Article ; Online: Biochemical analysis of histone deacetylase-independent transcriptional repression by MeCP2.
The Journal of biological chemistry
2013 Volume 288, Issue 10, Page(s) 7096–7104
Abstract: 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 ... ...
| Abstract | 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. |
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| MeSH term(s) | Chromatin/genetics ; Chromatin/metabolism ; DNA/genetics ; DNA/metabolism ; DNA Methylation ; Dinucleoside Phosphates/genetics ; Electrophoretic Mobility Shift Assay ; Gene Expression Regulation ; HeLa Cells ; Histone Deacetylase Inhibitors/pharmacology ; Histone Deacetylases/metabolism ; Humans ; Hydroxamic Acids/pharmacology ; Methyl-CpG-Binding Protein 2/genetics ; Methyl-CpG-Binding Protein 2/metabolism ; Mutation ; Promoter Regions, Genetic/genetics ; Protein Binding ; Transcription Initiation, Genetic/drug effects ; Transcription, Genetic/drug effects ; Transcription, Genetic/genetics | |||||
| Chemical Substances | Chromatin ; Dinucleoside Phosphates ; Histone Deacetylase Inhibitors ; Hydroxamic Acids ; Methyl-CpG-Binding Protein 2 ; cytidylyl-3'-5'-guanosine (2382-65-2) ; trichostatin A (3X2S926L3Z) ; DNA (9007-49-2) ; Histone Deacetylases (EC 3.5.1.98) | |||||
| Language | English | |||||
| Publishing date | 2013-01-24 | |||||
| Publishing country | United States | |||||
| Document type | Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't | |||||
| ZDB-ID | 2997-x | |||||
| ISSN | 1083-351X ; 0021-9258 | |||||
| ISSN (online) | 1083-351X | |||||
| ISSN | 0021-9258 | |||||
| DOI | 10.1074/jbc.M112.438697 | |||||
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| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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