Techniques for Evaluation of AR Transcriptional Output and Recruitment to DNA.

Steroid receptors are ligand activated transcription factors whose promoter specificity is regulated by a broad set of coregulators and pioneer factors. Corepressors and coactivators determine receptors' recruitment to specific regulatory elements and ultimately their transcriptional output. Using androgen receptor (AR) and NCOR1 corepressor as examples, this chapter describes experimental approaches to evaluate recruitment of steroid receptors and their coregulators to DNA and to determine coregulator contribution to the transcriptional output of the receptor. The chromatin immunoprecipitation assay, or ChIP, quantifies protein-DNA interaction in the cellular chromatin environment. Here, we describe a protocol to measure NCOR1 recruitment to AR binding sites of interest using ChIP. Gene Set Enrichment Analysis, GSEA, is a computational technique to determine whether a defined gene set is significantly represented among changes in gene expression between two biological groups. As an example, we examine whether AR repressed genes are significantly represented among genes altered by the NCOR1 knockout.