Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
J Biol Chem. 2023 Jul;299(7):104928. doi: 10.1016/j.jbc.2023.104928. Epub 2023 Jun 15.
A nucleosome is typically positioned with its proximal edge (NPE) ∼50 bp downstream from the transcription start site of metazoan RNA polymerase II promoters. This +1 nucleosome has distinctive characteristics, including the presence of variant histone types and trimethylation of histone H3 at lysine 4. To address the role of these features in transcription complex assembly, we generated templates with four different promoters and nucleosomes located at a variety of downstream positions, which were transcribed in vitro using HeLa nuclear extracts. Two promoters lacked TATA elements, but all supported strong initiation from a single transcription start site. In contrast to results with minimal in vitro systems based on the TATA-binding protein (TBP), TATA promoter templates with a +51 NPE were transcriptionally inhibited in extracts; activity continuously increased as the nucleosome was moved downstream to +100. Inhibition was much more pronounced for the TATA-less promoters: +51 NPE templates were inactive, and substantial activity was only seen with the +100 NPE templates. Substituting the histone variants H2A.Z, H3.3, or both did not eliminate the inhibition. However, addition of excess TBP restored activity on nucleosomal templates with TATA promoters, even with an NPE at +20. Remarkably, nucleosomal templates with histone H3 trimethylated at lysine 4 are active with an NPE at +51 for both TATA and TATA-less promoters. Our results strongly suggest that the +1 nucleosome interferes with promoter recognition by TFIID. This inhibition can be overcome with TBP alone at TATA promoters or through positive interactions with histone modifications and TFIID.
核小体通常位于其近端边缘（NPE）距真核生物 RNA 聚合酶 II 启动子转录起始位点下游约 50bp 处。这个+1 核小体具有独特的特征，包括存在变体组蛋白类型和组蛋白 H3 赖氨酸 4 的三甲基化。为了研究这些特征在转录复合物组装中的作用，我们生成了具有四种不同启动子和位于各种下游位置的核小体的模板，这些模板在使用 HeLa 核提取物进行体外转录时使用。两个启动子缺乏 TATA 元件，但都支持从单个转录起始位点进行强烈起始。与基于 TATA 结合蛋白 (TBP) 的最小体外系统的结果相反，TATA 启动子模板的+51 NPE 在提取物中被转录抑制；当核小体向下游移动到+100 时，活性持续增加。对于 TATA 缺失的启动子，抑制作用更为明显：+51 NPE 模板无活性，只有当核小体移动到+100 时才会有大量活性。替代组蛋白变体 H2A.Z、H3.3 或两者都不能消除抑制作用。然而，添加过量的 TBP 可以恢复 TATA 启动子核小体模板的活性，即使 NPE 位于+20。值得注意的是，对于 TATA 和 TATA 缺失的启动子，赖氨酸 4 三甲基化的核小体模板在 NPE 为+51 时是活跃的。我们的结果强烈表明+1 核小体干扰 TFIID 对启动子的识别。这种抑制作用可以通过 TBP 在 TATA 启动子上单独克服，也可以通过与组蛋白修饰和 TFIID 的正相互作用来克服。
