mSWI/SNF promotes Polycomb repression both directly and through genome-wide redistribution.

The mammalian SWI/SNF complex, or BAF complex, has a conserved and direct role in antagonizing Polycomb-mediated repression. Yet, BAF also promotes repression by Polycomb in stem cells and cancer. How BAF both antagonizes and promotes Polycomb-mediated repression remains unknown. Here, we utilize targeted protein degradation to dissect the BAF-Polycomb axis in mouse embryonic stem cells on short timescales. We report that rapid BAF depletion redistributes Polycomb repressive complexes PRC1 and PRC2 from highly occupied domains, like Hox clusters, to weakly occupied sites normally opposed by BAF. Polycomb redistribution from highly repressed domains results in their decompaction, gain of active epigenomic features and transcriptional derepression. Surprisingly, through dose-dependent degradation of PRC1 and PRC2, we identify a conventional role for BAF in Polycomb-mediated repression, in addition to global Polycomb redistribution. These findings provide new mechanistic insight into the highly dynamic state of the Polycomb-Trithorax axis.

Microarray expression profile analysis of circular RNAs and their potential regulatory role in bladder carcinoma.

Dysregulated circular RNAs (circRNAs) often contribute to the occurrence and development of various tumors; however, the function and mechanism of circRNAs are largely unknown in human bladder cancer (BC). In the present study, dysregulated circRNAs between BC and adjacent non­neoplastic bladder tissues were analyzed by circRNA microarray. We randomly selected 10 upregulated and five downregulated circRNAs for validation by quantitative real­time PCR. Bioinformatics analysis was further conducted to investigate the potential function of these differentially expressed circRNAs, with the differential expression of hsa_circRNA_100876, mir­136­5p, and mRNA­chromobox 4 (CBX4) subsequently verified. A total of 512 differentially expressed circRNAs were identified after scanning and normalization (340 upregulated and 172 downregulated circRNAs), with pathway and Gene Ontology analyses revealing their association with multiple significant cancer pathways. Construction of a circRNA­microRNA­mRNA network suggested additional potential roles of these circRNAs. The expression of hsa_circRNA_100876 and CBX4 was significantly negatively correlated with the expression of miR­136­5p. Additionally, hsa_circRNA_100876 was highly positively correlated with CBX4 expression. The results revealed that hsa_circRNA_100876 inhibition suppressed BC cell proliferation and it was associated with advanced T stage and lymphatic metastasis, and poor overall survival of BC patients. In conclusion, these differentially expressed circRNAs offer novel insights into potential biological markers or new therapeutic targets for the treatment of BC. Furthermore, hsa_circRNA_100876 may increase the expression of CBX4 by competing with miR­136­5p, ultimately promoting the malignant biological behavior of BC. Aberrantly expressed hsa_circRNA_100876 could be used as a potential non­invasive biomarker for the early detection and screening of BC.

Maintenance of Nucleolar Homeostasis by CBX4 Alleviates Senescence and Osteoarthritis.

CBX4, a component of polycomb repressive complex 1 (PRC1), plays important roles in the maintenance of cell identity and organ development through gene silencing. However, whether CBX4 regulates human stem cell homeostasis remains unclear. Here, we demonstrate that CBX4 counteracts human mesenchymal stem cell (hMSC) aging via the maintenance of nucleolar homeostasis. CBX4 protein is downregulated in aged hMSCs, whereas CBX4 knockout in hMSCs results in destabilized nucleolar heterochromatin, enhanced ribosome biogenesis, increased protein translation, and accelerated cellular senescence. CBX4 maintains nucleolar homeostasis by recruiting nucleolar protein fibrillarin (FBL) and heterochromatin protein KRAB-associated protein 1 (KAP1) at nucleolar rDNA, limiting the excessive expression of rRNAs. Overexpression of CBX4 alleviates physiological hMSC aging and attenuates the development of osteoarthritis in mice. Altogether, our findings reveal a critical role of CBX4 in counteracting cellular senescence by maintaining nucleolar homeostasis, providing a potential therapeutic target for aging-associated disorders.

Deregulated Polycomb functions in myeloproliferative neoplasms.

Polycomb proteins function in the maintenance of gene silencing via post-translational modifications of histones and chromatin compaction. Genetic and biochemical studies have revealed that the repressive function of Polycomb repressive complexes (PRCs) in transcription is counteracted by the activating function of Trithorax-group complexes; this balance fine-tunes the expression of genes critical for development and tissue homeostasis. The function of PRCs is frequently dysregulated in various cancer cells due to altered expression or recurrent somatic mutations in PRC genes. The tumor suppressive functions of EZH2-containing PRC2 and a PRC2-related protein ASXL1 have been investigated extensively in the pathogenesis of hematological malignancies, including myeloproliferative neoplasms (MPN). BCOR, a component of non-canonical PRC1, suppresses various hematological malignancies including MPN. In this review, we focus on recent findings on the role of PRCs in the pathogenesis of MPN and the therapeutic impact of targeting the pathological functions of PRCs in MPN.

CBX6 is negatively regulated by EZH2 and plays a potential tumor suppressor role in breast cancer.

Chromobox 6 (CBX6) is a subunit of Polycomb Repressive Complex 1 (PRC1) that mediates epigenetic gene repression and acts as an oncogene or tumor suppressor in a cancer type-dependent manner. The specific function of CBX6 in breast cancer is currently undefined. In this study, a comprehensive analysis of The Cancer Genome Atlas (TCGA) dataset led to the identification of CBX6 as a consistently downregulated gene in breast cancer. We provided evidence showing enhancer of zeste homolog 2 (EZH2) negatively regulated CBX6 expression in a Polycomb Repressive Complex 2 (PRC2)-dependent manner. Exogenous overexpression of CBX6 inhibited cell proliferation and colony formation, and induced cell cycle arrest along with suppression of migration and invasion of breast cancer cells in vitro. Microarray analyses revealed that CBX6 governs a complex gene expression program. Moreover, CBX6 induced significant downregulation of bone marrow stromal cell antigen-2 (BST2), a potential therapeutic target, via interactions with its promoter region. Our collective findings support a tumor suppressor role of CBX6 in breast cancer.

Emerging role of noncanonical polycomb repressive complexes in normal and malignant hematopoiesis.

Polycomb group (PcG) proteins are the key epigenetic regulators of normal hematopoiesis and the dysregulation of their functions is closely involved in the pathogenesis of hematological malignancies. These proteins function in the multimeric complexes called polycomb repressive complex (PRC) 1 and 2. In addition to canonical PRC1, four noncanonical PRC1 complexes have been identified. In contrast to canonical PRC1, which is recruited to its target sites in a manner dependent on H3K27me3, noncanonical PRC1 complexes are recruited to their target sites independently of H3K27me3. Among them, PRC1.1, consisting of PCGF1, RING1A/B, KDM2B, and BCL6 corepressor (BCOR) or BCLRL1, regulates diverse biological processes, including pluripotency, reprogramming, and hematopoiesis. PRC1.1 has been implicated in myelopoiesis and lymphopoiesis and is targeted by somatic gene mutations in various hematological malignancies. These findings revealed the more complex regulation of epigenetic cellular memory by PcG proteins than we expected and propose PRC1.1 as a novel therapeutic target in hematological malignancies.

PHD finger protein 1 (PHF1) is a novel reader for histone H4R3 symmetric dimethylation and coordinates with PRMT5-WDR77/CRL4B complex to promote tumorigenesis.

Histone post-translational modifications regulate chromatin structure and function largely through interactions with effector proteins that often contain multiple histone-binding domains. PHF1 [plant homeodomain (PHD) finger protein 1], which contains two kinds of histone reader modules, a Tudor domain and two PHD fingers, is an essential factor for epigenetic regulation and genome maintenance. While significant progress has been made in characterizing the function of the Tudor domain, the roles of the two PHD fingers are poorly defined. Here, we demonstrated that the N-terminal PHD finger of PHF1 recognizes symmetric dimethylation of H4R3 (H4R3me2s) catalyzed by PRMT5-WDR77. However, the C-terminal PHD finger of PHF1, instead of binding to modified histones, directly interacts with DDB1, the main component of the CUL4B-Ring E3 ligase complex (CRL4B), which is responsible for H2AK119 mono-ubiquitination (H2AK119ub1). We showed that PHF1, PRMT5-WDR77, and CRL4B reciprocally interact with one another and collaborate as a functional unit. Genome-wide analysis of PHF1/PRMT5/CUL4B targets identified a cohort of genes including E-cadherin and FBXW7, which are critically involved in cell growth and migration. We demonstrated that PHF1 promotes cell proliferation, invasion, and tumorigenesis in vivo and in vitro and found that its expression is markedly upregulated in a variety of human cancers. Our data identified a new reader for H4R3me2s and provided a molecular basis for the functional interplay between histone arginine methylation and ubiquitination. The results also indicated that PHF1 is a key factor in cancer progression, supporting the pursuit of PHF1 as a target for cancer therapy.

Signalling crosstalk during early tumorigenesis in the absence of Polycomb silencing.

In response to stress and injury a coordinated activation of conserved signalling modules, such as JNK and JAK/STAT, is critical to trigger regenerative tissue restoration. While these pathways rebuild homeostasis and promote faithful organ recovery, it is intriguing that they also become activated in various tumour conditions. Therefore, it is crucial to understand how similar pathways can achieve context-dependent functional outputs, likely depending on cellular states. Compromised chromatin regulation, upon removal of the Polycomb group member polyhomeotic, leads to tumour formation with ectopic activation of JNK signalling, mediated by egr/grnd, in addition to JAK/STAT and Notch. Employing quantitative analyses, we show that blocking ectopic signalling impairs ph tumour growth. Furthermore, JAK/STAT functions in parallel to JNK, while Notch relies on JNK. Here, we reveal a signalling hierarchy in ph tumours that is distinct from the regenerative processes regulated by these pathways. Absence of ph renders a permissive state for expression of target genes, but our results suggest that both loss of repression and the presence of activators may collectively regulate gene expression during tumorigenesis. Further dissecting the effect of signalling, developmental or stress-induced factors will thus elucidate the regulation of physiological responses and the contribution of context-specific cellular states.

[The biological complexity of Polycomb group proteins: the case of EZH2]. / Au cœur d'une complexité biologique - EZH2, une protéine du groupe Polycomb.

Polycomb Group proteins (PcG) are repressive epigenetic factors essential for development and involved in numerous cancer processes, yet their modes of action and recruitment to specific genomic loci are not fully understood. Recently, it has been shown that the PcG protein recruitment is a dynamic process, contrary to what was foreseen in the initial hierarchical model. In addition, EZH2, a key PcG protein, can be associated to transcribed genes, challenging the former function of PcG proteins as transcriptional repressors. Furthermore, the dual role of EZH2, which can act as an oncogene or a tumor suppressor depending on the cellular type, illustrates the functional complexity of PcG proteins.

Epigenetics of Huntington's Disease.

Huntington's disease (HD) is a genetic, fatal autosomal dominant neurodegenerative disorder typically occurring in midlife with symptoms ranging from chorea, to dementia, to personality disturbances (Philos Trans R Soc Lond Ser B Biol Sci 354:957-961, 1999). HD is inherited in a dominant fashion, and the underlying mutation in all cases is a CAG trinucleotide repeat expansion within exon 1 of the HD gene (Cell 72:971-983, 1993). The expanded CAG repeat, translated into a lengthened glutamine tract at the amino terminus of the huntingtin protein, affects its structural properties and functional activities. The effects are pleiotropic, as huntingtin is broadly expressed in different cellular compartments (i.e., cytosol, nucleus, mitochondria) as well as in all cell types of the body at all developmental stages, such that HD pathogenesis likely starts at conception and is a lifelong process (Front Neurosci 9:509, 2015). The rate-limiting mechanism(s) of neurodegeneration in HD still remains elusive: many different processes are commonly disrupted in HD cell lines and animal models, as well as in HD patient cells (Eur J Neurosci 27:2803-2820, 2008); however, epigenetic-chromatin deregulation, as determined by the analysis of DNA methylation, histone modifications, and noncoding RNAs, has now become a prevailing feature. Thus, the overarching goal of this chapter is to discuss the current status of the literature, reviewing how an aberrant epigenetic landscape can contribute to altered gene expression and neuronal dysfunction in HD.

Cbx8 Acts Non-canonically with Wdr5 to Promote Mammary Tumorigenesis.

Chromatin-mediated processes influence the development and progression of breast cancer. Using murine mammary carcinoma-derived tumorspheres as a functional readout for an aggressive breast cancer phenotype, we performed a loss-of-function screen targeting 60 epigenetic regulators. We identified the Polycomb protein Cbx8 as a key regulator of mammary carcinoma both in vitro and in vivo. Accordingly, Cbx8 is overexpressed in human breast cancer and correlates with poor survival. Our genomic analyses revealed that Cbx8 positively regulates Notch signaling by maintaining H3K4me3 levels on Notch-network gene promoters. Ectopic expression of Notch1 partially rescues tumorsphere formation in Cbx8-depleted cells. We find that Cbx8 associates with non-PRC1 complexes containing the H3K4 methyltransferase complex component WDR5, which together regulate Notch gene expression. Thus, our study implicates a key non-canonical role for Cbx8 in promoting breast tumorigenesis.

The roles of Polycomb group proteins in hematopoietic stem cells and hematological malignancies.

Polycomb group (PcG) proteins are epigenetic regulatory factors that maintain the repression of target gene expression through histone modification. PcG proteins control the repression of genes that regulate differentiation and the cell cycle in the maintenance of hematopoietic stem cells (HSC). Moreover, abnormalities in expression level and mutations in PcG genes have been reported in various types of cancer, including hematological malignancies. In this review, we present an overview of the roles of PcG proteins in HSC and various types of hematological malignancies.

The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function.

Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation.

Long Non-coding RNA ANRIL and Polycomb in Human Cancers and Cardiovascular Disease.

The long non-coding RNA CDKN2B-AS1, commonly referred to as the A ntisense N on-coding R NA in the I NK4 L ocus (ANRIL), is a 3.8-kb-long RNA transcribed from the short arm of human chromosome 9 on p21.3 that overlaps a critical region encompassing three major tumor suppressor loci juxtaposed to the INK4b-ARF-INK4a gene cluster and the methyl-thioadenosine phosphorylase (MTAP) gene. Genome-wide association studies have identified this region with a remarkable and growing number of disease-associated DNA alterations and single nucleotide polymorphisms, which corresponds to increased susceptibility to human disease. Recent attention has been devoted on whether these alterations in the ANRIL sequence affect its expression levels and/or its splicing transcript variation, and in consequence, global cellular homeostasis. Moreover, recent evidence postulates that ANRIL not only can regulate their immediate genomic neighbors in cis, but also has the capacity to regulate additional loci in trans. This action would further increase the complexity for mechanisms imposed through ANRIL and furthering the scope of this lncRNA in disease pathogenesis. In this chapter, we summarize the most recent findings on the investigation of ANRIL and provide a perspective on the biological and clinical significance of ANRIL as a putative biomarker, specifically, its potential role in directing cellular fates leading to cancer and cardiovascular disease.

Polycomb Group (PcG) Proteins and Human Cancers: Multifaceted Functions and Therapeutic Implications.

Polycomb group (PcG) proteins are transcriptional repressors that regulate several crucial developmental and physiological processes in the cell. More recently, they have been found to play important roles in human carcinogenesis and cancer development and progression. The deregulation and dysfunction of PcG proteins often lead to blocking or inappropriate activation of developmental pathways, enhancing cellular proliferation, inhibiting apoptosis, and increasing the cancer stem cell population. Genetic and molecular investigations of PcG proteins have long been focused on their PcG functions. However, PcG proteins have recently been shown to exert non-classical-Pc-functions, contributing to the regulation of diverse cellular functions. We and others have demonstrated that PcG proteins regulate the expression and function of several oncogenes and tumor suppressor genes in a PcG-independent manner, and PcG proteins are associated with the survival of patients with cancer. In this review, we summarize the recent advances in the research on PcG proteins, including both the Pc-repressive and non-classical-Pc-functions. We specifically focus on the mechanisms by which PcG proteins play roles in cancer initiation, development, and progression. Finally, we discuss the potential value of PcG proteins as molecular biomarkers for the diagnosis and prognosis of cancer, and as molecular targets for cancer therapy.

PcG and trxG in plants - friends or foes.

The highly-conserved Polycomb group (PcG) and trithorax group (trxG) proteins play major roles in regulating gene expression and maintaining developmental states in many organisms. However, neither the recruitment of Polycomb repressive complexes (PRC) nor the mechanisms of PcG and trxG-mediated gene silencing and activation are well understood. Recent progress in Arabidopsis research challenges the dominant model of PRC2-dependent recruitment of PRC1 to target genes. Moreover, evidence indicates that diverse forms of PRC1, with shared components, are a common theme in plants and mammals. Although trxG is known to antagonize PcG, emerging data reveal that trxG can also repress gene expression, acting cooperatively with PcG. We discuss these recent findings and highlight the employment of diverse epigenetic mechanisms during development in plants and animals.

Tumor-secreted Hsp90 subverts polycomb function to drive prostate tumor growth and invasion.

Prostate cancer remains the second highest contributor to male cancer-related lethality. The transition of a subset of tumors from indolent to invasive disease is associated with a poor clinical outcome. Activation of the epithelial to mesenchymal transition (EMT) genetic program is a major risk factor for cancer progression. We recently reported that secreted extracellular Hsp90 (eHsp90) initiates EMT in prostate cancer cells, coincident with its enhanced expression in mesenchymal models. Our current work substantially extended these findings in defining a pathway linking eHsp90 signaling to EZH2 function, a methyltransferase of the Polycomb repressor complex. EZH2 is also implicated in EMT activation, and its up-regulation represents one of the most frequent epigenetic alterations during prostate cancer progression. We have now highlighted a novel epigenetic function for eHsp90 via its modulation of EZH2 expression and activity. Mechanistically, eHsp90 initiated sustained activation of MEK/ERK, a signal critical for facilitating EZH2 transcriptional up-regulation and recruitment to the E-cadherin promoter. We further demonstrated that an eHsp90-EZH2 pathway orchestrates an expanded repertoire of EMT-related events including Snail and Twist expression, tumor cell motility, and anoikis resistance. To evaluate the role of eHsp90 in vivo, eHsp90 secretion was stably enforced in a prostate cancer cell line resembling indolent disease. Remarkably, eHsp90 was sufficient to induce tumor growth, suppress E-cadherin, and initiate localized invasion, events that are exquisitely dependent upon EZH2 function. In summary, our findings illuminate a hitherto unknown epigenetic function for eHsp90 and support a model wherein tumor eHsp90 functions as a rheostat for EZH2 expression and activity to orchestrate mesenchymal properties and coincident aggressive behavior.

Polycomb genes, miRNA, and their deregulation in B-cell malignancies.

Posttranslational modifications of histone proteins represent a fundamental means to define distinctive epigenetic states and regulate gene expression during development and differentiation. Aberrations in various chromatin-modulation pathways are commonly used by tumors to initiate and maintain oncogenesis, including lymphomagenesis. Recently, increasing evidence has demonstrated that polycomb group (PcG) proteins, a subset of histone-modifying enzymes known to be crucial for B-cell maturation and differentiation, play a central role in malignant transformation of B cells. PcG hyperactivity in B-cell lymphomas is caused by overexpression or recurrent mutations of PcG genes and deregulation of microRNAs (miRNAs) or transcription factors such as c-MYC, which regulate PcG expression. Interplays of PcG and miRNA deregulations often establish a vicious signal-amplification loop in lymphoma associated with adverse clinical outcomes. Importantly, aberrant enzymatic activities associated with polycomb deregulation, notably those caused by EZH2 gain-of-function mutations, have provided a rationale for developing small-molecule inhibitors as novel therapies. In this review, we summarize our current understanding of PcG-mediated gene silencing, interplays of PcG with other epigenetic regulators such as miRNAs during B-cell differentiation and lymphomagenesis, and recent advancements in targeted strategies against PcG as promising therapeutics for B-cell malignancies.

Role of PRC2-associated factors in stem cells and disease.

The Polycomb group (PcG) of proteins form chromatin-binding complexes with histone-modifying activity. The two main PcG repressive complexes studied (PRC1 and PRC2) are generally associated with chromatin in its repressed state. PRC2 is responsible for methylation of histone H3 at lysine 27 (H3K27me3), an epigenetic mark that is linked with numerous biological processes, including development, adult homeostasis and cancer. The core canonical complex PRC2, which contains the EZH1/2, SUZ12 and EED proteins, may be extended and functionally manipulated through interactions with several other proteins. In this review, we focus on these PRC2-associated proteins. As PRC2 functions are diverse, the variability conferred by these sub-stoichiometrically associated members may help to understand specific changes in PRC2 activity, chromatin recruitment and distribution required for gene repression.

The controversial role of the Polycomb group proteins in transcription and cancer: how much do we not understand Polycomb proteins?

Polycomb group proteins (PcGs) are a large protein family that includes diverse biochemical features assembled together in two large multiprotein complexes. These complexes maintain gene transcriptional repression in a cell type specific manner by modifying the surrounding chromatin to control development, differentiation and cell proliferation. PcGs are also involved in several diseases. PcGs are often directly or indirectly implicated in cancer development for which they have been proposed as potential targets for cancer therapeutic strategies. However, in the last few years a series of discoveries about the basic properties of PcGs and the identification of specific genetic alterations affecting specific Polycomb proteins in different tumours have converged to challenge old dogmas about PcG biological and molecular functions. In this review, we analyse these new data in the context of the old knowledge, highlighting the controversies and providing new models of interpretation and ideas that will perhaps bring some order among apparently contradicting observations.