DNA binding of methyl-CpG-binding protein MeCP2 in human MCF7 cells.
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.
Koch
Christoph
C
Institut für Biochemie und Molekularbiologie I, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany.
Strätling
Wolf H
WH
D016475:3T3 Cells; D000230:Adenocarcinoma / Q000473:pathology; D020087:Alu Elements; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000473:pathology; D045744:Cell Line, Tumor; D002503:Centromere; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone; D002877:Chromosomes, Human; D002999:Clone Cells; D018899:CpG Islands; D004276:DNA, Satellite / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D015723:Gene Library; D015894:Genome, Human; D006801:Humans; D045170:Matrix Attachment Regions; D008677:Metaphase; D051783:Methyl-CpG-Binding Protein 2; D051379:Mice; D018613:Microscopy, Confocal; D011233:Precipitin Tests; D012097:Repressor Proteins; D012235:Rhodamines
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004276:DNA, Satellite; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; C497392:MECP2 protein, human; C497393:Mecp2 protein, mouse; D051783:Methyl-CpG-Binding Protein 2; D012097:Repressor Proteins; D012235:Rhodamines