Genomic Androgen Receptor-Occupied Regions with Different Functions, Defined by Histone Acetylation, Coregulators and Transcriptional Capacity

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 �C 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, CEBP��, 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.

背景 雄激素受体（androgen receptor, AR）是一类类固醇激活的转录因子，可结合于特定DNA位点，在前列腺癌的发病机制中发挥关键作用。尽管已有大量研究明确了AR结合与有限数量基因座的靶基因表达之间的关联，但对这些位点进行高通量解析，能够帮助我们更深入地理解这一过程的复杂性。
方法与主要结果 本研究通过染色质免疫共沉淀（chromatin immunoprecipitation, ChIP）微阵列分析，将189个AR结合区域（androgen receptor occupied regions, ARORs）与1388个组蛋白H3乙酰化（histone H3 acetylation, AcH3）位点定位到一段占人类基因组3%的连续DNA片段上。在62个重复性极佳的ARORs中，有32个（52%）同时带有AcH3修饰标记。尽管在前列腺癌细胞中检测到的ARORs数量多于邻近的双氢睾酮（dihydrotestosterone, DHT）响应基因数量，但AcH3标记定义了一类与这类基因关联度显著更高的ARORs亚类：带有AcH3标记的ARORs侧翼基因中，有12%为DHT响应基因，而不带AcH3标记的ARORs侧翼基因中这一比例仅为1%。多数ARORs在荧光素酶报告基因实验中表现出增强子活性。对ARORs序列进行分析，并结合定点染色质免疫共沉淀实验，本研究鉴定出了AR转录共调控因子FoxA1、CEBPβ、NFI以及GATA2的结合位点；在小干扰RNA（siRNA）敲除实验中，这些因子对内源性AR靶基因的表达水平产生了各异的调控作用。
结论与意义 本研究认为，在给定的生理条件下，仅部分ARORs会发挥功能，且其作用机制具有多样性。这种多样性表明，同一转录因子可通过与染色质结构相关的不同机制，对基因表达进行差异化调控。