XIST directly regulates X-linked and autosomal genes in naive human pluripotent cells.

X chromosome inactivation (XCI) serves as a paradigm for RNA-mediated regulation of gene expression, wherein the long non-coding RNA XIST spreads across the X chromosome in cis to mediate gene silencing chromosome-wide. In female naive human pluripotent stem cells (hPSCs), XIST is in a dispersed configuration, and XCI does not occur, raising questions about XIST's function. We found that XIST spreads across the X chromosome and induces dampening of X-linked gene expression in naive hPSCs. Surprisingly, XIST also targets specific autosomal regions, where it induces repressive chromatin changes and gene expression dampening. Thereby, XIST equalizes X-linked gene dosage between male and female cells while inducing differences in autosomes. The dispersed Xist configuration and autosomal localization also occur transiently during XCI initiation in mouse PSCs. Together, our study identifies XIST as the regulator of X chromosome dampening, uncovers an evolutionarily conserved trans-acting role of XIST/Xist, and reveals a correlation between XIST/Xist dispersal and autosomal targeting.

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.

Modularity of PRC1 composition and chromatin interaction define condensate properties.

Polycomb repressive complexes (PRCs) play a key role in gene repression and are indispensable for proper development. Canonical PRC1 forms condensates in vitro and in cells that are proposed to contribute to the maintenance of repression. However, how chromatin and the various subunits of PRC1 contribute to condensation is largely unexplored. Using a reconstitution approach and single-molecule imaging, we demonstrate that nucleosomal arrays and PRC1 act synergistically, reducing the critical concentration required for condensation by more than 20-fold. We find that the exact combination of PHC and CBX subunits determines condensate initiation, morphology, stability, and dynamics. Particularly, PHC2's polymerization activity influences condensate dynamics by promoting the formation of distinct domains that adhere to each other but do not coalesce. Live-cell imaging confirms CBX's role in condensate initiation and highlights PHC's importance for condensate stability. We propose that PRC1 composition can modulate condensate properties, providing crucial regulatory flexibility across developmental stages.

H2AK119ub1 differentially fine-tunes gene expression by modulating canonical PRC1- and H1-dependent chromatin compaction.

Polycomb repressive complex 1 (PRC1) is a key transcriptional regulator in development via modulating chromatin structure and catalyzing histone H2A ubiquitination at Lys119 (H2AK119ub1). H2AK119ub1 is one of the most abundant histone modifications in mammalian cells. However, the function of H2AK119ub1 in polycomb-mediated gene silencing remains debated. In this study, we reveal that H2AK119ub1 has two distinct roles in gene expression, through differentially modulating chromatin compaction mediated by canonical PRC1 and the linker histone H1. Interestingly, we find that H2AK119ub1 plays a positive role in transcription through interfering with the binding of canonical PRC1 to nucleosomes and therefore counteracting chromatin condensation. Conversely, we demonstrate that H2AK119ub1 facilitates H1-dependent chromatin condensation and enhances the silencing of developmental genes in mouse embryonic stem cells, suggesting that H1 may be one of several possible pathways for H2AK119ub1 in repressing transcription. These results provide insights and molecular mechanisms by which H2AK119ub1 differentially fine-tunes developmental gene expression.

Alternative splicing decouples local from global PRC2 activity.

The Polycomb repressive complex 2 (PRC2) mediates epigenetic maintenance of gene silencing in eukaryotes via methylation of histone H3 at lysine 27 (H3K27). Accessory factors define two distinct subtypes, PRC2.1 and PRC2.2, with different actions and chromatin-targeting mechanisms. The mechanisms orchestrating PRC2 assembly are not fully understood. Here, we report that alternative splicing (AS) of PRC2 core component SUZ12 generates an uncharacterized isoform SUZ12-S, which co-exists with the canonical SUZ12-L isoform in virtually all tissues and developmental stages. SUZ12-S drives PRC2.1 formation and favors PRC2 dimerization. While SUZ12-S is necessary and sufficient for the repression of target genes via promoter-proximal H3K27me3 deposition, SUZ12-L maintains global H3K27 methylation levels. Mouse embryonic stem cells (ESCs) lacking either isoform exit pluripotency more slowly and fail to acquire neuronal cell identity. Our findings reveal a physiological mechanism regulating PRC2 assembly and higher-order interactions in eutherians, with impacts on H3K27 methylation and gene repression.

Imprinted X chromosome inactivation at the gamete-to-embryo transition.

In mammals, dosage compensation involves two parallel processes: (1) X inactivation, which equalizes X chromosome dosage between males and females, and (2) X hyperactivation, which upregulates the active X for X-autosome balance. The field currently favors models whereby dosage compensation initiates "de novo" during mouse development. Here, we develop "So-Smart-seq" to revisit the question and interrogate a comprehensive transcriptome including noncoding genes and repeats in mice. Intriguingly, de novo silencing pertains only to a subset of Xp genes. Evolutionarily older genes and repetitive elements demonstrate constitutive Xp silencing, adopt distinct signatures, and do not require Xist to initiate silencing. We trace Xp silencing backward in developmental time to meiotic sex chromosome inactivation in the male germ line and observe that Xm hyperactivation is timed to Xp silencing on a gene-by-gene basis. Thus, during the gamete-to-embryo transition, older Xp genes are transmitted in a "pre-inactivated" state. These findings have implications for the evolution of imprinting.

Pioneer and PRDM transcription factors coordinate bivalent epigenetic states to safeguard cell fate.

Pioneer transcription factors (TFs) regulate cell fate by establishing transcriptionally primed and active states. However, cell fate control requires the coordination of both lineage-specific gene activation and repression of alternative-lineage programs, a process that is poorly understood. Here, we demonstrate that the pioneer TF FOXA coordinates with PRDM1 TF to recruit nucleosome remodeling and deacetylation (NuRD) complexes and Polycomb repressive complexes (PRCs), which establish highly occupied, accessible nucleosome conformation with bivalent epigenetic states, thereby preventing precocious and alternative-lineage gene expression during human endoderm differentiation. Similarly, the pioneer TF OCT4 coordinates with PRDM14 to form bivalent enhancers and repress cell differentiation programs in human pluripotent stem cells, suggesting that this may be a common and critical function of pioneer TFs. We propose that pioneer and PRDM TFs coordinate to safeguard cell fate through epigenetic repression mechanisms.

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.

Methylation of histone H3 lysine 36 is a barrier for therapeutic interventions of head and neck squamous cell carcinoma.

Approximately 20% of head and neck squamous cell carcinomas (HNSCCs) exhibit reduced methylation on lysine 36 of histone H3 (H3K36me) due to mutations in histone methylase NSD1 or a lysine-to-methionine mutation in histone H3 (H3K36M). Whether such alterations of H3K36me can be exploited for therapeutic interventions is still unknown. Here, we show that HNSCC models expressing H3K36M can be divided into two groups: those that display aberrant accumulation of H3K27me3 and those that maintain steady levels of H3K27me3. The former group exhibits reduced proliferation, genome instability, and heightened sensitivity to genotoxic agents like PARP1/2 inhibitors. Conversely, H3K36M HNSCC models with constant H3K27me3 levels lack these characteristics unless H3K27me3 is elevated by DNA hypomethylating agents or inhibiting H3K27me3 demethylases KDM6A/B. Mechanistically, H3K36M reduces H3K36me by directly impeding the activities of the histone methyltransferase NSD3 and the histone demethylase LSD2. Notably, aberrant H3K27me3 levels induced by H3K36M expression are not a bona fide epigenetic mark because they require continuous expression of H3K36M to be inherited. Moreover, increased sensitivity to PARP1/2 inhibitors in H3K36M HNSCC models depends solely on elevated H3K27me3 levels and diminishing BRCA1- and FANCD2-dependent DNA repair. Finally, a PARP1/2 inhibitor alone reduces tumor burden in a H3K36M HNSCC xenograft model with elevated H3K27me3, whereas in a model with consistent H3K27me3, a combination of PARP1/2 inhibitors and agents that up-regulate H3K27me3 proves to be successful. These findings underscore the crucial balance between H3K36 and H3K27 methylation in maintaining genome instability, offering new therapeutic options for patients with H3K36me-deficient tumors.

Hyd/UBR5 defines a tumor suppressor pathway that links Polycomb repressive complex to regulated protein degradation in tissue growth control and tumorigenesis.

Tumor suppressor genes play critical roles in normal tissue homeostasis, and their dysregulation underlies human diseases including cancer. Besides human genetics, model organisms such as Drosophila have been instrumental in discovering tumor suppressor pathways that were subsequently shown to be highly relevant in human cancer. Here we show that hyperplastic disc (Hyd), one of the first tumor suppressors isolated genetically in Drosophila and encoding an E3 ubiquitin ligase with hitherto unknown substrates, and Lines (Lin), best known for its role in embryonic segmentation, define an obligatory tumor suppressor protein complex (Hyd-Lin) that targets the zinc finger-containing oncoprotein Bowl for ubiquitin-mediated degradation, with Lin functioning as a substrate adaptor to recruit Bowl to Hyd for ubiquitination. Interestingly, the activity of the Hyd-Lin complex is directly inhibited by a micropeptide encoded by another zinc finger gene, drumstick (drm), which functions as a pseudosubstrate by displacing Bowl from the Hyd-Lin complex, thus stabilizing Bowl. We further identify the epigenetic regulator Polycomb repressive complex1 (PRC1) as a critical upstream regulator of the Hyd-Lin-Bowl pathway by directly repressing the transcription of the micropeptide drm Consistent with these molecular studies, we show that genetic inactivation of Hyd, Lin, or PRC1 resulted in Bowl-dependent hyperplastic tissue overgrowth in vivo. We also provide evidence that the mammalian homologs of Hyd (UBR5, known to be recurrently dysregulated in various human cancers), Lin (LINS1), and Bowl (OSR1/2) constitute an analogous protein degradation pathway in human cells, and that OSR2 promotes prostate cancer tumorigenesis. Altogether, these findings define a previously unrecognized tumor suppressor pathway that links epigenetic program to regulated protein degradation in tissue growth control and tumorigenesis.

COOLAIR and PRC2 function in parallel to silence FLC during vernalization.

Noncoding transcription induces chromatin changes that can mediate environmental responsiveness, but the causes and consequences of these mechanisms are still unclear. Here, we investigate how antisense transcription (termed COOLAIR) interfaces with Polycomb Repressive Complex 2 (PRC2) silencing during winter-induced epigenetic regulation of Arabidopsis FLOWERING LOCUS C (FLC). We use genetic and chromatin analyses on lines ineffective or hyperactive for the antisense pathway in combination with computational modeling to define the mechanisms underlying FLC repression. Our results show that FLC is silenced through pathways that function with different dynamics: a COOLAIR transcription-mediated pathway capable of fast response and in parallel a slow PRC2 switching mechanism that maintains each allele in an epigenetically silenced state. Components of both the COOLAIR and PRC2 pathways are regulated by a common transcriptional regulator (NTL8), which accumulates by reduced dilution due to slow growth at low temperature. The parallel activities of the regulatory steps, and their control by temperature-dependent growth dynamics, create a flexible system for registering widely fluctuating natural temperature conditions that change year on year, and yet ensure robust epigenetic silencing of FLC.

The RPD3L deacetylation complex is required for facultative heterochromatin repression in Neurospora crassa.

Repression of facultative heterochromatin is essential for developmental processes in numerous organisms. Methylation of histone H3 lysine 27 (H3K27) by Polycomb repressive complex 2 is a prominent feature of facultative heterochromatin in both fungi and higher eukaryotes. Although this methylation is frequently associated with silencing, the detailed mechanism of repression remains incompletely understood. We utilized a forward genetics approach to identify genes required to maintain silencing at facultative heterochromatin genes in Neurospora crassa and identified three previously uncharacterized genes that are important for silencing: sds3 (NCU01599), rlp1 (RPD3L protein 1; NCU09007), and rlp2 (RPD3L protein 2; NCU02898). We found that SDS3, RLP1, and RLP2 associate with N. crassa homologs of the Saccharomyces cerevisiae Rpd3L complex and are required for repression of a subset of H3K27-methylated genes. Deletion of these genes does not lead to loss of H3K27 methylation but increases acetylation of histone H3 lysine 14 at up-regulated genes, suggesting that RPD3L-driven deacetylation is a factor required for silencing of facultative heterochromatin in N. crassa, and perhaps in other organisms.

COP1 controls light-dependent chromatin remodeling.

Light is a crucial environmental factor that impacts various aspects of plant development. Phytochromes, as light sensors, regulate myriads of downstream genes to mediate developmental reprogramming in response to changes in environmental conditions. CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) is an E3 ligase for a number of substrates in light signaling, acting as a central repressor of photomorphogenesis. The interplay between phytochrome B (phyB) and COP1 forms an antagonistic regulatory module that triggers extensive gene expression reprogramming when exposed to light. Here, we uncover a role of COP1 in light-dependent chromatin remodeling through the regulation of VIL1 (VIN3-LIKE 1)/VERNALIZATION 5, a Polycomb protein. VIL1 directly interacts with phyB and regulates photomorphogenesis through the formation of repressive chromatin loops at downstream growth-promoting genes in response to light. Furthermore, we reveal that COP1 governs light-dependent formation of chromatin loop and limiting a repressive histone modification to fine-tune expressions of growth-promoting genes during photomorphogenesis through VIL1.

PP2A inhibitor SET promotes mTORC1 and Bmi1 signaling through Akt activation and maintains the colony-formation ability of cancer cells.

Protein phosphatase 2A (PP2A) is an essential tumor suppressor, with its activity often hindered in cancer cells by endogenous PP2A inhibitory proteins like SE translocation (SET). SET/PP2A axis plays a pivotal role in the colony-formation ability of cancer cells and the stabilization of c-Myc and E2F1 proteins implicated in this process. However, in osteosarcoma cell line HOS, SET knock-down (KD) suppresses the colony-formation ability without affecting c-Myc and E2F1. This study aimed to unravel the molecular mechanism through which SET enhances the colony-formation ability of HOS cells and determine if it is generalized to other cancer cells. Transcriptome analysis unveiled that SET KD suppressed mTORC1 signaling. SET KD inhibited Akt phosphorylation, an upstream kinase for mTORC1. PP2A inhibitor blocked SET KD-mediated decrease in phosphorylation of Akt and a mTORC1 substrate p70S6K. A constitutively active Akt restored decreased colony-formation ability by SET KD, indicating the SET/PP2A/Akt/mTORC1 axis. Additionally, enrichment analysis highlighted that Bmi-1, a polycomb group protein, is affected by SET KD. SET KD decreased Bmi-1 protein by Akt inhibition but not by mTORC1 inhibition, and exogenous Bmi-1 expression rescued the reduced colony formation by SET KD. Four out of eight cancer cell lines exhibited decreased Bmi-1 by SET KD. Further analysis of these cell lines revealed that Myc activity plays a role in SET KD-mediated Bmi-1 degradation. These findings provide new insights into the molecular mechanism of SET-regulated colony-formation ability, which involved Akt-mediated activation of mTORC1/p70S6K and Bmi-1 signaling.

The competitive mechanism of EZH1 and EZH2 in promoting oral squamous cell carcinoma.

Enhancer of Zeste Homolog 1 (EZH1) and Enhancer of Zeste Homolog 2 (EZH2) are the key components of polycomb repressive complex 2 (PRC2); however, the roles of these proteins in oral squamous cell carcinoma (OSCC) have yet to be elucidated. In this study, we aimed to determine the respective roles of these proteins in OSCC by investigating the expression levels of EZH1 and EZH2 in OSCC tissues (N = 63) by immunohistochemistry. In addition, we used lentiviruses to construct stable OSCC cell lines that overexpressed EZH1 and EZH2. Then, we investigated these cell lines for cell viability, colony formation capacity, stemness, and epithelial-mesenchymal transition (EMT). Binding competition between EZH1 and EZH2 with PRC2 was further evaluated using Co-immunoprecipitation (Co-IP). Compared with normal tissues, the expression levels of EZH2 in OSCC tissues was up-regulated, while the expression of EZH1 was down-regulated. EZH2 enhanced cell viability, colony formation capacity, stemness, and EMT, while EZH1 did not. Furthermore, analysis indicated that EZH1 and EZH2 bound competitively to PRC2 and influenced the methylation status of H3K27. In conclusion, our findings verified that EZH1 and EZH2 play opposing roles in OSCC and that EZH1 and EZH2 compete as the key component of PRC2, thus affecting the characteristics of OSCC via the methylation of H3K27.

Identification of vernalization-related genes and cold memory element (CME) required for vernalization response in radish (Raphanus sativus L.).

Floral transition is accelerated by exposure to long-term cold like winter in plants, which is called as vernalization. Acceleration of floral transition by vernalization is observed in a diversity of biennial and perennial plants including Brassicaceae family plants. Scientific efforts to understand molecular mechanism underlying vernalization-mediated floral transition have been intensively focused in model plant Arabidopsis thaliana. To get a better understanding on floral transition by vernalization in radish (Raphanus sativus L.), we investigated transcriptomic changes taking place during vernalization in radish. Thousands of genes were differentially regulated along time course of vernalization compared to non-vernalization (NV) sample. Twelve major clusters of DEGs were identified based on distinctive expression profiles during vernalization. Radish FLC homologs were shown to exert an inhibition of floral transition when transformed into Arabidopsis plants. In addition, DNA region containing RY motifs located within a Raphanus sativus FLC homolog, RsFLC1 was found to be required for repression of RsFLC1 by vernalization. Transgenic plants harboring disrupted RY motifs were impaired in the enrichment of H3K27me3 on RsFLC1 chromatin, thus resulting in the delayed flowering in Arabidopsis. Taken together, we report transcriptomic profiles of radish during vernalization and demonstrate the requirement of RY motif for vernalization-mediated repression of RsFLC homologs in radish (Raphanus sativus L.).

Nucleocytoplasmic ß-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 ß-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 ß-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 ß-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.

Polycomb group proteins confer robustness to aposematic coloration in the milkweed bug, Oncopeltus fasciatus.

Aposematic coloration offers an opportunity to explore the molecular mechanisms underlying canalization. In this study, the role of epigenetic regulation underlying robustness was explored in the aposematic coloration of the milkweed bug, Oncopeltus fasciatus. Polycomb (Pc) and Enhancer of zeste (E(z)), which encode components of the Polycomb repressive complex 1 (PRC1) and PRC2, respectively, and jing, which encodes a component of the PRC2.2 subcomplex, were knocked down in the fourth instar of O. fasciatus. Knockdown of these genes led to alterations in scutellar morphology and melanization. In particular, when Pc was knocked down, the adults developed a highly melanized abdomen, head and forewings at all temperatures examined. In contrast, the E(z) and jing knockdown led to increased plasticity of the dorsal forewing melanization across different temperatures. Moreover, jing knockdown adults exhibited increased plasticity in the dorsal melanization of the head and the thorax. These observations demonstrate that histone modifiers may play a key role during the process of canalization to confer robustness in the aposematic coloration.

Sustained inactivation of the Polycomb PRC1 complex induces DNA repair defects and genomic instability in epigenetic tumors.

Cancer initiation and progression are typically associated with the accumulation of driver mutations and genomic instability. However, recent studies demonstrated that cancer can also be driven purely by epigenetic alterations, without driver mutations. Specifically, a 24-h transient downregulation of polyhomeotic (ph-KD), a core component of the Polycomb complex PRC1, is sufficient to induce epigenetically initiated cancers (EICs) in Drosophila, which are proficient in DNA repair and characterized by a stable genome. Whether genomic instability eventually occurs when PRC1 downregulation is performed for extended periods of time remains unclear. Here, we show that prolonged depletion of PH, which mimics cancer initiating events, results in broad dysregulation of DNA replication and repair genes, along with the accumulation of DNA breaks, defective repair, and widespread genomic instability in the cancer tissue. A broad misregulation of H2AK118 ubiquitylation and to a lesser extent of H3K27 trimethylation also occurs and might contribute to these phenotypes. Together, this study supports a model where DNA repair and replication defects accumulate during the tumorigenic transformation epigenetically induced by PRC1 loss, resulting in genomic instability and cancer progression.

Biallelic loss-of-function variants of EZH1 cause a novel developmental disorder with central precocious puberty.

Pathogenic variants of polycomb repressive complex-2 (PRC2) subunits are associated with overgrowth syndromes and neurological diseases. EZH2 is a major component of PRC2 and mediates the methylation of H3K27 trimethylation (H3K27me3). Germline variants of EZH2 have been identified as a cause of Weaver syndrome (WS), an overgrowth/intellectual disability (OGID) syndrome characterized by overgrowth, macrocephaly, accelerated bone age, intellectual disability (ID), and characteristic facial features. Germline variants of SUZ12 and EED, other components of PRC2, have also been reported in the WS or Weaver-like syndrome. EZH1 is a homolog of EZH2 that interchangeably associates with SUZ12 and EED. Recently, pathogenic variants of EZH1 have been reported in individuals with dominant and recessive neurodevelopmental disorders. We herein present sisters with biallelic loss-of-function variants of EZH1. They showed developmental delay, ID, and central precocious puberty, but not the features of WS or other OGID syndromes.