DNA Binding of Methyl-CpG-Binding Protein MeCP2 in Human MCF7 Cells

Abstract

MeCP2 has been identified as a chromatin-associated protein that recognizes MAR elements as well as methyl-CpGs. To characterize target sequences of MeCP2 in human cells, we employed two complementary methods. First, by use of a preparative chromatin immunoprecipitation protocol, we created from MCF7 cells a library enriched with sequences bound to MeCP2. A total of 154 representative clones were sequenced and analyzed. A large fraction of clones was found to be associated with retrotransposons, mostly with Alu repeats. A subgroup of four clones is derived from putative MARs; one clone is associated with a CpG island, and four clones contain alphoid repeats. Classical satellite DNAs II and III are not represented among clones, although they are heavily methylated. Second, using indirect immunofluorescence microscopy, we show that MeCP2 staining of human metaphase chromosomes has a dotted to knobby appearance with a reduced level of staining of centromeric regions of some chromosomes. On the other hand, an anti-5-methylcytosine antibody preferentially stained the juxtacentromeric regions of chromosomes 1, 9, and 16, which habor highly methylated, classical satellite DNAs, and methylated alphoid sequences in centromeric regions of several other chromosomes with reduced intensity. In interphase MCF7 cells, the distribution of MeCP2 exhibits a granular appearance throughout the nucleus. This distribution does not parallel that of methylated cytosine and heterochromatin. The selective binding behavior of MeCP2 revealed by these results (preference for murine major satellite DNA, Alu sequences, MARs, and CpG islands) is explained by its ability to recognize the sequence information (guanine bases) adjacent to CpG (TpG) as demonstrated in previous footprinting experiments.

Mitomycin C-induced pairing of heterochromatin reflects initiation of DNA repair and chromatid exchange formation

“…Paracentromeric heterochromatic bands have been reported to contain GC-rich repeats and harbor highly methylated DNA (Miller et al, 1974;Gosden et al, 1975;Meneveri et al, 1993). In line with this, it has been shown recently that an antibody raised against 5-methylcytosine preferentially stained the juxtacentromeric regions of chromosomes 1, 9, 16 and Y (Koch and Strätling, 2004). In addition to a nonuniform distribution of MMCinduced interchanges among human chromosomes, a striking preference for homologous interactions, particularly between chromosome 9 homologues, was observed in this study.…”

The Latency-Associated Nuclear Antigen Interacts with MeCP2 and Nucleosomes through Separate Domains

“…Accumulation of MeCP2 at the chromocenters of murine cells is well documented (1,10,32,35). Equivalent structures are hard to discern in human cells due to the more dispersed nature of the satellite repeats.…”

Analysis of protein domains and Rett syndrome mutations indicate that multiple regions influence chromatin-binding dynamics of the chromatin-associated protein MECP2 in vivo

“…For example, in MCF breast cancer cell (MCF-7), MECP2 is distributed throughout the nucleus and appears granular. It also does not completely correlate with CpG methylation and heterochromatic regions, and is excluded from classic satellite DNA in the interphase nucleus (Koch and Stratling, 2004).…”

“…MECP2 binds to both unmodified DNA as well as to methyl CpG dinucleotides, with increased affinity for symmetrically methylated DNA; thus, it is typically distributed throughout the nucleus and enriched in DAPI-bright heterochromatic regions in interphase murine nuclei (Koch and Stratling, 2004). In differentiating murine myocytes, MECP2 participates in the assembly of pericentromeric heterochromatin, and exogenous overexpression of MECP2 leads to aggregation of chromodomains in perinucleolar foci in a dose-dependent manner (Brero et al, 2005;Marchi et al, 2007).…”