Cytosine residues in the sequence 5'CpG (cytosine-guanine) are often postsynthetically methylated in animal genomes. CpG methylation is involved in long-term silencing of certain genes during mammalian development and in repression of viral genomes. The methyl-CpG-binding proteins MeCP1 and MeCP2 interact specifically with methylated DNA and mediate transcriptional repression. Here we study the mechanism of repression by MeCP2, an abundant nuclear protein that is essential for mouse embryogenesis. MeCP2 binds tightly to chromosomes in a methylation-dependent manner. It contains a transcriptional-repression domain (TRD) that can function at a distance in vitro and in vivo. We show that a region of MeCP2 that localizes with the TRD associates with a corepressor complex containing the transcriptional repressor mSin3A and histone deacetylases. Transcriptional repression in vivo is relieved by the deacetylase inhibitor trichostatin A, indicating that deacetylation of histones (and/or of other proteins) is an essential component of this repression mechanism. The data suggest that two global mechanisms of gene regulation, DNA methylation and histone deacetylation, can be linked by MeCP2.
MeCP2 is an abundant mammalian protein that binds to methylated CpG. We have found that native and recombinant MeCP2 repress transcription in vitro from methylated promoters but do not repress nonmethylated promoters. Repression is nonlinearly dependent on the local density of methylation, becoming significant at the density found in bulk vertebrate genomic DNA. Transient transfection using fusions with the GAL4 DNA binding domain identified a region of MeCP2 that is capable of long-range repression in vivo. Moreover, MeCP2 is able to displace histone H1 from preassembled chromatin that contains methyl-CpG. These properties, together with the abundance of MeCP2 and the high frequency of its 2 bp binding site, suggest a role as a global transcriptional repressor in vertebrate genomes.
DNA methylation plays an important role in mammalian development and correlates with chromatinassociated 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.Methylation of cytosines is essential for mammalian development and is associated with gene silencing (1). DNA methylation represses genes partly by recruitment of methyl-CpGbinding domain proteins, which selectively recognize methylated CpG dinucleotides. MeCP2 is the founder member of the methyl-CpG-binding domain proteins, which consists of a single polypeptide that contains a methyl-CpG-binding domain and a transcriptional repression domain. MeCP2 is capable of binding to a single symmetrically methylated CpG both in naked DNA and within chromatin (2, 3).It is now well established that MeCP2 silences transcription by recruiting the histone deacetylase (HDAC) 1 repressive machinery, which removes acetyl groups from histones resulting in gene silencing (4,5). However, the inhibition of histone deacetylase activity using drugs such as Trichostatin A only partially relieves MeCP2-mediated transcriptional repression. This partial relief indicates that additional mechanisms of repression by MeCP2 likely exist aside from the recruitment of histone deacetylase.Beside histone deacetylation, histone methylation is emerging as another key post-translational modification of histones and represents an important epigenetic mechanism for the organization of chromatin structure and the regulation of gene expression. In particular, methylation at lysine 9 of histone H3 is associated with gene silencing, and several enzymes that catalyze the addition of methyl groups to lysine 9 have recently been identified (6). Interestingly, recent data have shown that the retinoblastoma protein represses transcription through the recruitment of HDAC activity, but in a second step it recruits histone methylation activity specific for lysine 9 of histone H3 (7). By analogy to retinoblastoma, we considered in the present work whether MeCP2-mediated repression might also include, besides histone deacetylation, a second stage involving histone methylatio...
MeCP2 is a chromosomal protein which binds to DNA that is methylated at CpG. In situ immunofluorescence in mouse cells has shown that the protein is most concentrated in pericentromeric heterochromatin, suggesting that MeCP2 may play a role in the formation of inert chromatin. Here we have isolated a minimal methyl-CpG binding domain (MBD) from MeCP2. MBD is 85 amino acids in length, and binds exclusively to DNA that contains one or more symmetrically methylated CpGs. MBD has negligable non-specific affinity for DNA, confirming that non-specific and methyl-CpG specific binding domains of MeCP2 are distinct. In vitro footprinting indicates that MBD binding can protect a 12 nucleotide region surrounding a methyl-CpG pair, with an approximate dissociation constant of 10(-9) M.
CpG islands are short stretches of DNA containing a high density of non-methylated CpG dinucleotides, predominantly associated with coding regions. We have constructed an affinity matrix that contains the methyl-CpG binding domain from the rat chromosomal protein MeCP2, attached to a solid support. A column containing the matrix fractionates DNA according to its degree of CpG methylation, strongly retaining those sequences that are highly methylated. Using this column, we have developed a procedure for bulk isolation of CpG islands from human genomic DNA. As CpG islands overlap with approximately 60% of human genes, the resulting CpG island library can be used to isolate full-length cDNAs and to place genes on genomic maps.
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