Analysis of chromatin-immunopurified MeCP2-associated fragments

Assam El-Osta, Alan P. Wolffe

Research output: Contribution to journalArticleResearchpeer-review

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.

Original languageEnglish
Pages (from-to)733-737
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume289
Issue number3
DOIs
Publication statusPublished - 7 Dec 2001
Externally publishedYes

Cite this

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Analysis of chromatin-immunopurified MeCP2-associated fragments. / El-Osta, Assam; Wolffe, Alan P.

In: Biochemical and Biophysical Research Communications, Vol. 289, No. 3, 07.12.2001, p. 733-737.

Research output: Contribution to journalArticleResearchpeer-review

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AU - El-Osta, Assam

AU - Wolffe, Alan P.

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AB - 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.

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