With the demonstration that up to 80% of Rett syndrome cases are caused by mutation in a methyl DNA-binding protein, MeCP2, the analysis of neurological effects and mental retardation in this disease must focus on associated changes in chromatin. The working hypothesis is that chromatin remodeling based on methylation must be modified to turn on or off critical proteins active in brain function. Almost certainly, a multiprotein complex containing MeCP2 is involved, for it is becoming increasingly clear that chromatin remodeling ? like many reactions in the cell nucleus ? is carried out by such complexes. Often, one protein can be part of several complexes, with each complex having a unique function. In fact, MeCP2 has been shown to co-fractionate and coimmunoprecipitate with histone deacetylase HDAC1 and transcription corepressor mSin3A, proteins that are presumably part of the complex. However, the entire complex (or complexes) containing MeCP2 has not been completely purified or characterized. We want to apply purification procedures that we have developed and successfully used to analyze other disease-related chromatin remodeling complexes to identify the protein partners of MeCP2 in the cell. The proximal goal of this research is to understand the function of MeCP2 in vivo. Here we developed a highly specific MeCP2 antibody and immunopurified majority of endogenous MeCP2 from HeLa nuclear extract under near physiological salt conditions. Using this unbiased approach, we obtained 6 major polypeptides. Three of them were identified as MeCP2 by both mass spectrometry and immunoblotting analysis. Three others were identified as components a DNA-binding complex. Subsequent analysis showed that these proteins are likely contaminants that associate with MeCP2 through DNA. Histone deacetylase components Sin3A and HDAC1 were not detected in polypeptides immunoisolated by our MeCP2 antibody. Gel-filtration analysis indicated that some endogenous MeCP2 from human HeLa cells or a mouse brain cell line fractionates in high molecular weight complexes above 600 kd. Our studies suggest that most endogenous MeCP2 may exist in forms not related to histone deacetylase. Interestingly, we found that MeCP2 is present in three different forms. One of them is phosphorylated. We are now investigating whether phosphorylation of MeCP2 plays a role in regulating activity of MeCP2 and in etiology of Rett syndrome.
| Hu, Keping; Nan, Xinsheng; Bird, Adrian et al. (2006) Testing for association between MeCP2 and the brahma-associated SWI/SNF chromatin-remodeling complex. Nat Genet 38:962-4; author reply 964-7 |
