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methyl CpG binding domain protein


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  • An intrinsically disordered region of methyl-CpG binding domain protein 2 (MBD2) recruits the histone deacetylase core of the NuRD complex. 25753662

    The MBD2-NuRD (Nucleosome Remodeling and Deacetylase) complex is an epigenetic reader of DNA methylation that regulates genes involved in normal development and neoplastic diseases. To delineate the architecture and functional interactions of the MBD2-NuRD complex, we previously solved the structures of MBD2 bound to methylated DNA and a coiled-coil interaction between MBD2 and p66α that recruits the CHD4 nucleosome remodeling protein to the complex. The work presented here identifies novel structural and functional features of a previously uncharacterized domain of MBD2 (MBD2IDR). Biophysical analyses show that the MBD2IDR is an intrinsically disordered region (IDR). However, despite this inherent disorder, MBD2IDR increases the overall binding affinity of MBD2 for methylated DNA. MBD2IDR also recruits the histone deacetylase core components (RbAp48, HDAC2 and MTA2) of NuRD through a critical contact region requiring two contiguous amino acid residues, Arg(286) and Leu(287). Mutating these residues abrogates interaction of MBD2 with the histone deacetylase core and impairs the ability of MBD2 to repress the methylated tumor suppressor gene PRSS8 in MDA-MB-435 breast cancer cells. These findings expand our knowledge of the multi-dimensional interactions of the MBD2-NuRD complex that govern its function.
    Tipo de documento:
    Referencia
    Referencia del producto:
    05-814
    Nombre del producto:
    Anti-HDAC2 Antibody, clone 3F3

  • Dynamic changes in the epigenomic state and nuclear organization of differentiating mouse embryonic stem cells. 17397393

    Changes in nuclear organization and the epigenetic state of the genome are important driving forces for developmental gene expression. However, a strategy that allows simultaneous visualization of the dynamics of the epigenomic state and nuclear structure has been lacking to date. We established an experimental system to observe global DNA methylation in living mouse embryonic stem (ES) cells. The methylated DNA binding domain (MBD) and the nuclear localization signal (nls) sequence coding for human methyl CpG-binding domain protein 1 (MBD1) were fused to the enhanced green fluorescent protein (EGFP) reporter gene, and ES cell lines carrying the construct (EGFP-MBD-nls) were established. The EGFP-MBD-nls protein was used to follow DNA methylation in situ under physiological conditions. We also monitored the formation and rearrangement of methylated heterochromatin using EGFP-MBD-nls. Pluripotent mouse ES cells showed unique nuclear organization in that methylated centromeric heterochromatin coalesced to form large clusters around the nucleoli. Upon differentiation, the organization of these heterochromatin clusters changed dramatically. Time-lapse microscopy successfully captured a moment of dramatic change in chromosome positioning during the transition between two differentiation stages. Thus, this experimental system should facilitate studies focusing on relationships between nuclear organization, epigenetic status and cell differentiation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB3584
    Nombre del producto:
    Anti-Heterochromatin Protein-1 α Antibody, clone 2HP-1H5

  • HDAC1 regulates pluripotency and lineage specific transcriptional networks in embryonic and trophoblast stem cells. 22156375

    Epigenetic regulation of gene expression is important in maintaining self-renewal of embryonic stem (ES) and trophoblast stem (TS) cells. Histone deacetylases (HDACs) negatively control histone acetylation by removing covalent acetylation marks from histone tails. Because histone acetylation is a known mark for active transcription, HDACs presumably associate with inactive genes. Here, we used genome-wide chromatin immunoprecipitation to investigate targets of HDAC1 in ES and TS cells. Through evaluation of genes associated with acetylated histone H3 marks, and global expression analysis of Hdac1 knockout ES and trichostatin A-treated ES and TS cells, we found that HDAC1 occupies mainly active genes, including important regulators of ES and TS cells self-renewal. We also observed occupancy of methyl-CpG binding domain protein 3 (MBD3), a subunit of the nucleosome remodeling and histone deacetylation (NuRD) complex, at a subset of HDAC1-occupied sequences in ES cells, including the pluripotency regulators Oct4, Nanog and Kfl4. By mapping HDAC1 targets on a global scale, our results describe further insight into epigenetic mechanisms of ES and TS cells self-renewal.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • 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.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-199
    Nombre del producto:
    Anti-MBD2/3 Antibody

  • Disruption of Mbd5 in mice causes neuronal functional deficits and neurobehavioral abnormalities consistent with 2q23.1 microdeletion syndrome. 25001218

    2q23.1 microdeletion syndrome is characterized by intellectual disability, motor delay, autistic-like behaviors, and a distinctive craniofacial phenotype. All patients carry a partial or total deletion of methyl-CpG-binding domain protein 5 (MBD5), suggesting that haploinsufficiency of this gene is responsible for the phenotype. To confirm this hypothesis and to examine the role of MBD5 in vivo, we have generated and characterized an Mbd5 gene-trap mouse model. Our study indicates that the Mbd5(+/) (GT) mouse model recapitulates most of the hallmark phenotypes observed in 2q23.1 deletion carriers including abnormal social behavior, cognitive impairment, and motor and craniofacial abnormalities. In addition, neuronal cultures uncovered a deficiency in neurite outgrowth. These findings support a causal role of MBD5 in 2q23.1 microdeletion syndrome and suggest a role for MBD5 in neuronal processes. The Mbd5(+/) (GT) mouse model will advance our understanding of the abnormal brain development underlying the emergence of 2q23.1 deletion-associated behavioral and cognitive symptoms.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB377
    Nombre del producto:
    Anti-NeuN Antibody, clone A60

  • MBD1 is an Epigenetic Regulator of KEAP1 in Pancreatic Cancer. 26980696

    MBD1 (Methyl-CpG Binding Domain Protein 1) is highly expressed in pancreatic cancer. Nrf2 (NF-E2 p45-related factor 2) and the 'antioxidant response element' (ARE)-driven genes that NRF2 controls are frequently upregulated in pancreatic cancer and correlate with poor survival. Keap1 (Kelch-like ECH-associated protein 1) is a dominant negative regulator of NRF2 and is reported to be epigenetically regulated by promoter methylation. However, the role of MBD1 with antioxidant response and its association with KEAP1 has never been reported before and remains unclear.We investigated the role of MBD1 in antioxidant response and its regulatory function in KEAP1 transcription in pancreatic cancer cells.MBD1 was silenced to examine its role in antioxidant response. To explore the underlying mechanism, transcriptional and protein levels of KEAP1 was examined. The correlation between MBD1 and KEAP1 was confirmed in pancreatic cancer tissue samples by using immunohistochemistry (IHC). Dualluciferase reporter assay and Chromatin immunoprecipitation (ChIP) were used to elucidate he mechanism of MBD1 in KEAP1 transcriptional control. Moreover, co-immunoprecipitation (CoIP) assay was performed to uncover the regulatory role of MBD1 in KEAP1 transcription through its association with c-myc.MBD1 silencing decreased antioxidant response and the related ARE target genes through epigenetic regulation of KEAP1. MBD1 negatively correlated with KEAP1 in pancreatic cancer tissue samples. Moreover, c-myc was a MBD1 interaction partner in KEAP1 epigenetic regulation.MBD1 can induce antioxidant response in pancreatic cancer through down-regulation of KEAP1. c-myc plays a key role in MBD1 mediated epigenetic silencing of KEAP1.
    Tipo de documento:
    Referencia
    Referencia del producto:
    17-10085
    Nombre del producto:
    Magna ChIP™ A/G Chromatin Immunoprecipitation Kit

  • Generation and characterization of rat and mouse monoclonal antibodies specific for MeCP2 and their use in X-inactivation studies. 22140431

    Methyl CpG binding protein 2 (MeCP2) binds DNA, and has a preference for methylated CpGs and, hence, in cells, it accumulates in heterochromatin. Even though it is expressed ubiquitously MeCP2 is particularly important during neuronal maturation. This is underscored by the fact that in Rett syndrome, a neurological disease, 80% of patients carry a mutation in the MECP2 gene. Since the MECP2 gene lies on the X chromosome and is subjected to X chromosome inactivation, affected patients are usually chimeric for wild type and mutant MeCP2. Here, we present the generation and characterization of the first rat monoclonal MeCP2 specific antibodies as well as mouse monoclonal antibodies and a rabbit polyclonal antibody. We demonstrate that our antibodies are suitable for immunoblotting, (chromatin) immunoprecipitation and immunofluorescence of endogenous and ectopically expressed MeCP2. Epitope mapping revealed that most of the MeCP2 monoclonal antibodies recognize the C-terminal domain and one the N-terminal domain of MeCP2. Using slot blot analysis, we determined a high sensitivity of all antibodies, detecting amounts as low as 1 ng of MeCP2 protein. Moreover, the antibodies recognize MeCP2 from different species, including human, mouse, rat and pig. Lastly, we have validated their use by analyzing and quantifying X chromosome inactivation skewing using brain tissue of MeCP2 heterozygous null female mice. The new MeCP2 specific monoclonal antibodies described here perform well in a large variety of immunological applications making them a very valuable set of tools for studies of MeCP2 pathophysiology in situ and in vitro.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • 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.
    Tipo de documento:
    Referencia
    Referencia del producto:
    S7820