Purification of the MeCP2/Histone Deacetylase Complex from Xenopus laevis
DNA methylation has long been associated with stable transcriptional silencing and a repressive chromatin structure (reviewed in refs. 1,2). Differential methylation is associated with imprinting, carcinogenesis, silencing of repetitive DNA, and allows for differentiating cells to efficiently shut off unnecessary genes. In vertebrates, where 60-90% of genomic CpG dinucleotides are methylated, methylation-dependent repression is vital for proper embryonic development (3). Microinjection experiments using methylated DNA templates implicate chromatin structure as an underlying mechanism of methylation-dependent silencing (4,5). Methyl-specific transcriptional repression requires chromatin assembly, and can be partially relieved by the histone deacetylase inhibitor Trichostatin A. In addition, several proteins have been identified that specifically bind to methylated DNA (6-8). Two of these methyl-DNA binding proteins, MeCP1 and MeCP2, have been shown to mediate transcriptional repression (6,7). MeCP1 is a relatively uncharacterized complex that requires at least 12 symmetrical methyl-CpGs for DNA binding (6). MeCP2 is a single polypeptide containing a methyl-binding domain capable of binding a single methyl-CpG, and a transcriptional repression domain (9). Recently MeCP2 was shown to interact with the Sin3 corepressor and histone deacetylase (10,11). Changes in the acetylation state of the core histone tails correlates with changes in transcription (reviewed in refs. 12,13), and several transcriptional repression complexes containing histone deacetylases have recently been described (10,14,15). These data provide a direct link between methyl-dependent transcriptional repression and the modification of chromatin structure. Here, we describe techniques for purifying the MeCP2-contining histone deacetylase complex from Xenopus laevis oocytes.
KeywordsHistone Deacetylase Transcriptional Repression Histone Deacetylase Activity Histone Deacetylase Complex Oocyte Extract
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