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The Polycomb group protein (PcG) SUZ12 forms Polycomb repressive complexes together with histone methyltransferase EZH2. Although the complexes have been demonstrated to be involved in epigenetic maintenance of gene expression in a transcriptional repressive state, it is unclear how they are recruited to the target genes. Here we report that SUZ12 directly interacts with site-specific transcriptional repressor E2F6 and forms a complex together with EZH2. SUZ12 interacts with E2F6 selectively among the E2F family proteins and E2F6- containing SUZ12-EZH2 complex was biochemically purified from HEK293 cells stably expressing Flag-tagged SUZ12. Chromatin immunoprecipitation assays revealed the target genes of the E2F6-SUZ12-EZH2 complex. Contrary to expectation, the promoter regions of these genes are not or only weakly tri-methylated at histone H3-K27, and their expression is down-regulated by depletion of EZH2. Given that the transactivation function of SUZ12-EZH2 has been previously reported, the inhibitory effect on E2F6-mediated transcriptional repression by physical interaction can be considered a candidate mechanism of gene activation by these PcGs.

Abnormal activation of the Wnt signaling pathway is found in 90% of colorectal cancers (CRCs). Secreted frizzled-related protein 4 (sFRP4) serves as an antagonist of the canonical Wnt signaling pathway. Epigenetic alterations, including changes in DNA methylation and histone methylation, may influence the expression of sFRP4. Polycomb group (PcG) proteins are epigenetic transcriptional repressors that selectively repress gene expression by forming polycomb repressive complexes (PRCs). Enhancer of zeste homolog 2 (EZH2), the core component of PRC2, is a histone-lysine N-methyltransferase that interacts with DNA methyltransferases. In the present study, the promoter DNA methylation status of sFRP4 in CRC cell lines was analyzed and the underlying mechanisms of action governing this modification was investigated. Firstly, the DNA methylation status of the sFRP4 promoter in CRC cell lines was assessed using methylation-specific PCR. Subsequently, the mRNA and protein levels of sFRP4 were measured using real-time qPCR and western blot analysis, respectively, to determine whether the DNA methylation status of the sFRP4 promoter is correlated with its transcriptional levels. To screen for important epigenetic modifiers that may regulate the promoter DNA methylation status of sFRP4, the expression levels of PcG proteins were examined by gene array analysis. ChIP-qPCR was performed to test whether the selected PcG proteins directly bind the promoter region of sFRP4. Finally, the downregulated PcG proteins EZH2, chromobox 7 (CBX7) and jumonji and AT-rich interaction domain containing 2 (JARID2) were identified and their association with sFRP4 expression levels and Wnt/β-catenin signaling pathway activity were investigated. The present study revealed that sFRP4 was hypermethylated in the promoter region and downregulated during the progression of the CRC cell lines from Dukes A to Dukes C. Expression levels of PcG proteins EZH2, CBX7 and JARID2 were upregulated and positively associated with the aberrantly activated Wnt signaling pathway in the CRC cell lines. EZH2, CBX7 and JARID2 were all enriched in the sFRP4 promoter region in CRC cells. EZH2 downregulation did not affect the promoter DNA methylation status of sFRP4 but increased its expression levels and decreased CRC cell proliferation. DNA methylation controls the expression of sFRP4. EZH2 regulates sFRP4 expression without affecting the DNA hypermethylation of the sFRP4 promoter and influences CRC cell proliferation and Wnt/β-catenin signaling pathway activities., (Copyright: © Liu et al.)

Main Conclusion: The information on core components in maize polycomb repressive complex 2 (PRC2) are updated at a genome-wide scale, and the protein-protein interaction networks of PRC2 components are further provided in maize. The evolutionarily conserved polycomb group (PcG) proteins form multi-subunits polycomb repressive complexes (PRCs) that repress gene expression via chromatin condensation. In Arabidopsis, three distinct PRC2s have been identified, each determining a specific developmental program with partly functional redundancy. However, the core components and biological functions of PRC2 in cereals remain obscure. Here, we updated the information on maize PRC2 components at a genome-wide scale. Maize PRC2 subunits are highly duplicated, with five MSI1, three E(z), two ESC and two Su(z)12 homologs. ZmFIE1 is preferentially expressed in the endosperm, whereas the remaining are broadly expressed in many tissues. ZmCLF/MEZ1 and ZmFIE1 are maternally expressed imprinted genes, in contrast to the paternal-dominantly expression of ZmFIE2 in the endosperm. In maize, E(z) members likely provide a scaffold for assembling PRC2 complexes, whereas Su(z)12 and p55/MSI1-like proteins together reinforce the complex; ESC members probably determine its specificity: FIE1-PRC2 regulates endosperm cell development, whereas FIE2-PRC2 controls other cell types. The duplicated Brassicaceae-specific MEA and FIS2 also directly interact with maize PRC2 members. Together, this study establishes a roadmap for protein-protein interactions of maize PRC2 components, providing new insights into their functions in the growth and development of cereals.

Regulation of gene expression without changing the DNA sequence is governed by epigenetic mechanisms. Epigenetic regulation is important for changing chromatin structure in response to environmental cues as well as maintaining chromatin structure after cell division. The epigenetic machinery can reversibly change chromatin function, allowing a wide variety of biological processes in multicellular organisms to be controlled. Epigenetic regulation ensures spatial and temporal accuracy of the expression of developmentally regulated genes. So far, few studies have focused on the relationship between epigenetic regulation and mammalian sex development, despite this being an interesting area of research. Sex development consists of three sequential stages: the undifferentiated stage, gonadal differentiation into testes or ovaries, and differentiation of internal and external genitalia. Some genetic studies have revealed that epigenetic regulation is required for proper gonadal differentiation in mice. Particularly, the epigenetic machinery plays an integral part in sex determination, which is the first step of gonadal differentiation. Mammalian sex determination is triggered by activation of the mammalian sex-determining gene, Sry, in a spatially and temporally accurate manner. Several studies have demonstrated that expression of Sry is controlled not only by specific transcription factors but also by the epigenetic machinery. Here, we focus on the epigenetic regulation of Sry expression., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Enhancer of zeste homolog 2 (EZH2), the critical component of polycomb group protein family, has been demonstrated to be overexpressed in various types of human cancer, including hepatocellular carcinoma, breast, bladder and lung cancer. The mechanism of how EZH2 promotes oncogenesis has also been well studied. However, little is known about the role of EZH2 in colorectal cancer (CRC). The main purpose of the present study was to analyze the association between EZH2 expression and the clinicopathological features of CRC. Therefore, the mRNA and protein expression levels were analyzed in tumor tissues and adjacent non-cancerous tissues by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The expression of EZH2 was demonstrated to be significantly increased in tumor tissues compared with adjacent noncancerous tissues, according to the results of western blot analysis and RT-qPCR in the majority of cases. Patients with low EZH2 expression had a longer overall survival rate compared with those with high EZH2 expression. An analysis of the association between clinicopathological features and EZH2 expression indicated that high EZH2 expression was significantly associated with tumor stage, tumor size, histological differentiation and lymph node metastasis. Multivariate analysis demonstrated that high EZH2 expression was an independent predictor of overall survival. In conclusion, to the best of our knowledge, the data presented in the present study is the first to indicate that EZH2 is upregulated in CRC and may serve as a predictor of poor outcome for patients with CRC.

Yin yang 1 (YY1) is a ubiquitous transcription factor and mammalian polycomb group protein (PcG) with important functions to regulate embryonic development, lineage differentiation, and cell proliferation. YY1 mediates stable PcG-dependent transcriptional repression via recruitment of PcG proteins that catalyze histone modifications. Many questions remain unanswered regarding how cell- and tissue-specificity is achieved by PcG proteins. Here, we demonstrate that a conditional knockout of Yy1 in hematopoietic stem cells (HSCs) decreases long-term repopulating activity and ectopic YY1 expression expands HSCs. Although the YY1 PcG domain is required for Igκ chain rearrangement in B cells, the YY1 mutant lacking the PcG domain retained the capacity to stimulate HSC self-renewal. YY1 deficiency deregulated the genetic network governing HSC cell proliferation and impaired stem cell factor/c-Kit signaling, disrupting mechanisms conferring HSC quiescence. These results reveal a mechanism for how a ubiquitously expressed transcriptional repressor mediates lineage-specific functions to control adult hematopoiesis., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Polycomb group genes are a general class of regulators that are responsible for maintaining homeotic gene expression throughout cell division. Polycomb group expression plays an important role in oncogenesis of several types of human cancer. Melanoma nuclear protein 18 and B-cell-specific Moloney leukemia virus insert site 1 are key Polycomb group proteins. Studies have shown that melanoma nuclear protein 18 is a potential tumor suppression, and B-cell-specific Moloney leukemia virus insert site 1 is overexpressed in several human malignancies. However, the roles of melanoma nuclear protein 18 and B-cell-specific Moloney leukemia virus insert site 1 in esophageal squamous cell carcinoma are still unclear. In this study, we analyzed the expression levels of melanoma nuclear protein 18 and B-cell-specific Moloney leukemia virus insert site 1 in 89 esophageal cancer tissues and paired normal mucosal tissues using immunohistochemistry, Western blotting, and quantitative real-time polymerase chain reaction analyses. We found that the expression of melanoma nuclear protein 18 in the carcinoma tissues was significantly lower than that in the noncancerous mucosal tissues ( P < .05), and B-cell-specific Moloney leukemia virus insert site 1 expression in the carcinoma tissues was significantly higher than that in the noncancerous mucosal tissues ( P < .05). In addition, the expression of melanoma nuclear protein 18 was correlated with clinical stage, depth of invasion, and lymph node metastasis ( P < .05) but was not correlated with gender, age, degree of differentiation, or disease-free survival ( P > .05). B-cell-specific Moloney leukemia virus insert site 1 expression was strongly correlated with the degree of differentiation, clinical stage, and lymph node metastasis ( P <.05) but was not correlated with the gender, age, depth of invasion or disease-free survival ( P > .05). Moreover, there was a negative correlation between melanoma nuclear protein 18 and B-cell-specific Moloney leukemia virus insert site 1 expressions in esophageal squamous cell carcinoma ( P < .05). Our study suggests that melanoma nuclear protein 18 and B-cell-specific Moloney leukemia virus insert site 1 may play a crucial role in esophageal squamous cell carcinoma. Melanoma nuclear protein 18 or B-cell-specific Moloney leukemia virus insert site 1 may be a potential biomarker for diagnosis and prognosis of esophageal squamous cell carcinoma.

High expression of B-cell specific Moloney leukemia virus insert site 1 (Bmi-1) and peptidyl arginine deiminase IV (PADI4) is associated with esophageal carcinoma. However, few studies have investigated the association between the Bmi-1 and PADI4 genes. The aim of the present study was to evaluate the expression of Bmi-1 and PADI4 and identify the association between the Bmi-1 and PADI4 genes in esophageal squamous cell carcinoma (ESCC) tissues. Bmi-1 and PADI4 gene expression levels were measured using immunohistochemistry, western blotting and reverse transcription-quantitative polymerase chain reaction in ESCC tissues from 86 patients who had not received pre-operative chemoradiation. The results revealed that the Bmi-1 and PADI4 genes had increased expression in carcinoma tissues compared with pericarcinous tissue (P<0.05). Bmi-1 gene expression was revealed to be associated with differentiation degree, clinical stage and lymph node metastasis (P<0.05), but had no association with gender, age or depth of invasion (P>0.05). The expression of PADI4 was associated with clinical stage, depth of invasion and lymph node metastasis (P<0.05), but was not associated with gender, age or differentiation degree (P>0.05). In addition, there was a positive association between Bmi-1 and PADI4 gene expression in ESCC (P<0.05). These results indicated that Bmi-1 and PADI4 positively regulate carcinogenesis and progression of ESCC.

The polycomb group (PcG) proteins are a large and diverse family that epigenetically repress the transcription of key developmental genes. They form three broad groups of polycomb repressive complexes (PRCs) known as PRC1, PRC2 and Polycomb Repressive DeUBiquitinase, each of which modifies and/or remodels chromatin by distinct mechanisms that are tuned by having variable compositions of core and accessory subunits. Until recently, relatively little was known about how the various PcG proteins assemble to form the PRCs; however, studies by several groups have now allowed us to start piecing together the PcG puzzle. Here, we discuss some highlights of recent PcG structures and the insights they have given us into how these complexes regulate transcription through chromatin., (© 2017 The Author(s).)

Background: MicroRNAs (miRNAs) are short non-coding RNAs that are emerging as important post-transcriptional regulators of neuronal and synaptic development. The precise impact of miRNAs on presynaptic function and neurotransmission remains, however, poorly understood., Results: Here, we identify miR-27b-an abundant neuronal miRNA implicated in neurological disorders-as a global regulator of the presynaptic transcriptome. miR-27b influences the expression of three quarters of genes associated with presynaptic function in cortical neurons. Contrary to expectation, a large majority of these genes are up-regulated by miR-27b. This stimulatory effect is mediated by miR-27b-directed silencing of several transcriptional repressors that cooperate to suppress the presynaptic transcriptome. The strongest repressive activity appears to be mediated by Bmi1, a component of the polycomb repressive complex implicated in self-renewal of neural stem cells. miR-27b knockdown leads to reduced synaptogenesis and to a marked decrease in neural network activity, which is fully restored by RNAi-mediated silencing of Bmi1., Conclusions: We conclude that silencing of Bmi1 by miR-27b relieves repression of the presynaptic transcriptome and supports neurotransmission in cortical networks. These results expand the repressive activity of Bmi1 to genes involved in synaptic function and identify a unique post-transcriptional circuitry that stimulates expression of synaptic genes and promotes synapse differentiation.

Drosophila Jumonji/Jarid2 (dJmj) has been identified as a component of Polycomb repressive complex 2. However, it is suggested that dJmj has both PRC-dependent and -independent roles. Subcellular localization of dJmj during spermatogenesis is unknown. We therefore performed immunocytochemical analyses with specific antibodies to dJmj and tri-methylation at lysine 27 on histone H3 (H3K27me3). Interestingly, dJmj exclusively localizes at nucleolus in the late growth stage. Examination of the dJmj localization in various Polycomb group (PcG) mutant lines at the late growth stage allowed identification of some PcG genes, including Polycomb (Pc), to be responsible for dJmj recruitment to nucleolus. In addition, we found that size of nucleolus was decreased in some of these mutant lines. In a mutant of testis-specific TAF homolog (tTAF) that is responsible for nucleolus localization of Pc, dJmj signals were detected not only at nucleolus but also on the condensed chromatin in the late growth stage. Duolink In situ Proximity ligation assay clarified that Pc interacts with dJmj at nucleolus in the late growth stage. Furthermore, the level of H3K27me3 decreased in nuclei at this stage. Taken together, we conclude that tTAF is responsible for recruitments of dJmj to nucleolus in the late growth stage that appears to be mediated by Pc. Compartmentalization of dJmj in nucleolus together with some of PcG may be necessary to de-repress the expression of genes required to cellular growth and proliferation in the following meiotic divisions.

In tumor tissues, alterations of gene expression caused by aberrant epigenetic modifications confer phenotypic diversity on malignant cells. Although 3-deazaneplanocin A (DZNep) has been shown to reactivate tumor suppressor genes in several cancer cells, it remains unclear whether DZNep attenuates the malignant phenotypes of oral squamous cell carcinoma (OSCC) cells. In this study, we investigated the effect of DZNep on the expression of genes related to aggressive phenotypes, such as epithelial-mesenchymal transition, in OSCC cells. We found that DZNep reduced the cellular levels of polycomb group proteins (EZH2, SUZ12, BMI1, and RING1A) and the associated trimethylation of Lys27 on histone H3 and monoubiquitination of Lys119 on histone H2A in the poorly differentiated OSCC cell line SAS. Immunocytochemical staining demonstrated that DZNep induced the reorganization of filamentous actin and the membrane localization of E-cadherin associated with cell-cell adhesions. We also found an inhibitory effect of DZNep on cell proliferation using a WST assay. Finally, quantitative RT-PCR analysis demonstrated that genes involved in the aggressive phenotypes (TWIST2, EGFR, ACTA2, TGFB1, WNT5B, and APLIN) were down-regulated, whereas epithelial phenotype genes (CDH1, CLDN4, IVL, and TGM1) were up-regulated in SAS cells treated with DZNep. Collectively, our findings suggest that DZNep reverses the aggressive characteristics of OSCC cells through the dynamic regulation of epithelial plasticity via the reprogramming of gene expression patterns., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Nspc1 is an identified transcription repressor. However, transiently up-regulated or down-regulated Nspc1 in P19 embryonal carcinoma cells affects expression levels of Oct4, Sox2 and Nanog in a positive correlation. Luciferase activity assays verified that Nspc1 regulates the Oct4 promoter in a dose dependent manner. ChIP assay shows that Nspc1 activates Oct4 by directly binding to the (-1021 to -784) region of Oct4 promoter. Dominant negative analysis indicated the activation is dependent on the retinoid acid response element (RARE). We demonstrated Nspc1 has a positive role in maintaining the pluripotency of P19 cells by directly regulating Oct4., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Imprinted genes play important roles in placenta development and function. Parthenogenetic embryos, deficient in paternally expressed imprinted genes, lack extra-embryonic tissues of the trophoblast lineage. Parthenogenetic trophoblast stem cells (TSCs) are extremely difficult to derive, suggesting that an imprinted gene(s) is necessary for TSC establishment or maintenance. In a candidate study, we were able to narrow the list to one known paternally expressed gene, Sfmbt2. We show that mouse embryos inheriting a paternal Sfmbt2 gene trap null allele have severely reduced placentae and die before E12.5 due to reduction of all trophoblast cell types. We infected early embryos with lentivirus vectors expressing anti-Sfmbt2 shRNAs and found that TSC derivation was significantly reduced. Together, these observations support the hypothesis that loss of SFMBT2 results in defects in maintenance of trophoblast cell types necessary for development of the extra-embryonic tissues, the placenta in particular.

Polycomb Repressive Complex 2 (PRC2) mediates transcriptional silencing by catalyzing histone H3 lysine 27 trimethylation (H3K27me3), but its role in the maturation of postmitotic mammalian neurons remains largely unknown. We report that the PRC2 paralogs Ezh1 and Ezh2 are differentially expressed during hippocampal development. We show that depletion of Ezh2 leads to increased expression of PSD-95, a critical plasticity gene, and that reduced PSD-95 gene transcription is correlated with enrichment of Ezh2 at the PSD-95 gene promoter; however, the H3K27me3 epigenetic mark is not present at the PSD-95 gene promoter, likely due to the antagonizing effects of the H3S28P and H3K27Ac marks and the activity of the H3K27 demethylases JMJD3 and UTX. In contrast, increased PSD-95 gene transcription is accompanied by the presence of Ezh1 and elongation-engaged RNA Polymerase II complexes at the PSD-95 gene promoter, while knock-down of Ezh1 reduces PSD-95 transcription. These results indicate that Ezh1 and Ezh2 have antagonistic roles in regulating PSD-95 transcription., (© 2013.)

Flat-type "biliary intraepithelial neoplasia (BilIN)" and papillary-type "intraductal papillary neoplasm of the bile duct (IPN-B)" are proposed as precursors of invasive, perihilar intrahepatic cholangiocarcinoma (ICC). Three carcinogenetic pathways are proposed: BilIN progressing to tubular adenocarcinoma, and IPN-B progressing to tubular adenocarcinoma or to colloid carcinoma. Carcinogenesis via BilIN was characterized by mucin core protein 2-/cytokeratin 20-(MUC2-/CK20-) with MUC1 expression, while carcinogenesis via IPN-B leading to tubular adenocarcinoma was associated with MUC1 expression or that to colloid carcinoma with MUC1-negativity. In both the BilIN and IPNB series, the expression of p21, p53, and cyclin D1 was upregulated with histological progression. Interestingly, p53 expression was upregulated at the invasive stage of BilIN, but was low in noninvasive BilIN, while p53 expression was upregulated in IPN-B1 and reached a plateau in IPN-B2 and invasive ICC. Expression of p16(INK4a), which was frequent in BilIN1, was decreased in BilIN-2/3 and invasive carcinoma. EZH2 expression showed a stepwise increase from BilIN to invasive carcinoma. Membranous expression of β-catenin and E-cadherin was more markedly decreased in ICC with BilIN than in ICC with IPNB. Interestingly, disruption of the membranous distribution of β-catenin and E-cadherin seems to result in the invasion and metastasis of carcinoma cells of BilIN and IPN-B expressing MMP-7 and MT1-MMP. Increased expression of cyclin D1 and c-myc was more frequent in the IPNB lineage than BilIN lineage, possibly related to the Wnt signaling pathway associated with the nuclear accumulation of β-catenin. In conclusion, BilIN and IPN-B progress to invasive ICC through characteristic multistep processes.