Co-regulated gene expression by estrogen receptor-α and liver receptor homolog-1 is a feature of the estrogen response in breast cancer cells

Estrogen receptor α (ERα) is a nuclear receptor that is the driving transcription factor expressed in the majority of breast cancers. Recent studies have demonstrated that the liver receptor homolog-1 (LRH-1), another nuclear receptor, is ERα-regulated in breast cancer cells. Further, LRH-1 stimulates proliferation and promotes motility and invasion of breast cancer cells. To determine the mechanisms of LRH-1 action in breast cancer cells, we carried out gene expression microarray analysis following siRNA-mediated LRH-1 knockdown. Interestingly, gene ontology (GO) category enrichment analysis of the genes differentially regulated in the presence or absence of LRH-1 identified estrogen responsive genes as the most highly enriched GO categories. To further define LRH-1 target genes, we performed chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq) to identify genomic targets of LRH-1. Remarkably, ChIP-seq showed LRH-1 binding at many ERα binding sites. Analysis of select binding sites confirmed regulation of ERα-regulated genes by LRH-1 through binding to estrogen response elements, as exemplified by the TFF1/pS2 gene. Finally, LRH-1 over-expression stimulated ERα recruitment, whilst LRH-1 knockdown reduced ERα recruitment to ERα binding sites. Taken together, our findings establish a key role for LRH-1 in the regulation of ERα target genes in breast cancer cells and identify a mechanism in which co-operative binding of LRH-1 and ERα at estrogen response elements controls the expression of estrogen-responsive genes. MCF-7 cells were transfected with LRH-1 siRNA #2, #3, or with a non-targeting siRNA (siControl) for 72 hours. Following assessment of RNA integrity, four biological replicates for each siRNA treatment were used for microarray analysis.

雌激素受体α（ERα，Estrogen receptor α）是一类核受体，亦是多数乳腺癌中表达的核心驱动转录因子。近期研究证实，另一类核受体肝受体同源物1（LRH-1，liver receptor homolog-1）在乳腺癌细胞中受ERα调控。进一步研究表明，LRH-1可促进乳腺癌细胞增殖、迁移与侵袭。为阐明LRH-1在乳腺癌细胞中的作用机制，我们通过小干扰RNA（siRNA）介导LRH-1敲低后，开展了基因表达微阵列分析。有趣的是，对LRH-1存在与缺失条件下差异表达基因的基因本体（GO，gene ontology）类别富集分析显示，雌激素应答基因是富集程度最高的GO类别。为进一步明确LRH-1的靶基因，我们采用染色质免疫共沉淀结合高通量测序（ChIP-seq，chromatin immunoprecipitation coupled to massively parallel sequencing）技术鉴定LRH-1的基因组靶标。值得注意的是，ChIP-seq结果显示LRH-1可结合于大量ERα结合位点。对选定结合位点的分析证实，LRH-1可通过结合雌激素应答元件调控ERα靶基因，以TFF1/pS2基因为典型例证。最后，LRH-1过表达可促进ERα招募，而LRH-1敲低则会降低ERα向ERα结合位点的招募。综上，本研究结果证实LRH-1在乳腺癌细胞ERα靶基因调控中发挥关键作用，并阐明了LRH-1与ERα在雌激素应答元件上协同结合，进而调控雌激素应答基因表达的分子机制。本实验中，MCF-7细胞分别转染LRH-1 siRNA #2、#3或非靶向siRNA（si对照），培养72小时。在完成RNA完整性评估后，每组siRNA处理设置四个生物学重复，用于后续微阵列分析。