Discovery and Characterization of a Novel Cereblon-Recruiting PRC1 Bridged PROTAC Degrader.

Bridged PROTAC is a novel protein complex degrader strategy that exploits the target protein's binding partner to degrade undruggable proteins by inducing proximity to an E3 ubiquitin ligase. In this study, we discovered for the first time that cereblon (CRBN) can be employed for the bridged PROTAC approach and report the first-in-class CRBN-recruiting and EED-binding polycomb repressive complex 1 (PRC1) degrader, compound 1 (MS181). We show that 1 induces preferential degradation of PRC1 components, BMI1 and RING1B, in an EED-, CRBN-, and ubiquitin-proteosome system (UPS)-dependent manner. Compound 1 also has superior antiproliferative activity in multiple metastatic cancer cell lines over EED-binding PRC2 degraders and can be efficacious in VHL-defective cancer cells. Altogether, compound 1 is a valuable chemical biology tool to study the role of PRC1 in cancer. Importantly, we show that CRBN can be utilized to develop bridged PROTACs, expanding the bridged PROTAC technology for degrading undruggable proteins.

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.

Polycomb repression of Hox genes involves spatial feedback but not domain compaction or phase transition.

Polycomb group proteins have a critical role in silencing transcription during development. It is commonly proposed that Polycomb-dependent changes in genome folding, which compact chromatin, contribute directly to repression by blocking the binding of activating complexes. Recently, it has also been argued that liquid-liquid demixing of Polycomb proteins facilitates this compaction and repression by phase-separating target genes into a membraneless compartment. To test these models, we used Optical Reconstruction of Chromatin Architecture to trace the Hoxa gene cluster, a canonical Polycomb target, in thousands of single cells. Across multiple cell types, we find that Polycomb-bound chromatin frequently explores decompact states and partial mixing with neighboring chromatin, while remaining uniformly repressed, challenging the repression-by-compaction or phase-separation models. Using polymer simulations, we show that these observed flexible ensembles can be explained by 'spatial feedback'-transient contacts that contribute to the propagation of the epigenetic state (epigenetic memory), without inducing a globular organization.

Polycomb-mediated histone modifications and gene regulation.

Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) are transcriptional repressor complexes that play a fundamental role in epigenomic regulation and the cell-fate decision; these complexes are widely conserved in multicellular organisms. PRC1 is an E3 ubiquitin (ub) ligase that generates histone H2A ubiquitinated at lysine (K) 119 (H2AK119ub1), whereas PRC2 is a histone methyltransferase that specifically catalyzes tri-methylation of histone H3K27 (H3K27me3). Genome-wide analyses have confirmed that these two key epigenetic marks highly overlap across the genome and contribute to gene repression. We are now beginning to understand the molecular mechanisms that enable PRC1 and PRC2 to identify their target sites in the genome and communicate through feedback mechanisms to create Polycomb chromatin domains. Recently, it has become apparent that PRC1-induced H2AK119ub1 not only serves as a docking site for PRC2 but also affects the dynamics of the H3 tail, both of which enhance PRC2 activity, suggesting that trans-tail communication between H2A and H3 facilitates the formation of the Polycomb chromatin domain. In this review, we discuss the emerging principles that define how PRC1 and PRC2 establish the Polycomb chromatin domain and regulate gene expression in mammals.

YTHDF1 facilitates PRC1-mediated H2AK119ub in human ES cells.

Polycomb repressive complexes (PRCs) play critical roles in cell fate decisions during normal development as well as disease progression through mediating histone modifications such as H3K27me3 and H2AK119ub. How exactly PRCs recruited to chromatin remains to be fully illuminated. Here, we report that YTHDF1, the N6-methyladenine (m6 A) RNA reader that was previously known to be mainly cytoplasmic, associates with RNF2, a PRC1 protein that mediates H2AK119ub in human embryonic stem cells (hESCs). A portion of YTHDF1 localizes in the nuclei and associates with RNF2/H2AK119ub on a subset of gene loci related to neural development functions. Knock-down YTHDF1 attenuates H2AK119ub modification on these genes and promotes neural differentiation in hESCs. Our findings provide a noncanonical mechanism that YTHDF1 participates in PRC1 functions in hESCs.

Balanced Epigenetic Regulation of MHC Class I Expression in Tumor Cells by the Histone Ubiquitin Modifiers BAP1 and PCGF1.

MHC class I (MHC-I) molecules are critical for CD8+ T cell responses to viral infections and malignant cells, and tumors can downregulate MHC-I expression to promote immune evasion. In this study, using a genome-wide CRISPR screen on a human melanoma cell line, we identified the polycomb repressive complex 1 (PRC1) subunit PCGF1 and the deubiquitinating enzyme BAP1 as opposite regulators of MHC-I transcription. PCGF1 facilitates deposition of ubiquitin at H2AK119 at the MHC-I promoters to silence MHC-I, whereas BAP1 removes this modification to restore MHC-I expression. PCGF1 is widely expressed in tumors and its depletion increased MHC-I expression in multiple tumor lines, including MHC-Ilow tumors. In cells characterized by poor MHC-I expression, PRC1 and PRC2 act in parallel to impinge low transcription. However, PCGF1 depletion was sufficient to increase MHC-I expression and restore T cell-mediated killing of the tumor cells. Taken together, our data provide an additional layer of regulation of MHC-I expression in tumors: epigenetic silencing by PRC1 subunit PCGF1.

Transcriptional regulation of amino acid metabolism by KDM2B, in the context of ncPRC1.1 and in concert with MYC and ATF4.

INTRODUCTION: KDM2B encodes a JmjC domain-containing histone lysine demethylase, which functions as an oncogene in several types of tumors, including TNBC. This study was initiated to address the cancer relevance of the results of our earlier work, which had shown that overexpression of KDM2B renders mouse embryonic fibroblasts (MEFs) resistant to oxidative stress by regulating antioxidant mechanisms. METHODS: We mainly employed a multi-omics strategy consisting of RNA-Seq, quantitative TMT proteomics, Mass-spectrometry-based global metabolomics, ATAC-Seq and ChIP-seq, to explore the role of KDM2B in the resistance to oxidative stress and intermediary metabolism. These data and data from existing patient datasets were analyzed using bioinformatic tools, including exon-intron-split analysis (EISA), FLUFF and clustering analyses. The main genetic strategy we employed was gene silencing with shRNAs. ROS were measured by flow cytometry, following staining with CellROX and various metabolites were measured with biochemical assays, using commercially available kits. Gene expression was monitored with qRT-PCR and immunoblotting, as indicated. RESULTS: The knockdown of KDM2B in basal-like breast cancer cell lines lowers the levels of GSH and sensitizes the cells to ROS inducers, GSH targeting molecules, and DUB inhibitors. To address the mechanism of GSH regulation, we knocked down KDM2B in MDA-MB-231 cells and we examined the effects of the knockdown, using a multi-omics strategy. The results showed that KDM2B, functioning in the context of ncPRC1.1, regulates a network of epigenetic and transcription factors, which control a host of metabolic enzymes, including those involved in the SGOC, glutamate, and GSH metabolism. They also showed that KDM2B enhances the chromatin accessibility and expression of MYC and ATF4, and that it binds in concert with MYC and ATF4, the promoters of a large number of transcriptionally active genes, including many, encoding metabolic enzymes. Additionally, MYC and ATF4 binding sites were enriched in genes whose accessibility depends on KDM2B, and analysis of a cohort of TNBCs expressing high or low levels of KDM2B, but similar levels of MYC and ATF4 identified a subset of MYC targets, whose expression correlates with the expression of KDM2B. Further analyses of basal-like TNBCs in the same cohort, revealed that tumors expressing high levels of all three regulators exhibit a distinct metabolic signature that carries a poor prognosis. CONCLUSIONS: The present study links KDM2B, ATF4, and MYC in a transcriptional network that regulates the expression of multiple metabolic enzymes, including those that control the interconnected SGOC, glutamate, and GSH metabolic pathways. The co-occupancy of the promoters of many transcriptionally active genes, by all three factors, the enrichment of MYC binding sites in genes whose chromatin accessibility depends on KDM2B, and the correlation of the levels of KDM2B with the expression of a subset of MYC target genes in tumors that express similar levels of MYC, suggest that KDM2B regulates both the expression and the transcriptional activity of MYC. Importantly, the concerted expression of all three factors also defines a distinct metabolic subset of TNBCs with poor prognosis. Overall, this study identifies novel mechanisms of SGOC regulation, suggests novel KDM2B-dependent metabolic vulnerabilities in TNBC, and provides new insights into the role of KDM2B in the epigenetic regulation of transcription.

PRC1 directs PRC2-H3K27me3 deposition to shield adult spermatogonial stem cells from differentiation.

Spermatogonial stem cells functionality reside in the slow-cycling and heterogeneous undifferentiated spermatogonia cell population. This pool of cells supports lifelong fertility in adult males by balancing self-renewal and differentiation to produce haploid gametes. However, the molecular mechanisms underpinning long-term stemness of undifferentiated spermatogonia during adulthood remain unclear. Here, we discover that an epigenetic regulator, Polycomb repressive complex 1 (PRC1), shields adult undifferentiated spermatogonia from differentiation, maintains slow cycling, and directs commitment to differentiation during steady-state spermatogenesis in adults. We show that PRC2-mediated H3K27me3 is an epigenetic hallmark of adult undifferentiated spermatogonia. Indeed, spermatogonial differentiation is accompanied by a global loss of H3K27me3. Disruption of PRC1 impairs global H3K27me3 deposition, leading to precocious spermatogonial differentiation. Therefore, PRC1 directs PRC2-H3K27me3 deposition to maintain the self-renewing state of undifferentiated spermatogonia. Importantly, in contrast to its role in other tissue stem cells, PRC1 negatively regulates the cell cycle to maintain slow cycling of undifferentiated spermatogonia. Our findings have implications for how epigenetic regulators can be tuned to regulate the stem cell potential, cell cycle and differentiation to ensure lifelong fertility in adult males.

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.

Decoding the interaction between miR-19a and CBX7 focusing on the implications for tumor suppression in cancer therapy.

Cancer is a complex and multifaceted disease characterized by uncontrolled cell growth, genetic alterations, and disruption of normal cellular processes, leading to the formation of malignant tumors with potentially devastating consequences for patients. Molecular research is important in the diagnosis and treatment, one of the molecular mechanisms involved in various cancers is the fluctuation of gene expression. Non-coding RNAs, especially microRNAs, are involved in different stages of cancer. MicroRNAs are small RNA molecules that are naturally produced within cells and bind to the 3'-UTR of target mRNA, repressing gene expression by regulating translation. Overexpression of miR-19a has been reported in human malignancies. Upregulation of miR-19a as a member of the miR-17-92 cluster is key to tumor formation, cell proliferation, survival, invasion, metastasis, and drug resistance. Furthermore. bioinformatics and in vitro data reveal that the miR-19a-3p isoform binds to the 3'UTR of CBX7 and was identified as the miR-19a-3p target gene. CBX7 is known as a tumor suppressor. This review initially describes the regulation of mir-19a in multiple cancers. Accordingly, the roles of miR-19 in affecting its target gene expression CBX7 in carcinoma also be discussed.

Prognostic hub gene CBX2 drives a cancer stem cell-like phenotype in HCC revealed by multi-omics and multi-cohorts.

Hepatocellular carcinoma (HCC) is a malignant tumor with a high prevalence and fatality rate. CBX2 has been demonstrated to impact the development and advancement of various cancers, albeit it has received limited attention in relation to HCC. In this study, CBX2 and CEP55 were screened out with the refined triple regulatory networks constructed by total RNA-seq datasets (TCGA-LIHC, GSE140845) and a robust prognostic model. Aberrantly higher expression levels of CBX2 and CEP55 in HCC may be caused by CNV alterations, promoter hypo-methylation, open chromatin accessibility, and greater active marks such as H3K4me3, H3K4me1, and H3K27ac. Functionally, CBX2, which was highly correlated with CD44, shaped a cancer stem cell-like phenotype by positively regulating cell-cycle progression, proliferation, invasion, metastasis, wound healing, and radiation resistance, revealed by combining bulk RNA-seq and scRNA-seq datasets. CBX2 knockdown validated its role in affecting the cell cycle. Importantly, we revealed CBX2 could activate gene by cooperating with co-regulators or not rather than a recognizer of the repressive mark H3K27me3. For instance, we uncovered CBX2 bound to promoter of CTNNB1 and CEP55 to augment their expressions. CBX2 showed a highly positive correlation with CEP55 at pan-cancer level. In addition, CBX2 and CEP55 may enhance extracellular matrix reprograming via cancer-associated fibroblast. Surprisingly, patients with high expression of CBX2 or CEP55 exhibited a higher response to immunotherapy, indicating that CBX2 and CEP55 may be promising therapeutic targets for HCC patients.

Phosphorylation of USP27X by GSK3ß maintains the stability and oncogenic functions of CBX2.

Chromobox protein homolog 2 (CBX2) exerts a multifaceted impact on the progression of aggressive cancers. The proteasome-dependent pathway is crucial for modulating CBX2 regulation, while the specific regulatory roles and mechanisms of deubiquitinating enzymes targeting CBX2 remain poorly understood. Mass spectrometry analysis identified ubiquitin-specific peptidase 27X (USP27X) as a deubiquitinating enzyme that targets CBX2. Overexpression of USP27X significantly enhances CBX2 levels by promoting deubiquitination, while deficiency of USP27X leads to CBX2 degradation, thereby inhibiting tumorigenesis. Furthermore, it has been revealed that glycogen synthase kinase 3 beta (GSK3ß) can directly bind to and phosphorylate USP27X, thereby enhancing the interaction between USP27X and CBX2 and leading to further stabilization of the CBX2 protein. Clinically, the co-expression of high levels of USP27X and CBX2 in breast cancer tissues is indicative of a poor prognosis for patients with this disease. These findings collectively underscore the critical regulatory role played by USP27X in modulating CBX2, thereby establishing the GSK3ß-USP27X-CBX2 axis as a pivotal driver of malignant progression in breast cancer.

Augmenting L3MBTL2-induced condensates suppresses tumor growth in osteosarcoma.

Osteosarcoma is a highly aggressive cancer and lacks effective therapeutic targets. We found that L3MBTL2 acts as a tumor suppressor by transcriptionally repressing IFIT2 in osteosarcoma. L3MBTL2 recruits the components of Polycomb repressive complex 1.6 to form condensates via both Pho-binding pockets and polybasic regions within carboxyl-terminal intrinsically disordered regions; the L3MBTL2-induced condensates are required for its tumor suppression. Multi-monoubiquitination of L3MBTL2 by UBE2O results in its proteasomal degradation, and the UBE2O/L3MBTL2 axis was crucial for osteosarcoma growth. There is a reverse correlation between L3MBTL2 and UBE2O in osteosarcoma tissues, and higher UBE2O and lower L3MBTL2 are associated with poorer prognosis in osteosarcoma. Pharmacological blockage of UBE2O by arsenic trioxide can enhance L3MBTL2-induced condensates and consequently suppress osteosarcoma growth. Our findings unveil a crucial biological function of L3MBTL2-induced condensates in mediating tumor suppression, proposing the UBE2O-L3MBTL2 axis as a potential cancer therapeutic target in osteosarcoma.

Uncoupled evolution of the Polycomb system and deep origin of non-canonical PRC1.

Polycomb group proteins, as part of the Polycomb repressive complexes, are essential in gene repression through chromatin compaction by canonical PRC1, mono-ubiquitylation of histone H2A by non-canonical PRC1 and tri-methylation of histone H3K27 by PRC2. Despite prevalent models emphasizing tight functional coupling between PRC1 and PRC2, it remains unclear whether this paradigm indeed reflects the evolution and functioning of these complexes. Here, we conduct a comprehensive analysis of the presence or absence of cPRC1, nPRC1 and PRC2 across the entire eukaryotic tree of life, and find that both complexes were present in the Last Eukaryotic Common Ancestor (LECA). Strikingly, ~42% of organisms contain only PRC1 or PRC2, showing that their evolution since LECA is largely uncoupled. The identification of ncPRC1-defining subunits in unicellular relatives of animals and fungi suggests ncPRC1 originated before cPRC1, and we propose a scenario for the evolution of cPRC1 from ncPRC1. Together, our results suggest that crosstalk between these complexes is a secondary development in evolution.

Functional dissection of PRC1 subunits RYBP and YAF2 during neural differentiation of embryonic stem cells.

Polycomb repressive complex 1 (PRC1) comprises two different complexes: CBX-containing canonical PRC1 (cPRC1) and RYBP/YAF2-containing variant PRC1 (vPRC1). RYBP-vPRC1 or YAF2-vPRC1 catalyzes H2AK119ub through a positive-feedback model; however, whether RYBP and YAF2 have different regulatory functions is still unclear. Here, we show that the expression of RYBP and YAF2 decreases and increases, respectively, during neural differentiation of embryonic stem cells (ESCs). Rybp knockout impairs neural differentiation by activating Wnt signaling and derepressing nonneuroectoderm-associated genes. However, Yaf2 knockout promotes neural differentiation and leads to redistribution of RYBP binding, increases enrichment of RYBP and H2AK119ub on the RYBP-YAF2 cotargeted genes, and prevents ectopic derepression of nonneuroectoderm-associated genes in neural-differentiated cells. Taken together, this study reveals that RYBP and YAF2 function differentially in regulating mESC neural differentiation.

BMI1 Silencing Liposomes Suppress Postradiotherapy Cancer Stemness against Radioresistant Hepatocellular Carcinoma.

Radiotherapy causes DNA damage by direct ionization and indirect generation of reactive oxygen species (ROS) thereby destroying cancer cells. However, ionizing radiation (IR) unexpectedly elicits metastasis and invasion of cancer cells by inducing cancer stem cells' (CSCs) properties. As BMI1 is a crucial gene that causes radioresistance and an unfavorable prognosis of hepatocellular carcinoma (HCC), BMI1 inhibitor PTC-209 has been encapsulated in a ROS-responsive liposome (LP(PTC-209)) to be temporally and spatially delivered to radioresistant HCC tissue. The ROS generated during IR was not only considered to directly cause tumor cell death but also be used as a stimulator to trigger ROS-responsive drug release from LP(PTC-209). The PTC-209 released into resistant HCC tissue under radiotherapy further led to cancer stem cell (CSC) differentiation and then recovered radiosensitivity of HCC tumor. The suppression of the radioresistant performance of LP(PTC-209) has been proved on radiosensitive and radioresistant Hepa1-6 CSC tumor models, respectively. Our study clarified the relationship between radiotherapy and cancer stemness and provided insights to achieve complete suppression of radioresistant HCC tumor by inhibiting cancer stemness.

DNA elements tether canonical Polycomb Repressive Complex 1 to human genes.

Development of multicellular animals requires epigenetic repression by Polycomb group proteins. The latter assemble in multi-subunit complexes, of which two kinds, Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2), act together to repress key developmental genes. How PRC1 and PRC2 recognize specific genes remains an open question. Here we report the identification of several hundreds of DNA elements that tether canonical PRC1 to human developmental genes. We use the term tether to describe a process leading to a prominent presence of canonical PRC1 at certain genomic sites, although the complex is unlikely to interact with DNA directly. Detailed analysis indicates that sequence features associated with PRC1 tethering differ from those that favour PRC2 binding. Throughout the genome, the two kinds of sequence features mix in different proportions to yield a gamut of DNA elements that range from those tethering predominantly PRC1 or PRC2 to ones capable of tethering both complexes. The emerging picture is similar to the paradigmatic targeting of Polycomb complexes by Polycomb Response Elements (PREs) of Drosophila but providing for greater plasticity.

M phase-specific interaction between SBDS and RNF2 at the mitotic spindles regulates mitotic progression.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive inherited disorder caused by biallelic mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SBDS protein is involved in ribosome biogenesis; therefore SDS is classified as a ribosomopathy. SBDS is localized at mitotic spindles and stabilizes microtubules. Previously, we showed that SBDS interacts with ring finger protein 2 (RNF2) and is degraded through RNF2-dependent ubiquitination. In this study, we investigated when and where SBDS interacts with RNF2 and the effects of the interaction on cells. We found that SBDS co-localized with RNF2 on centrosomal microtubules in the mitotic phase (M phase), whereas SBDS and RNF2 localized to the nucleolus and nucleoplasm in the interphase, respectively. The microtubule-binding assay revealed that SBDS interacted directly with microtubules and RNF2 interacted with SBDS bound to microtubules. In addition, SBDS was ubiquitinated and degraded by RNF2 during the M phase. Moreover, RNF2 overexpression accelerated mitotic progression. These findings suggest that SBDS delays mitotic progression, and RNF2 releases cells from suppression through the ubiquitination and subsequent degradation of SBDS. The interaction between SBDS and RNF2 at mitotic spindles might be involved in mitotic progression as a novel regulatory cascade.