The Polycomb group (PcG) proteins mediate heritable silencing of developmental regulators in metazoans, participating in one of two distinct multimeric protein complexes, the Polycomb repressive ...complexes 1 (PRC1) and 2 (PRC2). Although PRC2 has been shown to share target genes with the core transcription network, including Oct3/4, to maintain embryonic stem (ES) cells, it is still unclear whether PcG proteins and the core transcription network are functionally linked. Here, we identify an essential role for the core PRC1 components Ring1A/B in repressing developmental regulators in mouse ES cells and, thereby, in maintaining ES cell identity. A significant proportion of the PRC1 target genes are also repressed by Oct3/4. We demonstrate that engagement of PRC1 at target genes is Oct3/4-dependent, whereas engagement of Oct3/4 is PRC1-independent. Moreover, upon differentiation induced by Gata6 expression, most of the Ring1A/B target genes are derepressed and the binding of Ring1A/B to their target loci is also decreased. Collectively, these results indicate that Ring1A/B-mediated Polycomb silencing functions downstream of the core transcriptional regulatory circuitry to maintain ES cell identity.
Sall4 is a mouse homolog of a causative gene of the autosomal dominant disorder Okihiro syndrome. We previously showed that the absence of Sall4 leads to lethality during peri‐implantation and that ...Sall4‐null embryonic stem (ES) cells proliferate poorly with intact pluripotency when cultured on feeder cells. Here, we report that, in the absence of feeder cells, Sall4‐null ES cells express the trophectoderm marker Cdx2, but are maintained for a long period in an undifferentiated state with minimally affected Oct3/4 expression. Feeder‐free Sall4‐null ES cells contribute solely to the inner cell mass and epiblast in vivo, indicating that these cells still retain pluripotency and do not fully commit to the trophectoderm. These phenotypes could arise from derepression of the Cdx2 promoter, which is normally suppressed by Sall4 and the Mi2/NuRD HDAC complex. However, proliferation was impaired and G1 phase prolonged in the absence of Sall4, suggesting another role for Sall4 in cell cycle control. Although Sall1, also a Sall family gene, is known to genetically interact with Sall4 in vivo, Sall1‐null ES cells have no apparent defects and no exacerbation is observed in ES cells lacking both Sall1 and Sall4, compared with Sall4‐null cells. This suggests a unique role for Sall4 in ES cells. Thus, though Sall4 does not contribute to the central machinery of the pluripotency, it stabilizes ES cells by repressing aberrant trophectoderm gene expression. STEM CELLS 2009;27:796–805
Cell lineages generated during development and tissue maintenance are derived from self‐renewing stem cells by differentiation of their committed progeny. Recent studies suggest that epigenetic ...mechanisms, and in particular the Polycomb group (PcG) of genes, play important roles in controlling stem cell self‐renewal. Here, we address PcG regulation of stem cell self‐renewal and differentiation through inactivation of Ring1B, a histone H2A E3 monoubiquitin ligase, in embryonic neural stem cells (NSCs) from the olfactory bulb of a conditional mouse mutant line. We show that neural stem/progenitor cell proliferation in vivo and in neurosphere assays is impaired, lacking Ring1B, and their self‐renewal and multipotential abilities, assessed as sphere formation and differentiation from single cells, are severely affected. We also observed unscheduled neuronal, but not glial, differentiation of mutant stem/progenitor cells under proliferating conditions, an alteration enhanced in cells also lacking Ring1A, the Ring1B paralog, some of which turned into morphologically identifiable neurons. mRNA analysis of mutant cells showed upregulation of some neuronal differentiation–related transcription factors and the cell proliferation inhibitor Cdkn1a/p21, as well as downregulation of effectors of the Notch signaling pathway, a known inhibitor of neuronal differentiation of stem/progenitor cells. In addition, differentiation studies of Ring1B‐deficient progenitors showed decreased oligodendrocyte formation in vitro and enhanced neurogenesis and reduced gliogenesis in vivo. These data suggest a role for Ring1B in maintenance of the undifferentiated state of embryonic neural stem/progenitor cells. They also suggest that Ring1B may modulate the differentiation potential of NSCs to neurons and glia. STEM CELLS 2009;27:1559–1570
To address the molecular mechanisms underlying Polycomb group (PcG)-mediated repression of Hox gene expression, we have focused on the binding patterns of PcG gene products to the flanking regions of ...the Hoxb8 gene in expressing and non-expressing tissues. In parallel, we followed the distribution of histone marks of transcriptionally active H3 acetylated on lysine 9 (H3-K9) and methylated on lysine 4 (H3-K4), and of transcriptionally inactive chromatin trimethylated on lysine 27 (H3-K27). Chromatin immunoprecipitation revealed that the association of PcG proteins, and H3-K9 acetylation and H3-K27 trimethylation around Hoxb8 were distinct in tissues expressing and not expressing the gene. We show that developmental changes of these epigenetic marks temporally coincide with the misexpression of Hox genes in PcG mutants. Functional analyses, using mutant alleles impairing the PcG class 2 component Rnf2 or the Suz12 mutation decreasing H3-K27 trimethylation, revealed that interactions between class 1 and class 2 PcG complexes, mediated by trimethylated H3-K27, play decisive roles in the maintenance of Hox gene repression outside their expression domain. Within the expression domains, class 2 PcG complexes appeared to maintain the transcriptionally active status via profound regulation of H3-K9 acetylation. The present study indicates distinct roles for class 2 PcG complexes in transcriptionally repressed and active domains of Hoxb8 gene.
The products of the Polycomb group of genes form complexes that maintain the state of transcriptional repression of several genes with relevance to development and in cell proliferation. We have ...identified Ring1B, the product of the Ring1B gene ( Rnf2 â Mouse Genome Informatics), by means of its interaction with the Polycomb group protein Mel18. We describe biochemical and genetic studies directed to understand the biological role of Ring1B. Immunoprecipitation studies indicate that Ring1B form part of protein complexes containing the products of other Polycomb group genes, such as Rae28/Mph1 and M33, and that this complexes associate to chromosomal DNA. We have generated a mouse line bearing a hypomorphic Ring1B allele, which shows posterior homeotic transformations of the axial skeleton and a mild derepression of some Hox genes ( Hoxb4 , Hoxb6 and Hoxb8 ) in cells anterior to their normal boundaries of expression in the mesodermal compartment. By contrast, the overexpression of Ring1B in chick embryos results in the repression of Hoxb9 expression in the neural tube. These results, together with the genetic interactions observed in compound Ring1B / Mel18 mutant mice, are consistent with a role for Ring1B in the regulation of Hox gene expression by Polycomb group complexes.
Cellular senescence is a program in normal cells triggered in response to various types of stress that cells experience when they are explanted into culture. In this study, functional analyses on the ...role of the class II polycomb complex in cellular senescence were performed using mouse embryo fibroblasts (MEFs) with a genetically deleted member of the complex, Mel18. Mel18-null MEFs undergo typical premature senescence accompanied by the up-regulation of ARF/p53/p16INK⁴a and decrease of Ring1b/Bmi1. Our results demonstrated that ARF or p53 deletion cancels the senescence in Mel18-null MEFs, and the fact that p16INK⁴a is up-regulated in double-null MEFs suggests that the ARF/p53 pathway plays a central role in stress-induced senescence. The in vivo binding of Ring1b and E2F3b to the ARF promoter decreased progressively in senescence, and Mel18 inactivation accelerated the exfoliation of Ring1b/E2F3b from the promoter sequence, indicating the cooperation of polycombs/E2F3b on ARF expression and cellular senescence. Taken together, it seems that class II polycomb proteins and E2F3b dually control cellular senescence via the ARF/p53 pathway.
Nucleosome depletion in the promoters has been indicated in yeasts, suggesting that nucleosome depletion in promoter might be a fundamental feature of eukaryotic transcriptional regulation. We ...compared the relationship between histone H3 acetylation at lysine 9 (K9) in promoter, gene expression level, and nucleosome density in the vicinity of the transcription start site (TSS), in HepG2 cells (human hepatocellular liver carcinoma cells). We found that the density of nucleosome is relatively low in the close vicinity of TSS flanked by H3 K9 significantly acetylated promoter, compared with that for genes without marked H3 K9 acetylation in promoter, regardless of their transcriptional activation status. Our results imply that the relative nucleosome depletion in the vicinity of TSS is not necessarily associated with active transcription, but with histone H3 K9 acetylation in promoter.
Abstract
The bursa of Fabricius is a gut-associated lymphoid organ that is essential for the generation of a diversified B cell repertoire in the chicken. We describe here a novel gene preferentially ...expressed in bursal B cells. The gene encodes an 85-kDa protein, designated BASH (B cell adaptor containing SH2 domain), that contains N-terminal acidic domains with SH2 domain-binding phosphotyrosine-based motifs, a proline-rich domain, and a C-terminal SH2 domain. BASH shows a substantial sequence similarity to SLP-76, an adaptor protein functioning in TCR-signal transduction. BASH becomes tyrosine-phosphorylated with the B cell Ag receptor (BCR) cross-link or by coexpression with Syk and Lyn and associates with signaling molecules including Syk and a putative chicken Shc homologue. Overexpression of BASH results in suppression of the NF-AT activation induced by BCR-cross-linking. These findings suggest that BASH is involved in BCR-mediated signal transduction and could play a critical role in B cell development in the bursa.
Cross-linking of the high-affinity IgE receptor (FcϵRI) on mast cells by IgE–antigen complex triggers signal transduction cascades leading to the release of inflammatory mediators and production of ...cytokines, which are critical for the development of allergic reactions. We have identified a novel member of the BASH/SLP-76 immunoreceptor-coupled adaptor family expressed in mast cells, termed MIST (for mast cell immunoreceptor signal transducer), which has later been found to be identical to a recently reported cytokine-dependent hemopoietic cell linker, Clnk. Upon FcϵRI cross-linking, MIST/Clnk is tyrosine phosphorylated and associates with signaling proteins, phospholipase Cγ, Vav, Grb2 and linker for activation of T cells (LAT). Overexpression of a mutant form of MIST/Clnk inhibited FcϵRI-mediated degranulation, increase in intracellular Ca2+, NF-AT activation and phosphorylation of LAT. As a crucial signaling component for FcϵRI-induced mast cell degranulation, MIST/Clnk might serve as a target for anti-allergic therapy.
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