Abstract
BackgroundThe androgen receptor (AR) is a steroid-activated transcription factor that binds at specific DNA locations and plays a key role in the etiology of prostate cancer. While numerous studies have identified a clear connection between AR binding and expression of target genes for a limited number of loci, high-throughput elucidation of these sites allows for a deeper understanding of the complexities of this process.Methodology/Principal FindingsWe have mapped 189 AR occupied regions (ARORs) and 1,388 histone H3 acetylation (AcH3) loci to a 3% continuous stretch of human genomic DNA using chromatin immunoprecipitation (ChIP) microarray analysis. Of 62 highly reproducible ARORs, 32 (52%) were also marked by AcH3. While the number of ARORs detected in prostate cancer cells exceeded the number of nearby DHT-responsive genes, the AcH3 mark defined a subclass of ARORs much more highly associated with such genes – 12% of the genes flanking AcH3+ARORs were DHT-responsive, compared to only 1% of genes flanking AcH3−ARORs. Most ARORs contained enhancer activities as detected in luciferase reporter assays. Analysis of the AROR sequences, followed by site-directed ChIP, identified binding sites for AR transcriptional coregulators FoxA1, CEBPβ, NFI and GATA2, which had diverse effects on endogenous AR target gene expression levels in siRNA knockout experiments.Conclusions/SignificanceWe suggest that only some ARORs function under the given physiological conditions, utilizing diverse mechanisms. This diversity points to differential regulation of gene expression by the same transcription factor related to the chromatin structure.
Highlights
The ‘textbook’ paradigm of gene regulation by steroid hormone receptors entails the binding of receptors to hormone response elements located 59-upstream of the transcription start sites (TSSs) of responsive genes, followed by the recruitment of non-DNAbinding coactivators or corepressors
AR occupied regions (ARORs) were classified as level 2 (L2) and not level 1 (L1) because of missing probes imposed by repetitive sequences and requirements of the NimbleGen design criteria
Over the past several decades a general concept of transcription factor (TF)-mediated gene regulation has emerged. It depicts a sequence of events, starting with TFs binding at specific regions of DNA, followed by the covalent modification of chromatin proteins to ‘open’ chromatin, and allowing the nearby docking of the transcription machinery
Summary
The ‘textbook’ paradigm of gene regulation by steroid hormone receptors entails the binding of receptors to hormone response elements located 59-upstream of the transcription start sites (TSSs) of responsive genes, followed by the recruitment of non-DNAbinding coactivators or corepressors These latter factors modify histones and interact with the basal transcriptional machinery to modulate transcriptional initiation [1]. The distribution of such sites, including androgen receptor (AR)occupied regions (ARORs) on chromosomes 21 & 22 [3], is poorly correlated with gene density For this reason, two studies that used genomic windows around the TSSs of annotated genes may have been limited in their capacity to assign ARORs in a genome-wide fashion [4,5] by missing functional ARORs far away on linear DNA. While numerous studies have identified a clear connection between AR binding and expression of target genes for a limited number of loci, high-throughput elucidation of these sites allows for a deeper understanding of the complexities of this process
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