title: Analysis of chromatin-immunopurified MeCP2-associated fragments.
abstract: As molecular biologists, we are continuing to unravel the interactions by which DNA binding proteins mediate the expression of genes. The chromatin immunoprecipitation (ChIP) technique provides us with an exquisite tool to investigate the interplay between chromatin structure and its role in regulating transcription, replication, and recombination in vivo. We describe a robust assay used to identify the molecular determinants associated with chromatin. In this article we illustrate the ChIP technique and use the transcriptionally silent-hypermethylated multidrug resistance (MDR1) gene as the platform for methyl-CpG binding protein 2 (MeCP2) localization on chromatin. Driven by the hypothesis that repression is strongly dependent on the methylation profile of the endogenous promoter, we demonstrate that MDR1 is targeted by MeCP2. Methylated MDR1 chromatin is highly enriched with MeCP2 and is in striking contrast to localization observed in cells in which MDR1 is transcriptionally active. In a distinct model system we discuss experimental methods used to immunopurify the MeCP2 repressor complex on chromatin and quantify protein-DNA association by competitive PCR approach.
authors.affiliation: Sir Donald & Lady Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, St. Andrews Place, East Melbourne, Victoria 3002, Australia. s.el-osta@pmci.unimelb.edu.au
authors.forename: A
authors.initials: A
authors.lastname: El-Osta
authors.forename: A P
authors.initials: AP
authors.lastname: Wolffe
mesh_terms: D002460:Cell Line; D002843:Chromatin / Q000737:chemistry*; D002868:Chromosomal Proteins, Non-Histone; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000276:immunology; D005557:Formaldehyde / Q000737:chemistry; D020868:Gene Silencing; D019450:Genes, MDR; D006801:Humans; D046911:Macromolecular Substances; D051783:Methyl-CpG-Binding Protein 2; D016133:Polymerase Chain Reaction / Q000379:methods; D011233:Precipitin Tests / Q000379:methods*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000032:analysis; D015203:Reproducibility of Results
chemical_list: D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C497392:MECP2 protein, human; D046911:Macromolecular Substances; D051783:Methyl-CpG-Binding Protein 2; D012097:Repressor Proteins; D005557:Formaldehyde