Study of Nuclear Receptor-Induced Transcription Complex Assembly and Histone Modification by Chromatin Immunoprecipitation Assays

Abstract

This chapter focuses on the study of nuclear receptor-induced transcription complex assembly and histone modification by chromatin immunoprecipitation (ChIP) assays. Nuclear receptor-mediated transcriptional regulation is a complex process involving the dynamic assembly of multiprotein complexes and modifications of chromosomal proteins, such as histones, at the target gene promoter. An increasingly used method to study this process in the context of natural chromosome structure is the chromatin immunoprecipitation assay. The ChIP assay employs a combination of formaldehyde induced in vivo cross-linking, immunoprecipitation, and sequence-specific DNA detection methods to observe the specific proteins associated with specific gene promoter regions in tissues or cultured cells. During the procedure of the ChIP assay a nuclear lysate is prepared from formaldehyde cross-linked cells that have been previously treated with or without hormones. ChIP assays allow the accurate assessment of the protein complexes associated with a given promoter during transcriptional regulation, spatially and temporally. The proteins of interest that can be examined by the use of specific antibodies include transcription factors that bind directly to the DNA, coactivators, corepressors, RNA polymerase and its associated proteins, and modified histones. ChIP assays have been applied to study nuclear receptor-mediated cofactor complex assembly and histone modifications at target gene promoters. The amount of antibody used in the immunoprecipitation depends on the affinity of the specific antibody, as well as the abundance of the chromatin-associated proteins and protein modifications.