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. MeCP2 has been reported 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 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. 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 found that the purified MeCP2 does not stably associate with any other proteins. In particular, we failed to detect the presence of histone deacetylase components Sin3A and HDAC1 in our purified MeCP2 fraction. Our studies suggest that most endogenous MeCP2 may exist in forms not related to histone deacetylase. Interestingly, we found that MeCP2 is constitutively phosphorylated at a specific site. We have identified this phosphorylation site on MeCP2 by mass spectrometry, and made a phosphorylation site-specific antibody. We are now collaborating with the group of M. Mattson to investigate whether phosphorylation at this specific site will play a role in regulating activity of MeCP2 in neurons.
| 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 |
