Biochemical analyses of nuclear receptor-dependent transcription with chromatin templates.

Chromatin, the physiological template for transcription, plays important roles in gene regulation by nuclear receptors (NRs). It can (1) restrict the binding of NRs or the transcriptional machinery to their genomic targets, (2) serve as a target of regulatory posttranslational modifications by NR coregulator proteins with histone-directed enzymatic activities, and (3) function as a binding scaffold for a variety of transcription-related proteins. The advent of in vitro or "cell-free" systems that accurately recapitulate ligand-dependent transcription by NRs with chromatin templates has allowed detailed analyses of these processes. Biochemical studies have advanced our understanding of the mechanisms of gene regulation, including the role of ligands, coregulators, and nucleosome remodeling. In addition, they have provided new insights about the dynamics of NR-mediated transcription. This chapter reviews the current methodologies for assembling, transcribing, and analyzing chromatin in vitro, as well as the new information that has been gained from these studies.