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.
Highlights
To test the activity of the purified Methyl-CpG-binding protein 2 (MeCP2), we used pUC-SCP-8CG as the reporter construct (Fig. 1B). This plasmid, which is on the basis of the pUC119 vector, contains a version of the super core promoter (SCP) [19] with eight CG dinucleotides in the region from Ϫ50 to ϩ50 relative to the ϩ1 transcription start site. pUC-SCP-8CG has a total of 217 CG dinucleotides, 209 from pUC119 and eight from the SCP-8CG core promoter
At a MeCP2-to-CG dinucleotide ratio of 1:2 (Fig. 2, 250 nM), which is approximately the same as that seen in mouse neurons [12], the magnitude of methyl-CG-specific repression was over 10-fold
We investigated whether transcriptional repression would be observed with a CG-deficient core promoter that is surrounded by CG-containing sequences
Summary
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. The abbreviations used are: MBD, methyl-CG binding domain; CG, cytosineguanine; TRD, transcription repression domain; HDAC, histone deacetylase; TSA, trichostatin A; PIC, preinitiation complex; ACF, ATP-utilizing chromatin assembly and remodeling factor; dslHairpin, double-stranded-like hairpin DNA; SCP, super core promoter. We established a biochemical transcription assay with purified full-length, wild-type human MeCP2 With this system, we observed potent CG-methylation-specific, HDAC-independent repression of transcription by MeCP2. The biochemical properties of MeCP2 suggest that HDAC-independent repression may be an important component of MeCP2 function in the brain
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