PcG Complexes Research Articles

YY1 Knockdown Relieves the Differentiation Block and Restores Apoptosis in AML Cells.

In this study we analyzed the expression of Yin and Yang 1 protein (YY1), a member of the noncanonical PcG complexes, in AML patient samples and AML cell lines and the effect of YY1 downregulation on the AML differentiation block. Our results show that YY1 is significantly overexpressed in AML patient samples and AML cell lines and that YY1 knockdown relieves the differentiation block. YY1 downregulation in two AML cell lines (HL-60 and OCI-AML3) and one AML patient sample restored the expression of members of the CEBP protein family, increased the expression of extrinsic growth factors/receptors and surface antigenic markers, induced morphological cell characteristics typical of myeloid differentiation, and sensitized cells to retinoic acid treatment and to apoptosis. Overall, our data show that YY1 is not a secondary regulator of myeloid differentiation but that, if overexpressed, it can play a predominant role in myeloid differentiation block.

Know when to fold 'em: Polycomb complexes in oncogenic 3D genome regulation.

Chromatin is spatially and temporally regulated through a series of orchestrated processes resulting in the formation of 3D chromatin structures such as topologically associating domains (TADs), loops and Polycomb Bodies. These structures are closely linked to transcriptional regulation, with loss of control of these processes a frequent feature of cancer and developmental syndromes. One such oncogenic disruption of the 3D genome is through recurrent dysregulation of Polycomb Group Complex (PcG) functions either through genetic mutations, amplification or deletion of genes that encode for PcG proteins. PcG complexes are evolutionarily conserved epigenetic complexes. They are key for early development and are essential transcriptional repressors. PcG complexes include PRC1, PRC2 and PR-DUB which are responsible for the control of the histone modifications H2AK119ub1 and H3K27me3. The spatial distribution of the complexes within the nuclear environment, and their associated modifications have profound effects on the regulation of gene transcription and the 3D genome. Nevertheless, how PcG complexes regulate 3D chromatin organization is still poorly understood. Here we glean insights into the role of PcG complexes in 3D genome regulation and compaction, how these processes go awry during tumorigenesis and the therapeutic implications that result from our insights into these mechanisms.

Histone Methylases and Demethylases Regulating Antagonistic Methyl Marks: Changes Occurring in Cancer.

Epigenetic regulation of gene expression is crucial to the determination of cell fate in development and differentiation, and the Polycomb (PcG) and Trithorax (TrxG) groups of proteins, acting antagonistically as complexes, play a major role in this regulation. Although originally identified in Drosophila, these complexes are conserved in evolution and the components are well defined in mammals. Each complex contains a protein with methylase activity (KMT), which can add methyl groups to a specific lysine in histone tails, histone 3 lysine 27 (H3K27), by PcG complexes, and H3K4 and H3K36 by TrxG complexes, creating transcriptionally repressive or active marks, respectively. Histone demethylases (KDMs), identified later, added a new dimension to histone methylation, and mutations or changes in levels of expression are seen in both methylases and demethylases and in components of the PcG and TrX complexes across a range of cancers. In this review, we focus on both methylases and demethylases governing the methylation state of the suppressive and active marks and consider their action and interaction in normal tissues and in cancer. A picture is emerging which indicates that the changes which occur in cancer during methylation of histone lysines can lead to repression of genes, including tumour suppressor genes, or to the activation of oncogenes. Methylases or demethylases, which are themselves tumour suppressors, are highly mutated. Novel targets for cancer therapy have been identified and a methylase (KMT6A/EZH2), which produces the repressive H3K27me3 mark, and a demethylase (KDM1A/LSD1), which demethylates the active H3K4me2 mark, are now under clinical evaluation.

The RNF26/CBX7 axis modulates the TNF pathway to promote cell proliferation and regulate sensitivity to TKIs in ccRCC.

Clear cell renal cell carcinoma (ccRCC) accounts for 85% of all malignant renal tumors. Currently, the pathogenesis of ccRCC is not fully understood. Chromobox (CBX) family proteins are the major subunits of PcG complexes and are implicated in regulating mammalian development. The CBX family consists of eight members, namely, CBX1-8. Numerous studies have highlighted that each CBX protein exhibits distinct functions and prognostic roles in specific cancer types. In this study, in silico analysis indicated that CBX7 was downregulated in ccRCC and correlated with favorable prognosis in a ccRCC cohort. Subsequent studies showed that CBX7 inhibited cancer cell proliferation and invasion. Then, we showed that CBX7 downregulated ETS1 to inactivate the tumor necrosis factor (TNF) signaling pathway, which inhibited tumor proliferation and enhanced the sensitivity of ccRCC cells to tyrosine kinase inhibitors (TKIs). Moreover, we found that CBX7 was a bona fide substrate of RNF26. RNF26 promoted the degradation of CBX7 and enhanced ccRCC tumor growth. Therefore, our results revealed a novel RNF26/CBX7 axis that modulates the TNF signaling pathway in ccRCC.

EAR domain-containing transcription factors trigger PRC2-mediated chromatin marking in Arabidopsis.

Polycomb group (PcG) complexes ensure that every cell in an organism expresses the genes needed at a particular stage, time, or condition. However, it is still not fully understood how PcG complexes PcG-repressive complex 1 (PRC1) and PRC2 are recruited to target genes in plants. Recent findings in Arabidopsis thaliana support the notion that PRC2 recruitment is mediated by different transcription factors (TFs). However, it is unclear how all these TFs interact with PRC2 and whether they also recruit PRC1 activity. Here, by using a system to bind selected TFs to a synthetic promoter lacking the complexity of PcG target promoters in vivo, we show that while binding of the TF VIVIPAROUS1/ABSCISIC ACID-INSENSITIVE3-LIKE1 recapitulates PRC1 and PRC2 marking, the binding of other TFs only renders PRC2 marking. Interestingly, all these TFs contain an Ethylene-responsive element binding factor-associated Amphiphilic Repression (EAR) domain that triggers both HISTONE DEACETYLASE COMPLEX and PRC2 activities, connecting two different repressive mechanisms. Furthermore, we show that different TFs can have an additive effect on PRC2 activity, which may be required to maintain long-term repression of gene expression.

How Polycomb-Mediated Cell Memory Deals With a Changing Environment: Variations in PcG complexes and proteins assortment convey plasticity to epigenetic regulation as a response to environment.

Cells and tissues are continuously exposed to a changing microenvironment, hence the necessity of a flexible modulation of gene expression that in complex organism have been achieved through specialized chromatin mechanisms. Chromatin-based cell memory enables cells to maintain their identity by fixing lineage specific transcriptional programs, ensuring their faithful transmission through cell division; in particular PcG-based memory system evolved to maintain the silenced state of developmental and cell cycle genes. In evolution the complexity of this system have increased, particularly in vertebrates, indicating combinatorial and dynamic properties of Polycomb proteins, in some cases even overflowing outside the cell nucleus. Therefore, their function may not be limited to the imposition of rigid states of genetic programs, but on the ability to recognize signals and allow plastic transcriptional changes in response to different stimuli. Here, we discuss the most novel PcG mediated memory functions in facing and responding to the challenges posed by a fluctuating environment.

Multiple roles of Ring 1 and YY1 binding protein in physiology and disease.

Ring 1 and YY1 binding protein (RYBP) was first identified in 1999, and its structure includes a conserved Npl4 Zinc finger motif at the N‐terminus, a central region that is characteristically enriched with arginine and lysine residues and a C‐terminal region enriched with serine and threonine amino acids. Over nearly 20 years, multiple studies have found that RYBP functions as an organ developmental adaptor. There is also evidence that RYBP regulates the expression of different genes involved in various aspects of biological processes, via a mechanism that is dependent on interactions with components of PcG complexes and/or through binding to different transcriptional factors. In addition, RYBP interacts directly or indirectly with apoptosis‐associated proteins to mediate anti‐apoptotic or pro‐apoptotic activity in both the cytoplasm and nucleus of various cell types. Furthermore, RYBP has also been shown to act as tumour suppressor gene in different solid tumours, but as an oncogene in lymphoma and melanoma. In this review, we summarize our current understanding of the functions of this multifaceted RYBP in physiological and pathological conditions, including embryonic development, apoptosis and cancer, as well as its role as a component of polycomb repressive complex 1.

Chromatin priming of genes in development: Concepts, mechanisms and consequences.

During ontogeny, cells progress through multiple alternate differentiation states by activating distinct gene regulatory networks. In this review, we highlight the important role of chromatin priming in facilitating gene activation during lineage specification and in maintaining an epigenetic memory of previous gene activation. We show that chromatin priming is part of a hugely diverse repertoire of regulatory mechanisms that genes use to ensure that they are expressed at the correct time, in the correct cell type, and at the correct level, but also that they react to signals. We also emphasize how increasing our knowledge of these principles could inform our understanding of developmental failure and disease.

Chromatin proteins and RNA are associated with DNA during all phases of mitosis.

Mitosis brings about major changes to chromosome and nuclear structure. We used recently developed proximity ligation assay-based techniques to investigate the association with DNA of chromatin-associated proteins and RNAs in Drosophila embryos during mitosis. All groups of tested proteins, histone-modifying and chromatin-remodeling proteins and methylated histones remained in close proximity to DNA during all phases of mitosis. We also found that RNA transcripts are associated with DNA during all stages of mitosis. Reduction of H3K27me3 levels or elimination of RNAs had no effect on the association of the components of PcG and TrxG complexes to DNA. Using a combination of proximity ligation assay-based techniques and super-resolution microscopy, we found that the number of protein–DNA and RNA–DNA foci undergoes significant reduction during mitosis, suggesting that mitosis may be accompanied by structural re-arrangement or compaction of specific chromatin domains.

Arabidopsis DNA polymerase ϵ recruits components of Polycomb repressor complex to mediate epigenetic gene silencing.

Arabidopsis ESD7 locus encodes the catalytic subunit of the DNA Pol ϵ involved in the synthesis of the DNA leading strand and is essential for embryo viability. The hypomorphic allele esd7-1 is viable but displays a number of pleiotropic phenotypic alterations including an acceleration of flowering time. Furthermore, Pol ϵ is involved in the epigenetic silencing of the floral integrator genes FT and SOC1, but the molecular nature of the transcriptional gene silencing mechanisms involved remains elusive. Here we reveal that ESD7 interacts with components of the PRC2 such as CLF, EMF2 and MSI1, and that mutations in ESD7 cause a decrease in the levels of the H3K27me3 mark present in the chromatin of FT and SOC1. We also demonstrate that a domain of the C-terminal region of ESD7 mediates the binding to the different PRC2 components and this interaction is necessary for the proper recruitment of PRC2 to FT and SOC1 chromatin. We unveil the existence of interplay between the DNA replication machinery and the PcG complexes in epigenetic transcriptional silencing. These observations provide an insight into the mechanisms ensuring that the epigenetic code at pivotal loci in developmental control is faithfully transmitted to the progeny of eukaryotic cells.

Injectable Ternary Nanocomplex Hydrogel for Long-Term Chemical Drug/Gene Dual Delivery.

We developed an injectable chemical drug/gene dual delivery platform system for long-term combination therapy. Ternary nanosized thermosensitive polymer/chemical drug/gene-(PCG) complexes showing sol-gel transition were prepared by simple mixing of three components via hydrophobic and ionic interaction. The PCG complex hydrogel showed sustained release of chemical drug and genes for 40 days without denaturalization of genes in vitro. Dual delivery-mediated anticancer effects of PCG complexes were shown in vitro and in an in vivo xenograft model for up to 1 month. Therefore, this dual delivery platform is expected to be a new effective system for treatment of diseases via long-term chemical drug/gene combination therapy.

Ectopic expression of EbFIE from apomictic Eulaliopsis binata in rice results in pleiotropic phenotypes likely due to interaction with OsCLF

FERTILIZATION INDEPENDENT ENDOSPERM (FIE) is a core component of PcG complexes and functions in plant phase transition and seed generation. However, understanding in its function of apomictic monocot plants remains blank. Here an FIE homology EbFIE, has been isolated from apomictic Graminae species Eulaliopsis binata. EbFIE shares higher homology to OsFIE2 than OsFIE1, and has been classified into the monocot FIE2 clade. In addition, the broad expression pattern of EbFIE is also similar to OsFIE2. While, ectopic expression of EbFIE in rice resulted in pleiotropic phenotypes similar to that of OsFIE1 over-expressing plants. Meanwhile, EbFIE could bind OsCLF in vitro as OsFIE1 but different with OsFIE2. Molecular models comparison indicated that both EbFIE and OsFIE1 had a smaller E(z) protein binding groove than OsFIE2. Further site-directed mutagenesis analysis revealed that single amino acid substitution of I194F in OsFIE2 could improve its OsCLF binding capacity. Taken together, our results suggested that EbFIE was a conserved FIE homolog belonging to monocot FIE2 clade, but due to the similarity in protein conformation with FIE1, EbFIE might play a broad role in vegetative and reproductive development regulation by interaction with CLF homolog.

Variable Requirements for DNA-Binding Proteins at Polycomb-Dependent Repressive Regions in Human HOX Clusters

Polycomb group (PcG)-mediated repression is an evolutionarily conserved process critical for cell fate determination and maintenance of gene expression during embryonic development. However, the mechanisms underlying PcG recruitment in mammals remain unclear since few regulatory sites have been identified. We report two novel prospective PcG-dependent regulatory elements within the human HOXB and HOXC clusters and compare their repressive activities to a previously identified element in the HOXD cluster. These regions recruited the PcG proteins BMI1 and SUZ12 to a reporter construct in mesenchymal stem cells and conferred repression that was dependent upon PcG expression. Furthermore, we examined the potential of two DNA-binding proteins, JARID2 and YY1, to regulate PcG activity at these three elements. JARID2 has differential requirements, whereas YY1 appears to be required for repressive activity at all 3 sites. We conclude that distinct elements of the mammalian HOX clusters can recruit components of the PcG complexes and confer repression, similar to what has been seen in Drosophila. These elements, however, have diverse requirements for binding factors, which, combined with previous data on other loci, speaks to the complexity of PcG targeting in mammals.

The role of maintenance proteins in the preservation of epithelial cell identity during mammary gland remodeling and breast cancer initiation

During normal postnatal mammary gland development and adult remodeling related to the menstrual cycle, pregnancy, and lactation, ovarian hormones and peptide growth factors contribute to the delineation of a definite epithelial cell identity. This identity is maintained during cell replication in a heritable but DNA-independent manner. The preservation of cell identity is fundamental, especially when cells must undergo changes in response to intrinsic and extrinsic signals. The maintenance proteins, which are required for cell identity preservation, act epigenetically by regulating gene expression through DNA methylation, histone modification, and chromatin remodeling. Among the maintenance proteins, the Trithorax (TrxG) and Polycomb (PcG) group proteins are the best characterized. In this review, we summarize the structures and activities of the TrxG and PcG complexes and describe their pivotal roles in nuclear estrogen receptor activity. In addition, we provide evidence that perturbations in these epigenetic regulators are involved in disrupting epithelial cell identity, mammary gland remodeling, and breast cancer initiation.

PcG-Mediated Higher-Order Chromatin Structures Modulate Replication Programs at the Drosophila BX-C

Polycomb group proteins (PcG) exert conserved epigenetic functions that convey maintenance of repressed transcriptional states, via post-translational histone modifications and high order structure formation. During S-phase, in order to preserve cell identity, in addition to DNA information, PcG-chromatin-mediated epigenetic signatures need to be duplicated requiring a tight coordination between PcG proteins and replication programs. However, the interconnection between replication timing control and PcG functions remains unknown. Using Drosophila embryonic cell lines, we find that, while presence of specific PcG complexes and underlying transcription state are not the sole determinants of cellular replication timing, PcG-mediated higher-order structures appear to dictate the timing of replication and maintenance of the silenced state. Using published datasets we show that PRC1, PRC2, and PhoRC complexes differently correlate with replication timing of their targets. In the fully repressed BX-C, loss of function experiments revealed a synergistic role for PcG proteins in the maintenance of replication programs through the mediation of higher-order structures. Accordingly, replication timing analysis performed on two Drosophila cell lines differing for BX-C gene expression states, PcG distribution, and chromatin domain conformation revealed a cell-type-specific replication program that mirrors lineage-specific BX-C higher-order structures. Our work suggests that PcG complexes, by regulating higher-order chromatin structure at their target sites, contribute to the definition and the maintenance of genomic structural domains where genes showing the same epigenetic state replicate at the same time.

CpG island structure and trithorax/polycomb chromatin domains in human cells

TrxG and PcG complexes play key roles in the epigenetic regulation of development through H3K4me3 and H3K27me3 modification at specific sites throughout the human genome, but how these sites are selected is poorly understood. We find that in pluripotent cells, clustered CpG-islands at genes predict occupancy of H3K4me3 and H3K27me3, and these "bivalent" chromatin domains precisely span the boundaries of CpG-island clusters. These relationships are specific to pluripotent stem cells and are not retained at H3K4me3 and H3K27me3 sites unique to differentiated cells. We show that putative transcripts from clustered CpG-islands predict stem-loop structures characteristic of those bound by PcG complexes, consistent with the possibility that RNA facilitates PcG recruitment or maintenance at these sites. These studies suggest that CpG-island structure plays a fundamental role in establishing developmentally important chromatin structures in the pluripotent genome, and a subordinate role in establishing TrxG/PcG chromatin structure at sites unique to differentiated cells.

Abstract B59: Polycomb group ring finger protein plays a role in gamma ionizing radiation-induced DNA damage response in MCF7 cell line.

Abstract Polycomb group (PcG) gene product is a family of proteins that were accumulated with complexes in nuclear and plays an important role in development and maintaining stem cell population of hematopoietic and neuronal cell lineages. PcG complexes can regulate the transcriptional activity of many genes by epigenetic silencing involved in embryogenesis. Recent reports showed the overexpressions of PcG proteins were correlated with the severity of many cancers. To investigate the underlying mechanisms of PcG ubiquitin ligase (PCGF1-6) on DNA damage response after exposure to gamma ionizing radiation, we generated the stable knockdown MCF7 breast cancer cell lines capable of silencing each of PcG ubiquitin ligase genes. Cell survival and proliferation analysis in stable PcG ubiquitin ligase knockdown MCF7 cell lines was compared to the analysis in control MCF7 cell lines after treatment of gamma ionizing radiation using MTT and colony forming assay. Furthermore, we found that loss of several PcG ubiquitin ligases sensitizes MCF7 cell lines to gamma ionizing radiation-mediated growth inhibition. We further demonstrate that several PCGF PcG ubiquitin ligases are localized at γ-H2AX foci after treatment of gamma ionizing radiation. These results demonstrate that PCGF family can coordinately regulate the cell resistance mechanism in DNA damage response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B59.

Chromatin regulated interchange between polycomb repressive complex 2 (PRC2)-Ezh2 and PRC2-Ezh1 complexes controls myogenin activation in skeletal muscle cells

BackgroundPolycomb group (PcG) genes code for chromatin multiprotein complexes that are responsible for maintaining gene silencing of transcriptional programs during differentiation and in adult tissues. Despite the large amount of information on PcG function during development and cell identity homeostasis, little is known regarding the dynamics of PcG complexes and their role during terminal differentiation.ResultsWe show that two distinct polycomb repressive complex (PRC)2 complexes contribute to skeletal muscle cell differentiation: the PRC2-Ezh2 complex, which is bound to the myogenin (MyoG) promoter and muscle creatine kinase (mCK) enhancer in proliferating myoblasts, and the PRC2-Ezh1 complex, which replaces PRC2-Ezh2 on MyoG promoter in post-mitotic myotubes. Interestingly, the opposing dynamics of PRC2-Ezh2 and PRC2-Ezh1 at these muscle regulatory regions is differentially regulated at the chromatin level by Msk1 dependent methyl/phospho switch mechanism involving phosphorylation of serine 28 of the H3 histone (H3S28ph). While Msk1/H3S28ph is critical for the displacement of the PRC2-Ezh2 complex, this pathway does not influence the binding of PRC2-Ezh1 on the chromatin. Importantly, depletion of Ezh1 impairs muscle differentiation and the chromatin recruitment of MyoD to the MyoG promoter in differentiating myotubes. We propose that PRC2-Ezh1 is necessary for controlling the proper timing of MyoG transcriptional activation and thus, in contrast to PRC2-Ezh2, is required for myogenic differentiation.ConclusionsOur data reveal another important layer of epigenetic control orchestrating skeletal muscle cell terminal differentiation, and introduce a novel function of the PRC2-Ezh1 complex in promoter setting.

Long Noncoding RNA, Polycomb, and the Ghosts Haunting INK4b-ARF-INK4a Expression

Polycomb group proteins (PcG) function as transcriptional repressors of gene expression. The important role of PcG in mediating repression of the INK4b-ARF-INK4a locus, by directly binding to the long noncoding RNA (lncRNA) transcript antisense noncoding RNA in the INK4 locus (ANRIL), was recently shown. INK4b-ARF-INK4a encodes 3 tumor-suppressor proteins, p15(INK4b), p14(ARF), and p16(INK4a), and its transcription is a key requirement for replicative or oncogene-induced senescence and constitutes an important barrier for tumor growth. ANRIL gene is transcribed in the antisense orientation of the INK4b-ARF-INK4a gene cluster, and different single-nucleotide polymorphisms are associated with increased susceptibility to several diseases. Although lncRNA-mediated regulation of INK4b-ARF-INK4a gene is not restricted to ANRIL, both polycomb repressive complex-1 (PRC1) and -2 (PRC2) interact with ANRIL to form heterochromatin surrounding the INK4b-ARF-INK4a locus, leading to its repression. This mechanism would provide an increased advantage for bypassing senescence, sustaining the requirements for the proliferation of stem and/or progenitor cell populations or inappropriately leading to oncogenesis through the aberrant saturation of the INK4b-ARF-INK4a locus by PcG complexes. In this review, we summarize recent findings on the underlying epigenetic mechanisms that link PcG function with ANRIL, which impose gene silencing to control cellular homeostasis as well as cancer development.

Polycomb eviction as a new distant enhancer function

Remote distal enhancers may be located tens or thousands of kilobases away from their promoters. How they control gene expression is still poorly understood. Here, we analyze the influence of a remote enhancer on the balance between repression (Polycomb-PcG) and activation (Trithorax-TrxG) of a developmentally regulated gene associated with a CpG island. We reveal its essential, nonredundant role in clearing the PcG complex and H3K27me3 from the CpG island. In the absence of the enhancer, the H3K27me3 demethylase (JMJD3) is not recruited to the CpG island. We propose a new role of long-range regulatory elements in removing repressive PcG complexes.