DNA Methylation Specifies Chromosomal Localization of MeCP2

Abstract

MeCP2 is a chromosomal protein that is concentrated in the centromeric heterochromatin of mouse cells. In vitro, the protein binds preferentially to DNA containing a single symmetrically methylated CpG. To find out whether the heterochromatic localization of MeCP2 depended on DNA methylation, we transiently expressed MeCP2-LacZ fusion proteins in cultured cells. Intact protein was targeted to heterochromatin in wild-type cells but was inefficiently localized in mutant cells with low levels of genomic DNA methylation. Deletions within MeCP2 showed that localization to heterochromatin required the 85-amino-acid methyl-CpG binding domain but not the remainder of the protein. Thus MeCP2 is a methyl-CpG-binding protein in vivo and is likely to be a major mediator of downstream consequences of DNA methylation.Widespread methylation of genomic DNA is a characteristic of the vertebrates. The target of methylation is the dinucleotide CpG, which acquires a methyl group at the 5 position on the cytosine ring. Functions of methylation have long been the subject of experiment and speculation, but recent experiments provide evidence for an essential role in development. Li et al. (16) disrupted the gene that encodes the DNA methyltransferase (MTase) enzyme in mice and found that homozygous mutant embryos had greatly reduced levels of methylation, were developmentally retarded, and died at midgestation. Undifferentiated embryonic stem (ES) cells, on the other hand, behaved normally in culture, despite very low levels of methylation, indicating that methylation may be less important at this totipotent stage. Why methylation is essential for development is not yet completely clear. One possibility is that methylation-mediated repression of genes is somehow built into the developmental program (discussed in reference 1).To understand the biology of DNA methylation, it is necessary to identify all components of the system. Several proteins that bind specifically to methylated DNA are known (reviewed in reference 31). Two of these, MeCP1 and MeCP2, bind to symmetrical methyl-CpG pairs in any sequence context and may therefore be of general significance (14,17,18). Studies of MeCP1 have implicated it in methylation-mediated gene inactivation (2, 3). The second protein, MeCP2, consists of a single chain of 492 amino acids in the rat, including an 85-amino-acid domain near the N terminus that encodes all of the specificity required for binding to methylated DNA (22). The mouse protein (484 amino acids) is very similar to its rat homolog, showing an overall identity of 95% at the amino acid level and absolute conservation of the methylated DNA binding domain (29). This methyl-CpG binding domain (MBD) can bind as a monomer to a single symmetrically methylated CpG pair. Immunofluorescence analysis of mouse chromosomes shows that MeCP2 is preferentially localized in pericentromeric heterochromatin (14), which is also the region of highest 5-methylcytosine concentration (19). The predominant DNA sequence in mouse heterochromatin is the m...

Essential Role of MeCP2 in the Regulation of Myofibroblast Differentiation during Pulmonary Fibrosis

“…24 However, it is unclear if this dichotomy depends on binding to methylated versus unmethylated sequences, despite abundant studies showing that MeCP2 preferentially binds to methylated DNA. 31 Nevertheless, the findings in this study favored the interpretation of a direct activator role for MeCP2 vis-à-vis the ␣-SMA gene in lung fibroblasts, which might be of importance in pathogenesis of pulmonary fibrosis.…”

MeCP2 binds to non-CG methylated DNA as neurons mature, influencing transcription and the timing of onset for Rett syndrome

“…The fact that doubling or tripling the levels of MeCP2 results in progressive neurological syndromes with increasing severity (MECP2 duplication and triplication syndromes, respectively) (6, 7) also raises questions about the binding patterns of MeCP2 and their functional consequences. Early studies found that MeCP2 interacts specifically with methylated cytosine in the CG context (mCG) (8)(9)(10). Recent in vivo studies in the adult mouse brain confirm the preference of MeCP2 for mCG over nonmethylated CG, although MeCP2 clearly binds widely across the entire genome (11).…”

Site-specific DNA methylation in the neurofibromatosis (NF1) promoter interferes with binding of CREB and SP1 transcription factors

“…The functional signi®cance of methylation in this region and its e ect on binding has yet to be determined. However, methylation within non-CpG island regions is thought to contribute to the maintenance of a heterochromatinized state which precludes binding of transcription factors (Tate and Bird, 1993;Antequera et al, 1990;Nan et al, 1996). Since it has been shown that CpG dinucleotides in bulk chromatin outside CpG-islands may become hypomethylated in some tumours, altered methylation within this NF1 region could compromise the gene's structural integrity and alter NF1 expression (Gra et al, 1997).…”