Polycomb Complex Research Articles

Aging of spermatogonial stem cells by Jnk-mediated glycolysis activation

Because spermatogonial stem cells (SSCs) are immortal by serial transplantation, SSC aging in intact testes is considered to be caused by a deteriorated microenvironment. Here, we report a cell-intrinsic mode of SSC aging by glycolysis activation. Using cultured SSCs, we found that aged SSCs proliferated more actively than young SSCs and showed enhanced glycolytic activity. Moreover, they remained euploid and exhibited stable androgenetic imprinting patterns with robust SSC activity despite having shortened telomeres. Aged SSCs showed increased Wnt7b expression, which was associated with decreased Polycomb complex 2 activity. Our results suggest that aberrant Wnt7b expression activated c-jun N-terminal kinase (JNK), which down-regulated mitochondria numbers by suppressing Ppargc1a Down-regulation of Ppargc1a probably decreased reactive oxygen species and enhanced glycolysis. Analyses of the Klotho-deficient aging mouse model and 2-y-old aged rats confirmed JNK hyperactivation and increased glycolysis. Therefore, not only microenvironment but also intrinsic activation of JNK-mediated glycolysis contributes to SSC aging.

Fluorescence imaging of biochemical relationship between ubiquitinated histone 2A and Polycomb complex protein BMI1

Several in vitro experiments have highlighted that the Polycomb group protein BMI1 plays a pivotal role in determining the biological functions of the Polycomb Repressor Complex 1 (PRC1), including its E3-ligase activity towards the Lys119 of histone H2A to yield ubiquitinated uH2A. The role of BMI1 in the epigenetic activity of PRC1 is particularly relevant in several cancers, particularly Non-Small Cell Lung Cancer (NSCLC). In this study, using indirect immunofluorescence protocols implemented on a confocal microscopy apparatus, we investigated the relationship between BMI1 and uH2A at different resolutions, in cultured (A549) and clinical NSCLC tissues, at the single cell level. In both cases, we observed a linear dependence of uH2A concentration upon BMI1 expression at the single nucleus level, indicating that the association of BMI1 to PRC1, which is needed for E3-ligase activity, occurs linearly in the physiological BMI1 concentration range. Additionally, in the NSCLC cell line model, a minor pool of uH2A may exist in absence of concurrent BMI1 expression, indicating non-exclusive, although predominant, role of BMI1 in the amplification of the E3-ligase activity of PRC1. A pharmacological downregulator of BMI1, PTC-209, was also tested in this context. Finally, the absence of significant colocalization (as measured by the Pearson's coefficient) between BMI1 and uH2A submicron clusters hints to a dynamic model where PRC1 resides transiently at ubiquitination sites. Beside unveiling subtle functional relationships between BMI1 and uH2A, these results also validate the use of uH2A as downstream “reporter” for BMI1 activity at the nuclear level in NSCLC contexts.

Regulatory networks between Polycomb complexes and non-coding RNAs in the central nervous system.

High-throughput sequencing has facilitated the identification of many types of non-coding RNAs (ncRNAs) involved in diverse cellular processes. NcRNAs as epigenetic mediators play key roles in neuronal development, maintenance, and dysfunction by controlling gene expression at multiple levels. NcRNAs may not only target specific DNA or RNA for gene silence but may also directly interact with chromatin-modifying proteins like Polycomb group (PcG) proteins to drive orchestrated transcriptional programs. Recent significant progress has been made in characterizing ncRNAs and PcG proteins involved in transcriptional, post-transcriptional, and epigenetic regulation. More importantly, dysregulation of ncRNAs, PcG proteins, and interplay among them is closely associated with the pathogenesis of central nervous system (CNS) disorders. In this review, we focus on the interplay between ncRNAs and PcG proteins in the CNS and highlight the functional roles of the partnership during neural development and diseases.

Storing memories: the distinct phases of Polycomb-mediated silencing of Arabidopsis FLC.

Polycomb-mediated epigenetic silencing is central to correct growth and development in higher eukaryotes. The evolutionarily conserved Polycomb repressive complex 2 (PRC2) transcriptionally silences target genes through a mechanism requiring the histone modification H3K27me3. However, we still do not fully understand what defines Polycomb targets, how their expression state is switched from epigenetically ON to OFF and how silencing is subsequently maintained through many cell divisions. An excellent system in which to dissect the sequence of events underlying an epigenetic switch is the Arabidopsis FLC locus. Exposure to cold temperatures progressively induces a PRC2-dependent switch in an increasing proportion of cells, through a mechanism that is driven by the local chromatin environment. Temporally distinct phases of this silencing mechanism have been identified. First, the locus is transcriptionally silenced in a process involving cold-induced antisense transcripts; second, nucleation at the first exon/intron boundary of a Polycomb complex containing cold-induced accessory proteins induces a metastable epigenetically silenced state; third, a Polycomb complex with a distinct composition spreads across the locus in a process requiring DNA replication to deliver long-term epigenetic silencing. Detailed understanding from this system is likely to provide mechanistic insights important for epigenetic silencing in eukaryotes generally.

Abstract 5188: Epigenetic changes mediated by polycomb repressive complex 2 are associated with cisplatin acquired resistance in testicular germ cell tumor

Abstract Testicular germ cell tumors (TGCTs) represent the most common carcinoma affecting young men. TGCTs serve as a paradigm of chemo-sensitive tumors as more than 80% of metastatic disease can be cured with cisplatin-based chemotherapy. However, therapy resistance can occur, which results in poor patient outcomes. The mechanisms of cisplatin resistant in these tumors remain largely undefined. To study the mechanism of cisplatin acquired resistance we generated several independent, acquired resistant clones of parental NT2/D1, 833K and 2102EP testicular cancer cells employing an in vitro protocol designed to mimic the standard-of-care cisplatin regimen used to treat TGCT patients. We further characterized 10 of these clonal lines and confirmed stable cisplatin resistance ranging from 5- to 30-fold. After confirming the cisplatin resistance, we performed RNA seq transcriptomic profiling, differential gene expression analysis, DAVID and GSEA. Remarkably, RNA-seq based transcriptional profiling revealed highly significant alterations shared between the majority of the resistant cells regardless of their parental cell of origin. This includes apparent amplification of chromosomal region 17q21-25 in 8 of 10 lines. Unexpectedly, 7 of 10 lines had a highly significant enrichment of genes regulated by H3K27 methylation and polycomb repressive complex 2 (PRC2). Transcription factor prediction analysis of genes differentially expressed between resistant and parental cells revealed that polycomb complex members were among the most highly ranked predicted transcription factors responsible for the gene expression changes. These include EZH2, SUZ12, JARID2 and BMI1. We will present our ongoing studies investigating the role of PRC2 and H3K27 methylation in mediating cisplatin resistance in these cells and in patient samples. Together our data suggests that repression of H3K27 methylation may be a mechanism of cisplatin acquired resistance in TGCTs and that restoration of PRC2 function could be a viable approach to overcome treatment failure. Citation Format: Ratnakar Singh, Zeeshan Fazal, Emmanuel Bikorimana, Andrea Corbet, Jennifer Rodriguez, Alyssa Steege, Sarah J. Freemantle, Michael J. Spinella. Epigenetic changes mediated by polycomb repressive complex 2 are associated with cisplatin acquired resistance in testicular germ cell tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5188.

Abstract 4517: Tumor-associated macrophages are reprogrammed by pancreatic ductal adenocarcinoma cells through tumor-induced DNA methylation on metabolic genes

Abstract Metabolism is shifted toward glycolysis with increased glucose uptake in anti-tumor M1 macrophages, and toward oxidative phosphorylation with significant decreased glucose uptake in pro-tumor M2 macrophages in Pancreatic ductal adenocarcinoma (PDA). Current study aims to investigate the mechanism by which tumor cells reprogram tumor associated macrophages metabolically and functionally. Using mouse bone marrow derived macrophages and human macrophages derived from peripheral blood mononuclear cells for contact and non-contact co-culture experiments with PDA tumor cells, we showed PDA tumor cells, through direct cell-cell contact confirmed by Lucifer yellow dye-coupling assay, induced DNA methylation and downregulation of a panel of glucose metabolism and oxidative phosphorylation genes selectively in M1 but not M2 macrophages by methylation specific PCR, methylation microarray analysis and qPCR, RNA-seq analysis. Glucose-response genes such as IL10 were subsequently activated. Through IL10 and its receptor IL-10R on tumor cells, macrophages enhanced the in vitro migration of tumor cells in trans-well migration assay verified by shRNA knockdown of IL10 and IL10R respectively. Exogenous infusion of both M1 and M2 macrophages promoted PDA metastasis in orthotopic mouse model after resident macrophage depletion, although pretreating with DNA demethylating agent Decitabine prevented M1, but not M2 macrophages in promoting metastasis, suggesting M1 macrophages were reprogrammed metabolically and functionally by tumor cells to become pro-cancerous. To interrogate the mechanism that regulates the tumor-induced DNA methylation in macrophages, we first examined chromatin remodeling complex Polycomb Repressive Complex 2 (PRC2), with its major component enhancer of zeste homolog 2 (EZH2) previously shown to recruit DNA methyltransferases to specific chromatin regions for epigenetic gene regulation. We found EZH2 protein was most significantly increased in M1 macrophages while Ser21 phosphorylated EZH2 (pSer21-EZH2) remained constant, indicating a decreased percentage of pSer21-EZH2/EZH2 in M1 macrophages co-cultured with tumor cells. Further identification of potential F-box containing ubiquitin ligases that interact with pSer21-EZH2 by Co-immunoprecipitation suggested EZH2 may be stabilized in M1 macrophages by tumor cells through inhibiting the ubiquitin-dependent proteolysis of pSer21-EZH2 in a phosphorylation dependent manner. This study thus far reveals a novel mechanism that reprograms M1 macrophages phenotypically into M2-like macrophages and functionally into pro-cancerous macrophages. Identification of cell surface receptors that mediate the direct tumor-macrophage contact are actively being sought, which will lead to the development of therapeutic blockades to reverse the macrophage reprogramming. Citation Format: Xingyi Pan, Mengwen Zhang, Kenji Fujiwara, Noelle R. Jurcak, Stephen Muth, Lei Zheng. Tumor-associated macrophages are reprogrammed by pancreatic ductal adenocarcinoma cells through tumor-induced DNA methylation on metabolic genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4517.

Aberrantly elevated Bmi1 promotes cervical cancer tumorigenicity and tumor sphere formation via enhanced transcriptional regulation of Sox2 genes.

The exact molecular mechanisms underlying cervical tumorigenesis are poorly understood. Polycomb complex protein Bmi1 (Bmi1) is involved in the malignant transformation and biological aggressiveness of several human carcinomas. Therefore, the present study assessed the expression of Bmi1 protein in human cervical cancer tissues and examined the mechanisms involved in cervical carcinogenesis. The expression of Bmi1 protein was examined by immunohistochemistry in cervical carcinoma tissues (n=71), high-grade squamous intraepithelial lesions (n=41) and normal cervical tissues (n=47). Expression of Bmi1 protein gradually increased across samples from the normal cervix (1/47; 2.12%), high-grade squamous intraepithelial lesions (5/42; 16.13%) and cervical carcinomas (31/71; 43.66%; P<0.05). Additionally, Bmi1 protein expression was associated with tumor histopathological grade. The effects of Bmi1 silencing and overexpression on tumor sphere formation and the tumorigenicity of cervical cancer cells were investigated. Overexpression of Bmi1 resulted in significantly attenuated tumor formation and tumor sphere formation. Consistently, Bmi1 silencing significantly inhibited tumor formation and tumor sphere formation. Furthermore, Bmi1 upregulated the expression of Sox2, and the dual-luciferase reporter assay and chromatin immunoprecipitation showed that Bmi1 transactivated Sox2 by binding to the two E-box motifs in the Sox2 promoter. In conclusion, aberrantly elevated Bmi1 promotes cervical cancer tumorigenicity and tumor sphere formation via enhanced transcriptional regulation of Sox2 genes as a potential oncogenic factor that participates in the carcinogenesis of cervical carcinomas.

Dangerous liaisons: interplay between SWI/SNF, NuRD, and Polycomb in chromatin regulation and cancer.

Changes in chromatin structure mediated by ATP-dependent nucleosome remodelers and histone modifying enzymes are integral to the process of gene regulation. Here, we review the roles of the SWI/SNF (switch/sucrose nonfermenting) and NuRD (nucleosome remodeling and deacetylase) and the Polycomb system in chromatin regulation and cancer. First, we discuss the basic molecular mechanism of nucleosome remodeling, and how this controls gene transcription. Next, we provide an overview of the functional organization and biochemical activities of SWI/SNF, NuRD, and Polycomb complexes. We describe how, in metazoans, the balance of these activities is central to the proper regulation of gene expression and cellular identity during development. Whereas SWI/SNF counteracts Polycomb, NuRD facilitates Polycomb repression on chromatin. Finally, we discuss how disruptions of this regulatory equilibrium contribute to oncogenesis, and how new insights into the biological functions of remodelers and Polycombs are opening avenues for therapeutic interventions on a broad range of cancer types.

Polycomb complex mediated epigenetic reprogramming alters TGF-β signaling via a novel EZH2/miR-490/TGIF2 axis thereby inducing migration and EMT potential in glioblastomas.

Recent advancement in understanding cancer etiology has highlighted epigenetic deregulation as an important phenomenon leading to poor prognosis in glioblastoma (GBM). Polycomb repressive complex 2 (PRC2) is one such important epigenetic modifier reportedly altered in GBM. However, its defined mechanism in tumorigenesis still remains elusive. In present study, we analyzed our in-house ChIPseq data for H3k27me3 modified miRNAs and identified miR-490-3p to be the most common target in GBM with significantly downregulated expression in glioma patients in both TCGA and GBM patient cohort. Our functional analysis delineates for the first time, a central role of PRC2 catalytic unit EZH2 in directly regulating expression of this miRNA and its host gene CHRM2 in GBM. In accordance, cell line treatment with EZH2 siRNA and 5-azacytidine also confirmed its coregulation by CpG and histone methylation based epigenetic mechanisms. Furthermore, induced overexpression of miR-490-3p in GBM cell lines significantly inhibited key hallmarks including cellular proliferation, colony formation and spheroid formation, as well as epithelial-to-mesenchymal transition (EMT), with downregulation of multiple EMT transcription factors and promigratory genes (MMP9, CCL5, PIK3R1, ICAM1, ADAM17 and NOTCH1). We also for the first time report TGFBR1 and TGIF2 as two direct downstream effector targets of miR-490-3p that are also deregulated in GBM. TGIF2, a novel target, was shown to promote migration and EMT that could partially be rescued by miR-490-3p overexpression. Overall, this stands as a first study that provides a direct link between epigenetic modulator EZH2 and oncogenic TGF-β signaling involving novel miR-490-3p/TGIF2/TGFBR1 axis, that being targetable might be promising in developing new therapeutic intervention strategies for GBM.

The Hox transcription factor Ubx stabilizes lineage commitment by suppressing cellular plasticity in Drosophila.

During development cells become restricted in their differentiation potential by repressing alternative cell fates, and the Polycomb complex plays a crucial role in this process. However, how alternative fate genes are lineage-specifically silenced is unclear. We studied Ultrabithorax (Ubx), a multi-lineage transcription factor of the Hox class, in two tissue lineages using sorted nuclei and interfered with Ubx in mesodermal cells. We find that depletion of Ubx leads to the de-repression of genes normally expressed in other lineages. Ubx silences expression of alternative fate genes by retaining the Polycomb Group protein Pleiohomeotic at Ubx targeted genomic regions, thereby stabilizing repressive chromatin marks in a lineage-dependent manner. Our study demonstrates that Ubx stabilizes lineage choice by suppressing the multipotency encoded in the genome via its interaction with Pho. This mechanism may explain why the Hox code is maintained throughout the lifecycle, since it could set a block to transdifferentiation in adult cells.

Defining CBX7-Dependent Chromatin Architecture with Rapid Small-Molecule Inhibition

While the repressive roles of Polycomb complexes have been well-established with in vitro systems, it remains unclear how subtypes of these complexes function in living cells. Conditional alleles have enabled studies of Polycomb Repressive Complex 1 (PRC1), but the available timescales on which inducible deletion occurs is not sufficient to define its direct regulatory targets. We have used short-term treatments with a new PRC1 probe UNC4976, that antagonizes recognition of H3K27me3 by the chromodomain-containing subunit CBX7. This methyl-lysine binding domain targeted strategy has enabled the discovery that hundreds of chromatin loop and contact domains are repressed by the chromodomain function of PRC1. Repressive chromatin architecture that is dependent specifically upon methyl-lysine recognition by the PRC1 chromodomain is rapidly de-repressed by 4 hours of CBX antagonism. Utilizing Hi-C experiments after compound treatment, and defining UNC4976-sensitive chromatin by ChIP-seq before major transcriptional changes occur, we characterize, for the first time, direct canonical PRC1-dependent chromatin architecture in human T lymphocytes.

Ash1 counteracts Polycomb repression independent of histone H3 lysine 36 methylation.

Polycomb repression is critical for metazoan development. Equally important but less studied is the Trithorax system, which safeguards Polycomb target genes from the repression in cells where they have to remain active. It was proposed that the Trithorax system acts via methylation of histone H3 at lysine 4 and lysine 36 (H3K36), thereby inhibiting histone methyltransferase activity of the Polycomb complexes. Here we test this hypothesis by asking whether the Trithorax group protein Ash1 requires H3K36 methylation to counteract Polycomb repression. We show that Ash1 is the only Drosophila H3K36-specific methyltransferase necessary to prevent excessive Polycomb repression of homeotic genes. Unexpectedly, our experiments reveal no correlation between the extent of H3K36 methylation and the resistance to Polycomb repression. Furthermore, we find that complete substitution of the zygotic histone H3 with a variant in which lysine 36 is replaced by arginine does not cause excessive repression of homeotic genes. Our results suggest that the model, where the Trithorax group proteins methylate histone H3 to inhibit the histone methyltransferase activity of the Polycomb complexes, needs revision.

Xist Deletional Analysis Reveals an Interdependency between Xist RNA and Polycomb Complexes for Spreading along the Inactive X

During X-inactivation, Xist RNA spreads along anentire chromosome to establish silencing. However, the mechanism and functional RNA elements involved in spreading remain undefined. By performing a comprehensive endogenous Xist deletion screen, we identify Repeat B as crucial for spreading Xist and maintaining Polycomb repressive complexes 1 and 2 (PRC1/PRC2) along the inactive X (Xi). Unexpectedly, spreading of these three factors is inextricably linked. Deleting Repeat B or its direct bindingpartner, HNRNPK, compromises recruitment of PRC1 and PRC2. In turn, ablating PRC1 or PRC2 impairs Xist spreading. Therefore, Xist and Polycomb complexes require each other to propagate along the Xi, suggesting a positive feedback mechanism between RNA initiator and protein effectors. Perturbing Xist/Polycomb spreading causes failure of de novo Xi silencing, with partial compensatory downregulation of the active X, and also disrupts topologicalXireconfiguration. Thus, Repeat B is a multifunctional element that integrates interdependent Xist/Polycomb spreading, silencing, and changes in chromosome architecture.

R-Loops Enhance Polycomb Repression at a Subset of Developmental Regulator Genes

SummaryR-loops are three-stranded nucleic acid structures that form during transcription, especially over unmethylated CpG-rich promoters of active genes. In mouse embryonic stem cells (mESCs), CpG-rich developmental regulator genes are repressed by the Polycomb complexes PRC1 and PRC2. Here, we show that R-loops form at a subset of Polycomb target genes, and we investigate their contribution to Polycomb repression. At R-loop-positive genes, R-loop removal leads to decreased PRC1 and PRC2 recruitment and Pol II activation into a productive elongation state, accompanied by gene derepression at nascent and processed transcript levels. Stable removal of PRC2 derepresses R-loop-negative genes, as expected, but does not affect R-loops, PRC1 recruitment, or transcriptional repression of R-loop-positive genes. Our results highlight that Polycomb repression does not occur via one mechanism but consists of different layers of repression, some of which are gene specific. We uncover that one such mechanism is mediated by an interplay between R-loops and RING1B recruitment.

Comprehensive Analysis of Chromatin States in Atypical Teratoid/Rhabdoid Tumor Identifies Diverging Roles for SWI/SNF and Polycomb in Gene Regulation

Biallelic inactivation of SMARCB1, encoding a member of the SWI/SNF chromatin remodeling complex, is the hallmark genetic aberration of atypical teratoid rhabdoid tumors (ATRT). Here, we report how loss of SMARCB1 affects the epigenome in these tumors. Using chromatin immunoprecipitation sequencing (ChIP-seq) on primary tumors for a series of active and repressive histone marks, we identified the chromatin states differentially represented in ATRTs compared with other brain tumors and non-neoplastic brain. Re-expression of SMARCB1 in ATRT cell lines enabled confirmation of our genome-wide findings for the chromatin states. Additional generation of ChIP-seq data for SWI/SNF and Polycomb group proteins and the transcriptional repressor protein REST determined differential dependencies of SWI/SNF and Polycomb complexes in regulation of diverse gene sets in ATRTs.

Classifying human promoters by occupancy patterns identifies recurring sequence elements, combinatorial binding, and spatial interactions

BackgroundCharacterizing recurring sequence patterns in human promoters has been a challenging undertaking even nowadays where a near-complete overview of promoters exists. However, with the more recent availability of genomic location (ChIP-seq) data, one can approach that question through the identification of characteristic patterns of transcription factor occupancy and histone modifications.ResultsBased on the ENCODE annotation and integration of sequence motifs as well as three-dimensional chromatin data, we have undertaken a re-analysis of occupancy and sequence patterns in human promoters. We identify clear groups of CAAT-box and E-box sequence motif containing promoters, as well as a group of promoters whose interaction with an enhancer appears to be mediated by CCCTC-binding factor (CTCF) binding on the promoter. We also extend our analysis to inactive promoters, showing that only a surprisingly small number of inactive promoters is repressed by the polycomb complex. We also identify combinatorial patterns of transcription factor interactions indicated by the ChIP-seq signals.ConclusionOur analysis defines subgroups of promoters characterized by stereotypic patterns of transcription factor occupancy, and combinations of specific sequence patterns which are required for their binding. This grouping provides new hypotheses concerning the assembly and dynamics of transcription factor complexes at their respective promoter groups, as well as questions on the evolutionary origin of these groups.

Untangling the Role of Polycomb Complexes in Chemotherapy Resistance.

In this issue, Maganti and colleagues described an epigenetic link between reduced abundance of Polycomb-related protein MTF2 and chemotherapy resistance in refractory acute myeloid leukemia. MTF2 deficiency impaired expression of the PRC2 complex and deposition of H3K27me3 at many genes, including the key target gene MDM2, leading to increased MDM2 expression that in turn depleted p53 and thereby conferred chemoresistance. Cancer Discov; 8(11); 1348-51. ©2018 AACR See related article by Maganti et al., p. 1376.

Ezh1 Targets Bivalent Genes to Maintain Self-Renewing Stem Cells in Ezh2-Insufficient Myelodysplastic Syndrome

SummaryPolycomb repressive complex (PRC) 2 represses transcription through histone H3K27 trimethylation (H3K27me3). We previously reported that the hematopoietic-cell-specific deletion of Ezh2, encoding a PRC2 enzyme, induced myelodysplastic syndrome (MDS) in mice, whereas the concurrent Ezh1 deletion depleted hematopoietic stem and progenitor cells (HSPCs). We herein demonstrated that mice with only one Ezh1 allele (Ezh1+/-Ezh2Δ/Δ) maintained HSPCs. A chromatin immunopreciptation sequence analysis revealed that residual PRC2 preferentially targeted genes with high levels of H3K27me3 and H2AK119 monoubiquitination (H2AK119ub1) in HSPCs (designated as Ezh1 core target genes), which were mostly developmental regulators, and maintained H3K27me3 levels in Ezh1+/-Ezh2Δ/Δ HSPCs. Even upon the complete depletion of Ezh1 and Ezh2, H2AK119ub1 levels were largely retained, and only a minimal number of Ezh1 core targets were de-repressed. These results indicate that genes marked with high levels of H3K27me3 and H2AK119ub1 are the core targets of polycomb complexes in HSPCs as well as MDS stem cells.

Promoter bivalency favors an open chromatin architecture in embryonic stem cells.

In embryonic stem cells (ESCs), developmental gene promoters are characterized by their bivalent chromatin state, with simultaneous modification by MLL2 and Polycomb complexes. Although essential for embryogenesis, bivalency is functionally not well understood. Here, we show that MLL2 plays a central role in ESC genome organization. We generate a catalog of bona fide bivalent genes in ESCs and demonstrate that loss of MLL2 leads to increased Polycomb occupancy. Consequently, promoters lose accessibility, long-range interactions are redistributed, and ESCs fail to differentiate. We pose that bivalency balances accessibility and long-range connectivity of promoters, allowing developmental gene expression to be properly modulated.

Retracted : Anticancer effects of miR‐200c in colorectal cancer through BMI1

Colorectal cancer (CRC) is one of the most leading cancer deaths throughout the world. MiR-200c has been shown to have a critical role in cancer initiation and progression. In this study, we investigated the miR-200c expression in CRC tissues and its effects on CRC cell lines which were mediated by polycomb complex protein (BMI1). Quantitative reverse transcription polymerase chain reaction (QRT-PCR) and immunohistochemistry were used to detect miR-200c and BMI1 expression in tumor tissues from 38 patients with CRC and 38 normal colon tissues. HCT-116 and SW-48 cells were transfected by locked nucleic acid (LNA)-anti-miR-200c. Western blot analysis and real-time PCR were applied to determine the BMI1 protein and microRNA (miRNA) levels. The apoptosis was analyzed via annexin/propidium iodide staining, and cell invasion was evaluated by transwell assay. MiR-200c was markedly downregulated in CRC tissues, whereas the protein expression of BMI1 in CRC tissues was upregulated compared with normal colon tissues. In the colon cancer cell lines, transfection of LNA-anti-miR-200c increased BMI1 gene and protein expression as well as the cell invasion. Downregulation of miR-200c by LNA decreased the apoptotic cells. The results from this study revealed that miR-200c may have antitumor effects through inhibition of BMI1 expression.