation dominated by ovarian estrogen and progesterone for receptivity establishment, the involvement of epigenetic mechanisms remains elusive. This study systematically profiles the transcriptome and genome-wide H3K27me3 distribution in the LE throughout the preimplantation. Combining genetic and pharmacological approaches targeting the PRC2 core enzyme Ezh1/2, we demonstrate that the defective remodeling of H3K27me3 in the preimplantation stage disrupts the differentiation of LE, and derails uterine receptivity, resulting in implantation failure. Specifically, crucial epithelial genes, Pgr, Gata2, and Sgk1, are transcriptionally silenced through de novo deposition of H3K27me3 for LE transformation, and their sustained expression in the absence of H3K27me3 synergistically confines the nuclear translocation of FOXO1. Further functional studies identify several actin-associated genes, including Arpin, Tmod1, and Pdlim2, as novel direct targets of H3K27me3. Their aberrantly elevated expression impedes the morphological remodeling of LE, a hindrance alleviated by treatment with cytochalasin D which depolymerizes F-actin. Collectively, this study uncovers a previously unappreciated epigenetic regulatory mechanism for the transcriptional silencing of key LE genes via H3K27me3, essential for LE differentiation and thus embryo implantation.

Inseparable RNA binding and chromatin modification activities of a nucleosome-interacting surface in EZH2.

Polycomb repressive complex 2 (PRC2) interacts with RNA in cells, but there is no consensus on how RNA regulates PRC2 canonical functions, including chromatin modification and the maintenance of transcription programs in lineage-committed cells. We assayed two separation-of-function mutants of the PRC2 catalytic subunit EZH2, defective in RNA binding but functional in methyltransferase activity. We find that part of the RNA-binding surface of EZH2 is required for chromatin modification, yet this activity is independent of RNA. Mechanistically, the RNA-binding surface within EZH2 is required for chromatin modification in vitro and in cells, through interactions with nucleosomal DNA. Contrarily, an RNA-binding-defective mutant exhibited normal chromatin modification activity in vitro and in lineage-committed cells, accompanied by normal gene repression activity. Collectively, we show that part of the RNA-binding surface of EZH2, rather than the RNA-binding activity per se, is required for the histone methylation in vitro and in cells, through interactions with the substrate nucleosome.