Biochemical analysis of histone deacetylase-independent transcriptional repression by MeCP2.
MeCP2 is an abundant methyl-cytosine-guanine (CG)-binding protein and transcriptional repressor. We developed a biochemical system that exhibits CG methylation-specific transcriptional repression by purified human MeCP2. MeCP2 represses transcription by histone deacetylase (HDAC)-dependent and HDAC-independent mechanisms. Our system appears to recreate the HDAC-independent component of MeCP2-mediated repression and occurs via inhibition of the assembly of transcription preinitiation complexes. At a ratio of approximately one molecule of MeCP2 per two methyl-CG dinucleotides, as found in mammalian neurons, the magnitude of methylation-specific repression was greater than 10-fold. Notably, the HDAC inhibitor trichostatin A had no effect on MeCP2-mediated repression with either naked DNA or chromatin templates. We designed a CG-deficient core promoter that is resistant to MeCP2-mediated repression when placed in a plasmid lacking CG dinucleotides. By using this CG-deficient reporter as a reference, we found that eight CG dinucleotides in the core promoter region are sufficient for strong methylation-specific repression by MeCP2. In contrast, MeCP2 does not repress a construct with 13 CG dinucleotides located ∼1.7 kbp upstream of the promoter. Furthermore, by analysis of C-terminally truncated MeCP2 proteins, we found that binding of MeCP2 to methyl-CG dinucleotides is not sufficient for transcriptional repression. Hence, MeCP2-mediated repression is not due to the simple steric blockage of the transcriptional machinery. These experiments suggest that MeCP2 can function as a global methyl-CG-specific, HDAC-independent repressor. This HDAC-independent mechanism of MeCP2-mediated repression may be important in cells, such as mammalian neurons, that have high levels of CG methylation and MeCP2.
Section of Molecular Biology, University of California San Diego, La Jolla, California 92093, USA.
Joshua W M
JW
Theisen
James S
JS
Gucwa
Timur
T
Yusufzai
Mai T
MT
Khuong
James T
JT
Kadonaga
D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D015226:Dinucleoside Phosphates / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D006367:HeLa Cells; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006655:Histone Deacetylases / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D051783:Methyl-CpG-Binding Protein 2 / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D061785:Transcription Initiation, Genetic / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics*
D002843:Chromatin; D004247:DNA; D015226:Dinucleoside Phosphates; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases; D006877:Hydroxamic Acids; D051783:Methyl-CpG-Binding Protein 2; C015772:CpG dinucleotide; C012589:trichostatin A