Epigenetic mechanisms of androgen regulated gene expression

This project seeks advance understanding of the basic biology of androgen regulation of transcription. Androgens and the androgen receptors are essential for normal embryonic development, male puberty and lifelong heath. Conversely, defects in androgen signalling are associated with a diversity of disorders in men and women including male under-masculinization, poly-cystic ovary syndrome in women, muscle wasting and many cancers. The physiological actions of androgens are mediated by the androgen receptor (AR) which are ligand dependent transcription factors. The AR recruits a diverse range of epigenetic coregulators to regulate gene expression. Approximately 400 proteins are believed to act as AR coregulators, however the exact role for these coregulators remain poorly understood. There is evidence that distinct androgen-responsive gene sub-classes exist in the genome and that epigenetic contexts underpin the ability of transcription factors such as the AR to regulate distinct gene networks in different cell types. Our hypothesis is that a key subset of AR-epigenetic coregulators termed lysine demethylases (KDMs) underpin both the cell and loci specific transcriptional effects regulated by the AR. To test this hypothesis we will use biochemical, molecular biology and bioinformatics approaches to define the role of two crucial AR-KDM coregulators, KDM5B and KDM7A.

Specifically we will use (i) well established biochemical approaches, including yeast-2-hybdrid and co-immunoprecipitation to determine the composition and mechanisms of interaction of these specific AR-KDM complexes. (ii) We will use siRNA approaches to functionally deplete specific KDMs in cultured androgen responsive human cell line cultures to determine the transcriptional functions of these coregulators as specific androgen regulated loci. We will use pharmaco-inhibitors of the KDMs to further investigate the role of these proteins in androgen regulated gene expression. (iii) We will use site-directed mutagenesis to recreate novel AR-mutations known to induce androgen insensitive phenotypes and will use yeast-2-hybrid to test whether these mutations disrupt AR interaction with KDM and related coregulators. (iv) We will examine the expression of KDMs in human tissue specimens and relate this to the expression of known AR-target genes, to confirm the mechanistic data obtained in cell lines is relevant in vivo. (v) We will use bioinformatics to exploit existing genomewide transcriptomic and epigenetic datasets to underpin, inform and guide the analysis of the role of these complexes at distinct AR-regulated loci. Collectively these approaches will determine the mechanistic roles of AR-KDM coregulator complexes in regulating distinct gene sub-types.

This mechanistic knowledge is required to underpin the development of the next generation of epigenetic-targeting pharmaceuticals to influence androgen signalling in distinct cellular contexts and to inform the basic understanding of the mechanism of androgen signalling in fetal and pubertal development.