PIAS1 is a target gene selective androgen receptor coregulator in prostate cancer cell chromatin. Homo sapiens

To study the importance of PIAS1 (protein inhibitor of activated STAT1) for the androgen-regulated transcriptome of VCaP prostate cancer cells, we silenced its expression by RNAi. Transcriptome analyses revealed that a subset of the androgen-regulated genes is significantly influenced, either activated or repressed, by PIAS1 depletion. The depletion also exposed a completely new set of genes to androgen regulation, suggesting that PIAS1 can mask genes from androgen receptor (AR). Pathway analyses of gene expression data suggest involvement of PIAS1 in VCaP cell proliferation. According to genome-wide ChIP-seq analyses, PIAS1 interacts with the AR on chromatin harboring also SUMO2/3, as androgen exposure multiplied the occupancy of PIAS1 in the chromatin, and resulted in nearly complete overlap with AR chromatin binding events. PIAS1 interacted also with pioneer factor FOXA1 in the chromatin. Our results strongly suggest that PIAS1 is a genuine chromatin-bound coregulator of AR which functions in a target gene selective fashion in prostate cancer cells. Overall design: All ChIP-seq samples were collected from cells treated with vehicle (EtOH) or 10 nM R1881 (synthetic androgen methyltrienolone) for 2 h. In addition, AR and FOXA1 ChIP-seq samples were collected from cells exposed to control siRNA (siNON, Dharmacon Non-targeting pool) or PIAS1 siRNA (siPIAS1, Dharmacon ON-TARGETplus SMARTpool for human PIAS1). IgG sample was collected from EtOH- and R1881-treated cells (siNON and siPIAS1 cells) and used as background control. Biological duplicate samples of the AR (siNON-R1881), PIAS1 (vehicle- and R1881-treated), FOXA1 (vehicle- and R1881-treated), RNA polymerase 2 (Pol2) (R1881-treated) and SUMO2/3 (R1881-treated) ChIP-seq samples were analyzed by using Solexa/Illumina 1.5 System and AR (siPIAS1-R1881), FOXA1 (siNON-R1881- and siPIAS1-R1881-treated) and SUMO2/3 (vehicle-treated) samples were analyzed using Illumina HiSeq 2000 platform. Single IgG sample was analyzed using Solexa/Illumina 1.5 System and single H3K4me2 (R1881-treated) sample with Illumina HiSeq 2000 platform 1.9.

为研究PIAS1（protein inhibitor of activated STAT1，活化STAT1蛋白抑制剂）对VCaP前列腺癌细胞雄激素调控转录组的重要作用，我们通过RNA干扰（RNA interference，RNAi）敲低其表达。转录组分析结果显示，PIAS1敲除会显著影响部分雄激素调控基因的表达，包括激活或抑制其转录。此外，PIAS1的敲除还使得全新的一组基因受到雄激素调控，提示PIAS1可将部分基因屏蔽于雄激素受体（androgen receptor，AR）的调控之外。基因表达数据的通路分析表明，PIAS1参与VCaP细胞的增殖过程。全基因组染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing，ChIP-seq）分析显示，PIAS1与携带SUMO2/3修饰的染色质上的AR存在相互作用：雄激素处理会使PIAS1在染色质上的结合占据量显著增加，且其结合位点与AR的染色质结合位点几乎完全重合。PIAS1还可与染色质上的先驱因子FOXA1发生相互作用。我们的研究结果有力表明，PIAS1是一种真正的染色质结合型AR共调控因子，在前列腺癌细胞中以靶基因选择性的方式发挥功能。
实验整体设计：所有ChIP-seq样本均取自经溶剂对照（乙醇，EtOH）或10 nM R1881（合成雄激素甲群勃龙，methyltrienolone）处理2小时的细胞。此外，我们还收集了经对照小干扰RNA（siNON，Dharmacon非靶向RNA混合池）或PIAS1小干扰RNA（siPIAS1，Dharmacon ON-TARGETplus人PIAS1专用SMARTpool）处理的细胞的AR与FOXA1 ChIP-seq样本。IgG对照样本取自经乙醇和R1881处理的细胞（siNON与siPIAS1组细胞），用作背景对照。AR（siNON-R1881组）、PIAS1（溶剂对照与R1881处理组）、FOXA1（溶剂对照与R1881处理组）、RNA聚合酶Ⅱ（Pol2，R1881处理组）以及SUMO2/3（R1881处理组）的ChIP-seq生物学重复样本均采用Solexa/Illumina 1.5测序系统进行分析；而AR（siPIAS1-R1881组）、FOXA1（siNON-R1881与siPIAS1-R1881处理组）以及SUMO2/3（溶剂对照处理组）的样本则采用Illumina HiSeq 2000测序平台进行分析。单个IgG对照样本采用Solexa/Illumina 1.5测序系统进行分析，单个H3K4me2（R1881处理组）样本则采用Illumina HiSeq 2000平台进行分析。