TRPS1 modulates chromatin accessibility to regulate estrogen receptor (ER) binding and ER target gene expression in luminal breast cancer cells (Time course PRO-seq)

Breast cancer is the most frequently diagnosed cancer in women. The most common subtype is luminal breast cancer, which is typically driven by the estrogen receptor alpha (ER), a transcription factor (TF) that activates many genes required for proliferation. Multiple effective therapies target this pathway, but individuals often develop resistance. Thus, there is a need to identify additional targets that regulate ER activity and contribute to breast tumor progression. TRPS1 is a repressive GATA-family TF that is overexpressed in breast tumors. Common genetic variants in the TRPS1 locus are associated with breast cancer risk, and luminal breast cancer cell lines are particularly sensitive to TRPS1 knockout. However, we do not know how TRPS1 regulates target genes to mediate these breast cancer patient and cellular outcomes. We introduced an inducible degron tag into the native TRPS1 locus within a luminal breast cancer cell line to identify the direct targets of TRPS1 and determine how TRPS1 mechanistically regulates gene expression. We acutely deplete over eighty percent of TRPS1 from chromatin within 30 minutes of inducing degradation. We find that TRPS1 regulates transcription of hundreds of genes, including those related to estrogen signaling. TRPS1 directly regulates chromatin structure, which causes ER to redistribute in the genome. ER redistribution causes direct repression of ER targets and indirectly activates dozens of ER target genes. Downstream from these primary effects, TRPS1 depletion represses cell cycle-related gene sets and reduces cell doubling rate. Finally, we show that high TRPS1 activity, calculated using a gene expression signature defined by primary TRPS1-regulated genes, is associated with worse breast cancer patient prognosis. Taken together, these data suggest a model in which TRPS1 modulates the activity of other TFs, both activating and repressing transcription of genes related to cancer cell fitness. Overall design: T47D cells with TRPS1 tagged with an inducible degron tag were treated with drugs to induce TRPS1 degradation for 0, 0.5, 1, 2, 4, and 24 hours, and collected for PRO-seq

乳腺癌是女性中确诊率最高的恶性肿瘤，最常见的亚型为管腔型乳腺癌，其通常由雌激素受体α（ER）驱动——ER是一类转录因子（TF），可激活增殖所需的众多基因。目前已有多种靶向该通路的有效治疗手段，但患者常出现耐药性。因此，亟需识别调控ER活性并参与乳腺肿瘤进展的其他靶点。TRPS1属于GATA家族的转录抑制因子，在乳腺肿瘤中呈过表达状态。TRPS1基因位点的常见遗传变异与乳腺癌发病风险相关，且管腔型乳腺癌细胞系对TRPS1敲除尤为敏感。然而，目前尚不清楚TRPS1如何调控靶基因，进而介导上述乳腺癌患者及细胞层面的表型。本研究将可诱导降解标签插入管腔型乳腺癌细胞系的内源TRPS1基因位点，以鉴定TRPS1的直接靶标，并阐明TRPS1调控基因表达的分子机制。在诱导降解后的30分钟内，我们即可将染色质上超过80%的TRPS1蛋白清除。研究发现，TRPS1可调控数百个基因的转录，其中包括与雌激素信号通路相关的基因。TRPS1可直接调控染色质结构，进而导致ER在基因组中发生重新分布。ER的重新分布会直接抑制ER靶基因的表达，并间接激活数十个ER靶基因。在这些初始效应之后，TRPS1敲除会抑制细胞周期相关基因集的表达，并降低细胞倍增速率。最后，我们通过基于TRPS1直接调控基因构建的基因表达特征计算得到的高TRPS1活性，与乳腺癌患者较差的预后显著相关。综上，本研究数据支持如下模型：TRPS1可调控其他转录因子的活性，双向调控与肿瘤细胞存活相关基因的转录。总体实验设计：将带有可诱导降解标签的TRPS1的T47D细胞经药物处理以诱导TRPS1降解，处理时长分别为0、0.5、1、2、4及24小时，随后收集样本进行PRO-seq测序。