Methylation of cytosine at CpG dinucleotides is a common feature of many higher eukaryotic genomes. DNA methylation plays important roles in diverse biological processes including embryogenesis, genomic imprinting, X-chromosome inactivation, and cancer. A major biological consequence of DNA methylation is gene silencing. However, the mechanism underlying methylated DNA silencing is not known. In an effort to understand how core histone acetylation and deacetylation regulate transcription, we previously purified and characterized two histone deacetylase complexes, the Sin3 and the NuRD complexes. Both complexes have been shown to be involved in methylated DNA silencing. To understand the relationship between nucleosome remodeling, histone deacetylation and methylated DNA silencing, we have purified the methyl-CpG specific binding protein complex MeCP1. Our preliminary studies revealed that in addition to the methyl-CpG binding protein MBD2, the MeCP1 complex contains all the known NuRD components indicating NuRD can be targeted to methylated DNA. We propose to extend this study with the following specific aims: 1. Cloning and characterization of the p66 and p68 components of the MeCP1 complex. We will isolate the cDNAs encoding p66 and p68. Their roles in methyl-CpG binding, nucleosome remodeling, and histone deacetylation will be analyzed. 2. Functional characterization of the MeCP1 complex. We will investigate chromosomal localization of the MeCP1 complex in response to DNA demethylation. We will also characterize the ATPase, nucleosome remodeling and histone deacetylase activities of the MeCP1 complex. 3. Identification of MeCP1 targeted genes. We will identify genes that are targeted by the MeCP1 complex using chromatin immunoprecipitation (CHIP) approach. The identified candidate genes will be verified by Northern blot analysis using the MTA2 null cells. 4. Understanding the mechanism of transcriptional repression by MeCP1. We will investigate the transcription repression mechanism of MeCP1 using a reconstituted in vitro chromatin transcription system. The proposed studies will provide a thorough characterization of the MeCP1 complex. They will also reveal the potential targets and repression mechanism of the MeCP1 complex.
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