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

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. Overall design: Methyl-Midi SeqTM (MMS) or Reduced Representation Hydroxymethylation Profiling (RRHP) in MCF7 cells in response to treatment with either a non-targeting siRNA control, or siRNA against TET2.

雌激素受体α（Estrogen receptor-a, ER）在雌激素受体阳性（ER+）乳腺癌中驱动肿瘤发生与转移。GATA3是一种与ER功能密切相关的转录因子，但GATA3在ER转录活性中的作用尚不清楚。本研究通过定量多重快速内源蛋白免疫沉淀质谱（quantitative multiplexed rapid immunoprecipitation mass spectrometry of endogenous proteins, qPLEX-RIME）分析GATA3缺失后ER复合物的变化，以期明确GATA3对ER复合物的贡献。出乎意料的是，在GATA3缺失的情况下，几乎无蛋白质从ER复合物中解离，唯一的显著变化为TET2从ER复合物中缺失。在乳腺癌细胞及患者来源异种移植（Patient-Derived Xenograft, PDX）组织中，TET2结合事件几乎完全构成ER结合事件的子集，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的调控关系，本研究检测了TET2在ER+乳腺癌细胞中调控DNA修饰的作用。结果显示，TET2敲低并未引起全基因组DNA甲基化水平的明显变化，但TET2缺失后，甲基化DNA氧化为5-羟甲基胞嘧啶（5-hydroxymethylcytosine, 5hmC）的过程显著减弱，且该过程发生于ER增强子区域。上述结果表明TET2参与ER结合位点处5hmC的生成与维持，为TET2介导的ER靶基因调控提供了潜在机制。实验整体设计：在MCF7细胞中采用Methyl-Midi SeqTM（MMS）或简化代表性羟甲基化谱分析（Reduced Representation Hydroxymethylation Profiling, RRHP），分别用非靶向siRNA对照或靶向TET2的siRNA进行处理。