Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Mol Cell. 2013 Jul 25;51(2):185-99. doi: 10.1016/j.molcel.2013.06.007. Epub 2013 Jul 11.
Chromatin immunoprecipitation studies have mapped protein occupancies at many genomic loci. However, a detailed picture of the complexity of coregulators (CoRs) bound to a defined enhancer along with a transcription factor is missing. To address this, we used biotin-DNA pull-down assays coupled with mass spectrometry-immunoblotting to identify at least 17 CoRs from nuclear extracts bound to 17β-estradiol (E2)-liganded estrogen receptor-α on estrogen response elements (EREs). Unexpectedly, these complexes initially are biochemically stable and contain certain atypical corepressors. Addition of ATP dynamically converts these complexes to an "activated" state by phosphorylation events, primarily mediated by DNA-dependent protein kinase. Importantly, a "natural" ERE-containing enhancer and nucleosomal EREs recruit similar complexes. We further discovered the mechanism whereby H3K4me3 stimulates ERα-mediated transcription as compared with unmodified nucleosomes. H3K4me3 templates promote specific CoR dynamics in the presence of ATP and AcCoA, as manifested by CBP/p300 and SRC-3 dismissal and SAGA and TFIID stabilization/recruitment.
染色质免疫沉淀研究已经绘制了许多基因组位置上的蛋白质占据图谱。然而，与特定增强子结合的共调节因子 (CoR) 以及转录因子的复杂情况的详细图片仍然缺失。为了解决这个问题，我们使用生物素-DNA 下拉测定法结合质谱免疫印迹法，从核提取物中鉴定出至少 17 个 CoR，这些 CoR 与雌激素受体-α上的 17β-雌二醇 (E2) 配体结合在雌激素反应元件 (ERE) 上。出乎意料的是，这些复合物最初在生化上是稳定的，并包含某些非典型的核心抑制剂。添加 ATP 通过磷酸化事件（主要由 DNA 依赖性蛋白激酶介导）将这些复合物动态转化为“激活”状态。重要的是，“天然”含有 ERE 的增强子和核小体 ERE 招募类似的复合物。我们进一步发现了与未修饰核小体相比，H3K4me3 刺激 ERα 介导转录的机制。H3K4me3 模板在 ATP 和 AcCoA 的存在下促进特定的 CoR 动力学，表现为 CBP/p300 和 SRC-3 的去除以及 SAGA 和 TFIID 的稳定/募集。
