Genomic analyses of TF binding, histone acetylation and gene expression reveal classes of E2-regulated promoters

To explore the global mechanisms of estrogen-regulated transcription, we used chromatin immunoprecipitation coupled with DNA microarrays to determine the localization of RNA polymerase II (Pol II), estrogen receptor alpha (ERalpha), steroid receptor coactivator proteins (SRC), and acetylated histones H3/H4 (AcH) at estrogen-regulated promoters in MCF-7 cells with or without estradiol (E2) treatment. In addition, we correlated factor occupancy with gene expression and the presence of transcription factor binding elements. Using this integrative approach, we defined a set of 58 direct E2 target genes based on E2-regulated Pol II occupancy and classified their promoters based on factor binding, histone modification, and transcriptional output. Many of these direct E2 target genes exhibit interesting modes of regulation and biological activities, some of which may be relevant to the onset and proliferation of breast cancers. Our studies indicate that about one-third of these direct E2 target genes contain promoter-proximal ERalpha-binding sites, which is considerably more than previous estimates. Some of these genes represent possible novel targets for regulation through the ERalpha/AP-1 tethering pathway. Our studies have also revealed several previously uncharacterized global features of E2-regulated gene expression, including strong positive correlations between Pol II occupancy and AcH levels, as well as between the E2-dependent recruitment of ERalpha and SRC at the promoters of E2-stimulated genes. Furthermore, our studies have revealed new mechanistic insights into E2-regulated gene expression, including the absence of SRC binding at E2-repressed genes and the presence of constitutively bound, promoter-proximally paused Pol IIs at some E2-regulated promoters. These mechanistic insights are likely to be relevant for understanding gene regulation by a wide variety of nuclear receptors. Keywords: MCF7 cells, E2, Estrogen, RNA, ER, RNA Polymerase II, SRC, Acetylated histones, ChIP-chip See Materials and Methods in the associated publication for more details

为探究雌激素调控转录的全局机制，我们采用染色质免疫共沉淀结合DNA微阵列技术，对经或未经过雌二醇（estradiol, E2）处理的MCF-7细胞中，RNA聚合酶II（RNA polymerase II, Pol II）、雌激素受体α（estrogen receptor alpha, ERα）、类固醇受体辅激活蛋白（steroid receptor coactivator proteins, SRC）以及乙酰化组蛋白H3/H4（acetylated histones H3/H4, AcH）在雌激素调控型启动子区域的定位情况进行了检测。此外，我们将转录因子结合占有率与基因表达水平以及转录因子结合元件的存在状态进行了关联分析。借助这一整合分析策略，我们基于受E2调控的Pol II结合占有率，确定了包含58个直接E2靶基因的基因集，并依据转录因子结合模式、组蛋白修饰特征以及转录输出结果对这些靶基因的启动子进行了分类。上述直接E2靶基因中有诸多展现出独特的调控模式与生物学活性，其中部分可能与乳腺癌的发生与增殖密切相关。本研究发现，约三分之一的此类直接E2靶基因包含启动子近端ERα结合位点，这一比例远高于此前的预估。其中部分基因可能是通过ERα/AP-1拴系通路进行调控的新型靶标。本研究还揭示了E2调控基因表达的多项此前未被阐明的全局特征，包括Pol II结合占有率与AcH水平之间的显著正相关，以及在E2激活基因的启动子区域，ERα与SRC的E2依赖性招募之间的显著正相关。此外，本研究还为E2调控的基因表达提供了新的机制视角：例如在E2抑制型基因的启动子区域不存在SRC结合，而部分E2调控型启动子区域存在组成型结合且启动子近端暂停的Pol II。这些机制见解有望为理解多种核受体介导的基因调控提供参考。关键词：MCF-7细胞、E2、雌激素、RNA、ER、RNA聚合酶II、SRC、乙酰化组蛋白、ChIP-chip 详细实验方法请参见相关发表文献的材料与方法部分。