Abstract
Sequence motifs are short, recurring patterns in DNA that can mediate sequence-specific binding for proteins such as transcription factors or DNA modifying enzymes. The androgen response element (ARE) is a palindromic, dihexameric motif present in promoters or enhancers of genes targeted by the androgen receptor (AR). Using chromatin immunoprecipitation sequencing (ChIP-Seq) we refined AR-binding and AREs at a genome-scale in androgen-insensitive and androgen-responsive prostate cancer cell lines. Model-based searches identified more than 120,000 ChIP-Seq motifs allowing for expansion and refinement of the ARE. We classified AREs according to their degeneracy and their transcriptional involvement. Additionally, we quantified ARE utilization in response to somatic copy number amplifications, AR splice-variants, and steroid treatment. Although imperfect AREs make up 99.9% of the motifs, the degree of degeneracy correlates negatively with validated transcriptional outcome. Weaker AREs, particularly ARE half sites, benefit from neighboring motifs or cooperating transcription factors in regulating gene expression. Taken together, ARE full sites generate a reliable transcriptional outcome in AR positive cells, despite their low genome-wide abundance. In contrast, the transcriptional influence of ARE half sites can be modulated by cooperating factors.
Highlights
Prostate cancer is the most common malignancy in American men[1,2]
We attempted to identify androgen response element (ARE) locations within our chromatin immunoprecipitation (ChIP)-Seq data using the find individual motif occurrences (FIMO) tool[41] with a position site specific matrix as it is defined in motif databases (Supplementary Table S1). This proved problematic as the ARE identified by currently available database models (e.g. Jaspar model MA0007.2)[51] did not resemble the palindromic ARE sequence described in the literature and were shifted in frame[22,52,53,54]
The lenient model is distinct from the ideal model such that it allowed for limited mismatch at individual base positions with the core hexamers of full site AREs as frequently observed in binding experiments
Summary
Prostate cancer is the most common malignancy in American men[1,2]. localized prostate cancer is curable by surgery, the primary treatment for metastatic prostate cancer remains androgen deprivation therapy (ADT). Known alterations of the AR include somatic gene amplification and/or over-expression that increase AR mediated response to low androgen levels, AR mutations that change ligand specificity to allow for activation by other steroids, and generation of AR splice variants (AR-Vs) that lack the LBD and are constitutively active even in the absence of androgen. The CWR22Rv1 prostate cancer cell line expresses AR full-length (AR(FL)) with a duplicated DBD in exon 317–19 and an AR splice variant, AR(V), lacking a LBD, becoming constitutively active[4,8,11,20]. The purpose of our AR ChIP-Seq study is to further characterize the ARE and identify cooperation with adjacent transcription binding motifs in androgen-responsive and androgen-insensitive prostate cancer cell lines
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