Sengupta Surojeet, Biarnes Michael C, Clarke Robert, Jordan V Craig
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, 3970 Reservoir Rd NW, Washington, DC, 20057, USA,
Breast Cancer Res Treat. 2015 Apr;150(2):265-78. doi: 10.1007/s10549-015-3319-1. Epub 2015 Feb 27.
Estrogen (E2)-induced transcription requires coordinated recruitment of estrogen receptor α (ER) and multiple factors at the promoter of activated genes. However, the precise mechanism by which this complex stimulates the RNA polymerase II activity required to execute transcription is largely unresolved. We investigated the role of bromodomain (BRD) containing bromodomain and extra-terminal (BET) proteins, in E2-induced growth and gene activation. JQ1, a specific BET protein inhibitor, was used to block BET protein function in two different ER-positive breast cancer cell lines (MCF7 and T47D). Real-time PCR and ChIP assays were used to measure RNA expression and to detect recruitment of various factors on the genes, respectively. Protein levels were measured by Western blotting. JQ1 suppressed E2-induced growth and transcription in both MCF7 and T47D cells. The combination of E2 and JQ1 down-regulated the levels of ER protein in MCF7 cells but the loss of ER was not responsible for JQ1-mediated inhibition of E2 signaling. JQ1 did not disrupt E2-induced recruitment of ER and co-activator (SRC3) at the E2-responsive DNA elements. The E2-induced increase in histone acetylation was also not altered by JQ1. However, JQ1 blocked the E2-induced transition of RNA polymerase II from initiation to elongation by stalling it at the promoter region of the responsive genes upstream of the transcription start site. This study establishes BET proteins as the key mediators of E2-induced transcriptional activation. This adds another layer of complexity to the regulation of estrogen-induced gene activation that can potentially be targeted for therapeutic intervention.
雌激素（E2）诱导的转录需要雌激素受体α（ER）和多个因子在激活基因的启动子处协同募集。然而，这种复合物刺激执行转录所需的RNA聚合酶II活性的精确机制在很大程度上尚未解决。我们研究了含溴结构域和额外末端结构域（BET）的蛋白质在E2诱导的生长和基因激活中的作用。JQ1是一种特异性BET蛋白抑制剂，用于在两种不同的ER阳性乳腺癌细胞系（MCF7和T47D）中阻断BET蛋白功能。实时PCR和染色质免疫沉淀（ChIP）分析分别用于测量RNA表达和检测各种因子在基因上的募集情况。蛋白质水平通过蛋白质印迹法进行测量。JQ1抑制了MCF7和T47D细胞中E2诱导的生长和转录。E2与JQ1的组合下调了MCF7细胞中ER蛋白的水平，但ER的缺失并非JQ1介导的E2信号抑制的原因。JQ1并未破坏E2诱导的ER和共激活因子（SRC3）在E2反应性DNA元件处的募集。JQ1也未改变E2诱导的组蛋白乙酰化增加。然而，JQ1通过将RNA聚合酶II滞留在转录起始位点上游的反应基因启动子区域，阻断了E2诱导的RNA聚合酶II从起始到延伸的转变。本研究确定BET蛋白是E2诱导的转录激活的关键介质。这为雌激素诱导的基因激活调控增加了另一层复杂性，这可能成为治疗干预的潜在靶点。
