Chromatin Immunoprecipitation Assay for Analyzing Transcription Factor Activity at the Level of Peripheral Myelin Gene Promoters
Disruption of epigenetic regulators of transcription is a central mechanism of oncogenesis. Differential gene expression is facilitated by transcriptional regulatory mechanisms and chromatin modifications through DNA-protein interactions. One of the widely used assays to study this is chromatin immunoprecipitation (ChIP) assay, which enables the analysis of association between regulatory molecules, specific promoters, and histone modifications within the context of the cell. This is of immense value as ChIP assays can provide a glimpse of the regulatory mechanisms involved in gene expression in vivo. It is also a powerful technique for analyzing histone modifications as well as binding sites for proteins that bind either directly or indirectly to DNA. The basic steps in this protocol are fixation, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. Although ChIP is a versatile tool, this procedure requires the optimization of the various reaction conditions. Here, we present a detailed application of the ChIP assay to study the differential recruitment of transcriptional factors at the level of peripheral myelin gene promoters.
Key wordsChromatin immunoprecipitation ChIP assay Quantitative real-time PCR TCF/LEF Transcription factor activity Regulation of gene expression Peripheral myelin genes MPZ PMP22 DNA-protein interactions Transcription factor recruitment β-Catenin
I would like to thank Ms. Nadine J. Makhoul and Dr. Jamal Al Ali for the critical reading of the manuscript.
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