TET2 is a component of the ER complex and controls 5mC to 5hmC conversion at ER cis-regulatory regions [RNA-Seq]

Estrogen receptor-a (ER) drives tumour development and metastasis in ER positive (ER+) breast cancer. GATA3 is a transcription factor that has been closely linked to ER function, but the role of GATA3 in ER transcriptional activity is not clear. We sought to identify the contribution of GATA3 to the ER complex by conducting quantitative multiplexed rapid immunoprecipitation mass spectrometry of endogenous proteins (qPLEX-RIME) to assess changes to the ER complex in response to GATA3 depletion. Unexpectedly, very few proteins were dissociated from the ER complex in the absence of GATA3, with the only major change being depletion of TET2 from the ER complex. In breast cancer cells and Patient-Derived Xenograft (PDX) tissue, TET2 binding events were shown to constitute a near-total subset of ER binding events, and loss of TET2 was functionally associated with reduced activation of proliferative pathways. To investigate the TET2-ER relationship, the role of TET2 in regulating DNA modifications in ER+ breast cancer cells was examined. TET2 knockdown did not appear to result in changes to global DNA methylation, however, oxidation of methylated DNA to 5-hydroxymethylcytosine (5hmC) was significantly reduced after TET2 depletion and these events occurred at ER enhancers. These findings implicate TET2 in the production and maintenance of 5hmC at ER sites, providing a potential mechanism for TET2-mediated regulation of ER target genes. RNA-seq in human breast cancer cell line MCF7 in response to siRNA-mediated knockdown of ER, GATA3 or TET2.

雌激素受体α（Estrogen receptor-α, ER）驱动雌激素受体阳性（ER+）乳腺癌的肿瘤发生与转移。GATA3是一种与ER功能密切相关的转录因子，但其在ER转录活性中的作用尚不明确。本研究旨在明确GATA3对ER复合物的贡献，通过定量多重快速内源蛋白免疫沉淀质谱（qPLEX-RIME）技术评估GATA3敲低后ER复合物的组成变化。出乎意料的是，在GATA3缺失的情况下，极少有蛋白从ER复合物中解离，唯一显著变化为TET2从ER复合物中耗竭。在乳腺癌细胞及患者来源异种移植（Patient-Derived Xenograft, PDX）组织中，研究显示TET2的结合事件几乎构成ER结合事件的全部子集，且TET2缺失与增殖通路激活减弱存在功能关联。为探究TET2与ER的相互关系，本研究检测了TET2在ER+乳腺癌细胞中调控DNA修饰的作用。TET2敲低并未对全局DNA甲基化水平产生明显影响，但在TET2耗竭后，甲基化DNA氧化为5-羟甲基胞嘧啶（5-hydroxymethylcytosine, 5hmC）的过程显著受阻，且该现象发生于ER增强子区域。上述发现表明TET2参与ER位点处5hmC的生成与维持，为TET2介导调控ER靶基因的潜在机制提供了依据。本研究还针对经siRNA介导敲低ER、GATA3或TET2的人乳腺癌细胞系MCF7开展了RNA测序（RNA-seq）。