Abstract B44: Chromatin structure impacts androgen receptor transcriptional specificity in prostate cancer cell lines
Authors: Tewari Alok K. , Song Lingyun , Furey Terrence S. , Crawford Gregory E. , Febbo Phillip G. 
Published: May 2012

Androgen receptor (AR) regulated gene expression and the phenotypic impact of AR activity varies significantly between different prostate cancer (PC) models. Specifically, in genetically engineered prostate epithelial cells, AR activation causes slowed growth and differentiation whereas in patient-derived AR-positive prostate cancer cell lines, AR activity increases proliferation. While the factors modulating AR transcriptional specificity remain poorly understood, recent reports have underscored AR co-regulators and AR-dependent enhancer sequences as modulators of AR activity. We hypothesized that differences in chromatin structure may also contribute significantly to AR transcriptional specificity and phenotypic effect.

We used genome-wide DNase I hypersensitivity (HS) analysis coupled with tiled microarrays (DNase-chip) and ultra-high throughput sequencing (DNase-seq) to identify regions of open chromatin that are markers for various gene regulatory elements. DNaseI HS analysis was conducted in both LNCaP cells, which increase their proliferation in response to androgen, and LHSR-AR cells, a genetically engineered prostate epithelial cell line that differentiates in response to androgen. Each cell line was processed using our established DNaseI HS protocol prior to and after androgen stimulation. For DNase-chip, hypersensitive sites were identified using a custom microarray that included proximal promoter and adjacent regions of the top 100 genes differentially regulated by AR activation in each cell line. DNase-seq analysis was conducted on the entire genome of each cell line. Parzen scores based upon the frequency of detected genomic sequence were generated and used as a relative measure of DNA accessibility.

Our results demonstrate significant differences in chromatin structure between our two androgen-responsive models. DNase-chip analysis of specific genes such as KLK3 displays both overlapping and cell-type specific regions of chromatin availability that correlate with AR-regulated expression. KLK3 is differentially transcribed only in LNCaP, not LHSR-AR, in response to stimulation. Accordingly, a HS site matching a known AR enhancer element is found four kilobases upstream of the KLK3 TSS in LNCaP, but not in LHSR-AR. While chromatin structure explains some of the differences between these cell lines, it is not sufficient to account for all the differences observed. DNase-seq analysis in LNCaP cells suggests that there little immediate impact of AR activity on chromatin structure (approximately 77% of peaks are shared between stimulated and un-stimulated samples). In the proximal promoter regions of AR-regulated genes, the Parzen score increases in response to stimulation (Wilcoxon p &lt; 0.001), but the degree of change is relatively modest and may not be biologically significant. In addition, the number of peaks within the 100kb surrounding the TSS of AR-regulated genes did not change with androgen stimulation (604 un-stimulated, 613 stimulated, 75% in common). Finally, we examined the overlap between HS peaks and previously published putative AR binding sites. Of 279 suspected binding sites on chromosomes 19–22, none corresponded to a HS peak in unstimulated LNCaP cells, but 147 overlapped a HS peak in stimulated LNCaP cells. Overall, our LNCaP DNase-seq data suggests that the promoter regions of AR-regulated genes are primed for transcription, but that AR activation causes key alterations to the distal chromatin landscape. Further work is required to understand the mechanism behind phenotype-specific differences in the chromatin landscape and its effects on AR transcriptional specificity.

Citation Information: Cancer Res 2009;69(23 Suppl):B44.