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  • The stress oncoprotein LEDGF/p75 interacts with the methyl CpG binding protein MeCP2 and influences its transcriptional activity. 22275515

    The lens epithelium-derived growth factor p75 (LEDGF/p75) is a transcription coactivator that promotes resistance to oxidative stress- and chemotherapy-induced cell death. LEDGF/p75 is also known as the dense fine speckles autoantigen of 70 kDa (DFS70) and has been implicated in cancer, HIV-AIDS, autoimmunity, and inflammation. To gain insights into mechanisms by which LEDGF/p75 protects cancer cells against stress, we initiated an analysis of its interactions with other transcription factors and the influence of these interactions on stress gene activation. We report here that both LEDGF/p75 and its short splice variant LEDGF/p52 interact with MeCP2, a methylation-associated transcriptional modulator, in vitro and in various human cancer cells. These interactions were established by several complementary approaches: transcription factor protein arrays, pull-down and AlphaScreen assays, coimmunoprecipitation, and nuclear colocalization by confocal microscopy. MeCP2 was found to interact with the N-terminal region shared by LEDGF/p75 and p52, particularly with the PWWP-CR1 domain. Like LEDGF/p75, MeCP2 bound to and transactivated the Hsp27 promoter (Hsp27pr). LEDGF/p75 modestly enhanced MeCP2-induced Hsp27pr transactivation in U2OS osteosarcoma cells, whereas this effect was more pronounced in PC3 prostate cancer cells. LEDGF/p52 repressed Hsp27pr activity in U2OS cells. Interestingly, siRNA-induced silencing of LEDGF/p75 in U2OS cells dramatically elevated MeCP2-mediated Hsp27pr transactivation, whereas this effect was less pronounced in PC3 cells depleted of LEDGF/p75. These results suggest that the LEDGF/p75-MeCP2 interaction differentially influences Hsp27pr activation depending on the cellular and molecular context. These findings are of significance in understanding the contribution of this interaction to the activation of stress survival genes.
    Document Type:
    Reference
    Product Catalog Number:
    07-013
    Product Catalog Name:
    Anti-MeCP2 Antibody

  • The methyl-CpG-binding protein MeCP2 links DNA methylation to histone methylation. 12427740

    DNA methylation plays an important role in mammalian development and correlates with chromatin-associated gene silencing. The recruitment of MeCP2 to methylated CpG dinucleotides represents a major mechanism by which DNA methylation can repress transcription. MeCP2 silences gene expression partly by recruiting histone deacetylase (HDAC) activity, resulting in chromatin remodeling. Here, we show that MeCP2 associates with histone methyltransferase activity in vivo and that this activity is directed against Lys(9) of histone H3. Two characterized repression domains of MeCP2 are involved in tethering the histone methyltransferase to MeCP2. We asked if MeCP2 can deliver Lys(9) H3 methylation to the H19 gene, whose activity it represses. We show that the presence of MeCP2 on nucleosomes within the repressor region of the H19 gene (the differentially methylated domain) coincides with an increase in H3 Lys(9) methylation. Our data provide evidence that MeCP2 reinforces a repressive chromatin state by acting as a bridge between two global epigenetic modifications, DNA methylation and histone methylation.
    Document Type:
    Reference
    Product Catalog Number:
    07-013
    Product Catalog Name:
    Anti-MeCP2 Antibody

  • DNA methylation represses IFN-gamma-induced and signal transducer and activator of transcription 1-mediated IFN regulatory factor 8 activation in colon carcinoma cells. 19074829

    IFN regulatory factor 8 (IRF8) is both constitutively expressed and IFN-gamma inducible in hematopoietic and nonhematopoietic cells. We have shown that IRF8 expression is silenced by DNA methylation in human colon carcinoma cells, but the molecular mechanism underlying methylation-dependent IRF8 silencing remains elusive. In this study, we observed that IRF8 protein level is inversely correlated with the methylation status of the IRF8 promoter and the metastatic phenotype in human colorectal carcinoma specimens in vivo. Demethylation treatment or knocking down DNMT1 and DNMT3b expression rendered the tumor cells responsive to IFN-gamma to activate IRF8 transcription in vitro. Bisulfite genomic DNA sequencing revealed that the entire CpG island of the IRF8 promoter is methylated. Electrophoresis mobility shift assay revealed that DNA methylation does not directly inhibit IFN-gamma-activated phosphorylated signal transducer and activator of transcription 1 (pSTAT1) binding to the IFN-gamma activation site element in the IRF8 promoter in vitro. Chromatin immunoprecipitation assay revealed that pSTAT1 is associated with the IFN-gamma activation site element of the IRF8 promoter in vivo regardless of the methylation status of the IRF8 promoter. However, DNA methylation results in preferential association of PIAS1, a potent inhibitor of pSTAT1, with pSTAT1 in the methylated IRF8 promoter region. Silencing methyl-CpG binding domain protein 1 (MBD1) expression resulted in IRF8 activation by IFN-gamma in human colon carcinoma cells with methylated IRF8 promoter. Our data thus suggest that human colon carcinoma cells silence IFN-gamma-activated IRF8 expression through MBD1-dependent and PIAS1-mediated inhibition of pSTAT1 function at the methylated IRF8 promoter.
    Document Type:
    Reference
    Product Catalog Number:
    S7820

  • The methylated-DNA binding protein MBD2 enhances NGFI-A (egr-1)-mediated transcriptional activation of the glucocorticoid receptor. 25135974

    Variations in maternal care in the rat influence the epigenetic state and transcriptional activity of glucocorticoid receptor (GR) gene in the hippocampus. The mechanisms underlying this maternal effect remained to be defined, including the nature of the relevant maternally regulated intracellular signalling pathways. We show here that increased maternal licking/grooming (LG), which stably enhances hippocampal GR expression, paradoxically increases hippocampal expression of the methyl-CpG binding domain protein-2 (MBD2) and MBD2 binding to the exon 17 GR promoter. Knockdown experiments of MBD2 in hippocampal primary cell culture show that MBD2 is required for activation of exon 17 GR promoter. Ectopic co-expression of nerve growth factor-inducible protein A (NGFI-A) with MBD2 in HEK 293 cells with site-directed mutagenesis of the NGFI-A response element within the methylated exon 17 GR promoter supports the hypothesis that MBD2 collaborates with NGFI-A in binding and activation of this promoter. These data suggest a possible mechanism linking signalling pathways, which are activated by behavioural stimuli and activation of target genes.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Genome-wide DNA methylation profiles in hematopoietic stem and progenitor cells reveal overrepresentation of ETS transcription factor binding sites. 22684279

    DNA methylation is an essential epigenetic mark that is required for normal development. Knockout of the DNA methyltransferase enzymes in the mouse hematopoietic compartment reveals that methylation is critical for hematopoietic differentiation. To better understand the role of DNA methylation in hematopoiesis, we characterized genome-wide DNA methylation in primary mouse hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), and erythroblasts (ERYs). Methyl binding domain protein 2 (MBD) enrichment of DNA followed by massively parallel sequencing (MBD-seq) was used to map genome-wide DNA methylation. Globally, DNA methylation was most abundant in HSCs, with a 40% reduction in CMPs, and a 67% reduction in ERYs. Only 3% of peaks arise during differentiation, demonstrating a genome-wide decline in DNA methylation during erythroid development. Analysis of genomic features revealed that 98% of promoter CpG islands are hypomethylated, while 20%-25% of non-promoter CpG islands are methylated. Proximal promoter sequences of expressed genes are hypomethylated in all cell types, while gene body methylation positively correlates with gene expression in HSCs and CMPs. Elevated genome-wide DNA methylation in HSCs and the positive association between methylation and gene expression demonstrates that DNA methylation is a mark of cellular plasticity in HSCs. Using de novo motif discovery, we identified overrepresented transcription factor consensus binding motifs in methylated sequences. Motifs for several ETS transcription factors, including GABPA and ELF1, are overrepresented in methylated regions. Our genome-wide survey demonstrates that DNA methylation is markedly altered during myeloid differentiation and identifies critical regions of the genome and transcription factor programs that contribute to hematopoiesis.
    Document Type:
    Reference
    Product Catalog Number:
    17-10085
    Product Catalog Name:
    Magna ChIP™ A/G Chromatin Immunoprecipitation Kit

  • MeCP2 knockdown reveals DNA methylation-independent gene repression of target genes in living cells and a bias in the cellular location of target gene products. 18506103

    MeCP2, a methyl-CpG binding domain (MBD) protein, is known to bind to methylated CpG sites via a conserved MBD, leading to transcriptional repression. However, studies in cell-free system for gene repression and MeCP2 binding have suggested that DNA methylation-independent repression also occurs in living cells. It has been difficult to characterize the target genes of MeCP2 because a limited number have been identified to date. In this context, we screened for MeCP2 target genes using knockdown (KD) experiments combined with microarray gene expression analyses. Of the 49 genes that showed a more than three-fold increase in expression in two independent KD experiments conducted with different siRNA sets, unexpectedly, half (24 genes) did not contain promoter CpG islands (CGIs). Of seven selected genes that did contain CGIs, only two were methylated at the CGI, bound MeCP2 before KD, and reduced MeCP2 after KD. For three, MeCP2 was observed to bind to the unmethylated CGI before KD, and for one MeCP2 was reduced after KD. Another two genes neither had DNA methylation nor bound MeCP2 before KD. Gene ontology analysis suggested that MeCP2 represses a certain group of genes. These results suggest that in addition to the canonical gene repression function, MeCP2 can repress gene expression by binding to unmethylated DNA in particular genes in living cells.
    Document Type:
    Reference
    Product Catalog Number:
    07-199
    Product Catalog Name:
    Anti-MBD2/3 Antibody

  • Maintenance of paternal methylation and repression of the imprinted H19 gene requires MBD3. 17708683

    Paternal repression of the imprinted H19 gene is mediated by a differentially methylated domain (DMD) that is essential to imprinting of both H19 and the linked and oppositely imprinted Igf2 gene. The mechanisms by which paternal-specific methylation of the DMD survive the period of genome-wide demethylation in the early embryo and are subsequently used to govern imprinted expression are not known. Methyl-CpG binding (MBD) proteins are likely candidates to explain how these DMDs are recognized to silence the locus, because they preferentially bind methylated DNA and recruit repression complexes with histone deacetylase activity. MBD RNA and protein are found in preimplantation embryos, and chromatin immunoprecipitation shows that MBD3 is bound to the H19 DMD. To test a role for MBDs in imprinting, two independent RNAi-based strategies were used to deplete MBD3 in early mouse embryos, with the same results. In RNAi-treated blastocysts, paternal H19 expression was activated, supporting the hypothesis that MBD3, which is also a member of the Mi-2/NuRD complex, is required to repress the paternal H19 allele. RNAi-treated blastocysts also have reduced levels of the Mi-2/NuRD complex protein MTA-2, which suggests a role for the Mi-2/NuRD repressive complex in paternal-specific silencing at the H19 locus. Furthermore, DNA methylation was reduced at the H19 DMD when MBD3 protein was depleted. In contrast, expression and DNA methylation were not disrupted in preimplantation embryos for other imprinted genes. These results demonstrate new roles for MBD3 in maintaining imprinting control region DNA methylation and silencing the paternal H19 allele. Finally, MBD3-depleted preimplantation embryos have reduced cell numbers, suggesting a role for MBD3 in cell division.
    Document Type:
    Reference
    Product Catalog Number:
    06-599
    Product Catalog Name:
    Anti-acetyl-Histone H3 Antibody