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Identification and characterization of a family of mammalian methyl-CpG binding proteins.
ABSTRACT: Methylation at the DNA sequence 5'-CpG is required for mouse development. MeCP2 and MBD1 (formerly PCM1) are two known proteins that bind specifically to methylated DNA via a related amino acid motif and that can repress transcription. We describe here three novel human and mouse proteins (MBD2, MBD3, and MBD4) that contain the methyl-CpG binding domain. MBD2 and MBD4 bind specifically to methylated DNA in vitro. Expression of MBD2 and MBD4 tagged with green fluorescent protein in mouse cells shows that both proteins colocalize with foci of heavily methylated satellite DNA. Localization is disrupted in cells that have greatly reduced levels of CpG methylation. MBD3 does not bind methylated DNA in vivo or in vitro. MBD1, MBD2, MBD3, and MBD4 are expressed in somatic tissues, but MBD1 and MBD2 expression is reduced or absent in embryonic stem cells which are known to be deficient in MeCP1 activity. The data demonstrate that MBD2 and MBD4 bind specifically to methyl-CpG in vitro and in vivo and are therefore likely to be mediators of the biological consequences of the methylation signal.
Project description:The methylation status of the CpG island located within the ribosomal RNA (rRNA) promoter in human hepatocellular carcinomas and pair-matched liver tissues was analyzed by bisulfite genomic sequencing. Significant hypomethylation of methyl-CpGs in the rRNA promoter was observed in the tumor samples compared with matching normal tissues, which was consistent with the relatively high level of rRNA synthesis in rapidly proliferating tumors. To study the effect of CpG methylation on RNA polymerase I (pol I)-transcribed rRNA genes, we constructed pHrD-IRES-Luc (human rRNA promoter-luciferase reporter). In this plasmid, Kozak sequence of the pGL3-basic vector was replaced by the internal ribosome entry site (IRES) of encephalomyocarditis viral genome to optimize pol I-driven reporter gene expression. Transfection of this plasmid into HepG2 (human) cells revealed reduced pol I-driven luciferase activity with an increase in methylation density at the promoter. Markedly reduced luciferase activity in Hepa (mouse) cells compared with HepG2 (human) cells showed that pHrD-IRES-Luc is transcribed by pol I. Site-specific methylation of human rRNA promoter demonstrated that methylation of CpG at the complementary strands located in the promoter (-9, -102, -347 with respect to the +1 site) inhibited luciferase activity, whereas symmetrical methylation of a CpG in the transcribed region (+152) did not affect the promoter activity. Immunofluorescence studies showed that the methyl-CpG-binding proteins, MBD1, MBD2, MBD3, and MeCP2, are localized both in the nuclei and nucleoli of HepG2 cells. Transient overexpression of MBD2 suppressed luciferase activity specifically from the methylated rRNA promoter, whereas MBD1 and MBD3 inhibited rRNA promoter activity irrespective of the methylation status. Chromatin immunoprecipitation analysis confirmed predominant association of MBD2 with the endogenous methylated rRNA promoter, which suggests a selective role for MBD2 in the methylation-mediated inhibition of ribosomal RNA gene expression.
Project description:BACKGROUND:Methylation at CpG dinucleotides in genomic DNA is a fundamental epigenetic mechanism of gene expression control in vertebrates. Proteins with a methyl-CpG-binding domain (MBD) can bind to single methylated CpGs and most of them are involved in transcription control. So far, five vertebrate MBD proteins have been described as MBD family members: MBD1, MBD2, MBD3, MBD4 and MECP2. RESULTS:We performed database searches for new proteins containing an MBD and identified six amino acid sequences which are different from the previously described ones. Here we present a comparison of their MBD sequences, additional protein motifs and the expression of the encoding genes. A calculated unrooted dendrogram indicates the existence of at least four different groups of MBDs within these proteins. Two of these polypeptides, KIAA1461 and KIAA1887, were only present as predicted amino acid sequences based on a partial human cDNA. We investigated their expression by Northern blot analysis and found transcripts of ~8 kb and ~5 kb respectively, in all eight normal tissues studied. CONCLUSIONS:Eleven polypeptides with a MBD could be identified in mouse and man. The analysis of protein domains suggests a role in transcriptional regulation for most of them. The knowledge of additional existing MBD proteins and their expression pattern is important in the context of Rett syndrome.
Project description:Cytosine methylation is an epigenetic and regulatory mark that functions in part through recruitment of chromatin remodeling complexes containing methyl-CpG binding domain (MBD) proteins. Two MBD proteins, Mbd2 and Mbd3, were previously shown to bind methylated or hydroxymethylated DNA, respectively; however, both of these findings have been disputed. Here, we investigated this controversy using experimental approaches and re-analysis of published data and find no evidence for methylation-independent functions of Mbd2 or Mbd3. We show that chromatin localization of Mbd2 and Mbd3 is highly overlapping and, unexpectedly, we find Mbd2 and Mbd3 are interdependent for chromatin association. Further investigation reveals that both proteins are required for normal levels of cytosine methylation and hydroxymethylation in murine embryonic stem cells. Furthermore, Mbd2 and Mbd3 regulate overlapping sets of genes that are also regulated by DNA methylation/hydroxymethylation factors. These findings reveal an interdependent regulatory mechanism mediated by the DNA methylation machinery and its readers.
Project description:The heterogeneous collection of nucleosome remodelling and deacetylation (NuRD) complexes can be grouped into the MBD2- or MBD3-containing complexes MBD2-NuRD and MBD3-NuRD. MBD2 is known to bind to methylated CpG sequences in vitro in contrast to MBD3. Although functional differences have been described, a direct comparison of MBD2 and MBD3 in respect to genome-wide binding and function has been lacking. Here, we show that MBD2-NuRD, in contrast to MBD3-NuRD, converts open chromatin with euchromatic histone modifications into tightly compacted chromatin with repressive histone marks. Genome-wide, a strong enrichment for MBD2 at methylated CpG sequences is found, whereas CpGs bound by MBD3 are devoid of methylation. MBD2-bound genes are generally lower expressed as compared with MBD3-bound genes. When depleting cells for MBD2, the MBD2-bound genes increase their activity, whereas MBD2 plus MBD3-bound genes reduce their activity. Most strikingly, MBD3 is enriched at active promoters, whereas MBD2 is bound at methylated promoters and enriched at exon sequences of active genes.
Project description:MBD2 and MBD3 are closely related proteins with consensus methyl-CpG binding domains. MBD2 is a transcriptional repressor that specifically binds to methylated DNA and is a component of the MeCP1 protein complex. In contrast, MBD3 fails to bind methylated DNA in murine cells, and is a component of the Mi-2/NuRD corepressor complex. We show by gene targeting that the two proteins are not functionally redundant in mice, as Mbd3-/- mice die during early embryogenesis, whereas Mbd2-/- mice are viable and fertile. Maternal behavior of Mbd2-/- mice is however defective and, at the molecular level, Mbd2-/- mice lack a component of MeCP1. Mbd2-mutant cells fail to fully silence transcription from exogenous methylated templates, but inappropriate activation of endogenous imprinted genes or retroviral sequences was not detected. Despite their differences, Mbd3 and Mbd2 interact genetically suggesting a functional relationship. Genetic and biochemical data together favor the view that MBD3 is a key component of the Mi-2/NuRD corepressor complex, whereas MBD2 may be one of several factors that can recruit this complex to DNA.
Project description:DNA methylation is the major modification of eukaryotic genomes and plays an essential role in mammalian gene regulation. In general, cytosine-phosphatidyl-guanosine (CpG)-methylated promoters are transcriptionally repressed and nuclear proteins such as MECP2, MBD1, MBD2, and MBD4 bind CpG-methylated DNA and contribute to epigenetic silencing. Methylation of viral DNA also regulates gene expression of Epstein-Barr virus (EBV), which is a model of herpes virus latency. In latently infected human B cells, the viral DNA is CpG-methylated, the majority of viral genes is repressed and virus synthesis is therefore abrogated. EBV's BZLF1 encodes a transcription factor of the AP-1 family (Zta) and is the master gene to overcome viral gene repression. In a genome-wide screen, we now identify and characterize those viral genes, which Zta regulates. Among them are genes essential for EBV's lytic phase, which paradoxically depend on strictly CpG-methylated promoters for their Zta-induced expression. We identified novel DNA recognition motifs, termed meZRE (methyl-Zta-responsive element), which Zta selectively binds in order to 'read' DNA in a methylation- and sequence-dependent manner unlike any other known protein. Zta is a homodimer but its binding characteristics to meZREs suggest a sequential, non-palindromic and bipartite DNA recognition element, which confers superior DNA binding compared to CpG-free ZREs. Our findings indicate that Zta has evolved to transactivate cytosine-methylated, hence repressed, silent promoters as a rule to overcome epigenetic silencing.
Project description:DNA methylation is thought to induce transcriptional silencing through the combination of two mechanisms: the repulsion of transcriptional activators unable to bind their target sites when methylated, and the recruitment of transcriptional repressors with specific affinity for methylated DNA. The Methyl CpG Binding Domain proteins MeCP2, MBD1 and MBD2 belong to the latter category. Here, we present MBD2 ChIPseq data obtained from the endogenous MBD2 in an isogenic cellular model of oncogenic transformation of human mammary cells. In immortalized (HMEC-hTERT) or transformed (HMLER) cells, MBD2 was found in a large proportion of methylated regions and associated with transcriptional silencing. A redistribution of MBD2 on methylated DNA occurred during oncogenic transformation, frequently independently of local DNA methylation changes. Genes downregulated during HMEC-hTERT transformation preferentially gained MBD2 on their promoter. Furthermore, depletion of MBD2 induced an upregulation of MBD2-bound genes methylated at their promoter regions, in HMLER cells. Among the 3,160 genes downregulated in transformed cells, 380 genes were methylated at their promoter regions in both cell lines, specifically associated by MBD2 in HMLER cells, and upregulated upon MBD2 depletion in HMLER. The transcriptional MBD2-dependent downregulation occurring during oncogenic transformation was also observed in two additional models of mammary cell transformation. Thus, the dynamics of MBD2 deposition across methylated DNA regions was associated with the oncogenic transformation of human mammary cells.
Project description:5-Methylcytosine (mC) exists in CpG dinucleotides of mammalian DNA and plays key roles in chromatin regulation during development and disease. As a main regulatory pathway, fully methylated CpG are recognized by methyl-CpG-binding domain (MBD) proteins that act in concert with chromatin remodelers, histone deacetylases and methyltransferases to trigger transcriptional downregulation. In turn, MBD mutations can alter CpG binding, and in case of the MBD protein MeCP2 can cause the neurological disorder Rett syndrome (RTT). An additional layer of complexity in CpG recognition is added by ten-eleven-translocation (TET) dioxygenases that oxidize mC to 5-hydroxymethyl-, 5-formyl- and 5-carboxylcytosine, giving rise to fifteen possible combinations of cytosine modifications in the two CpG strands. We report a comprehensive, comparative interaction analysis of the human MBD proteins MeCP2, MBD1, MBD2, MBD3, and MBD4 with all CpG combinations and observe individual preferences of each MBD for distinct combinations. In addition, we profile four MeCP2 RTT mutants and reveal that although interactions to methylated CpGs are similarly affected by the mutations, interactions to oxidized mC combinations are differentially affected. These findings argue for a complex interplay between local TET activity/processivity and CpG recognition by MBDs, with potential consequences for the transcriptional landscape in normal and RTT states.
Project description:Methyl-binding domain (MBD) family proteins specifically bind double-stranded, methylated DNA which makes them useful for DNA methylation analysis. We displayed three of the core members MBD1, MBD2 and MBD4 on the surface of Saccharomyces cerevisiae cells. Using the yeast display platform, we determined the equilibrium dissociation constant of human MBD2 (hMBD2) to be 5.9 ± 1.3 nM for binding to singly methylated DNA. The measured affinity for DNA with two methylated sites varied with the distance between the sites. We further used the yeast display platform to evolve the hMBD2 protein for improved binding affinity. Affecting five amino acid substitutions doubled the affinity of the wild-type protein to 3.1 ± 1.0 nM. The most prevalent of these mutations, K161R, occurs away from the DNA-binding site and bridges the N- and C-termini of the protein by forming a new hydrogen bond. The F208Y and L170R mutations added new non-covalent interactions with the bound DNA strand. We finally concatenated the high-affinity MBD variant and expressed it in Escherichia coli as a green fluorescent protein fusion. Concatenating the protein from 1× to 3× improved binding 6-fold for an interfacial binding application.
Project description:Although highly homologous to other methylcytosine-binding domain (MBD) proteins, MBD3 does not selectively bind methylated DNA, and thus the functional role of MBD3 remains in question. To explore the structural basis of its binding properties and potential function, we characterized the solution structure and binding distribution of the MBD3 MBD on hydroxymethylated, methylated, and unmethylated DNA. The overall fold of this domain is very similar to other MBDs, yet a key loop involved in DNA binding is more disordered than previously observed. Specific recognition of methylated DNA constrains the structure of this loop and results in large chemical shift changes in NMR spectra. Based on these spectral changes, we show that MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated cytosine-guanosine dinucleotides (CpGs), yet does not distinguish between hydroxymethylated and unmethylated sites. Measuring residual dipolar couplings for the different bound states clearly shows that the MBD3 structure does not change between methylation-specific and nonspecific binding modes. Furthermore, residual dipolar couplings measured for MBD3 bound to methylated DNA can be described by a linear combination of those for the methylation and nonspecific binding modes, confirming the preferential localization to methylated sites. The highly homologous MBD2 protein shows similar but much stronger localization to methylated as well as unmethylated CpGs. Together, these data establish the structural basis for the relative distribution of MBD2 and MBD3 on genomic DNA and their observed occupancy at active and inactive CpG-rich promoters.