Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
Department of Biochemistry, The University of Iowa, Iowa City, IA 52242, USA.
Nucleic Acids Res. 2020 Aug 20;48(14):7767-7785. doi: 10.1093/nar/gkaa531.
To better understand human RNA polymerase II (Pol II) promoters in the context of promoter-proximal pausing and local chromatin organization, 5' and 3' ends of nascent capped transcripts and the locations of nearby nucleosomes were accurately identified through sequencing at exceptional depth. High-quality visualization tools revealed a preferred sequence that defines over 177 000 core promoters with strengths varying by >10 000-fold. This sequence signature encompasses and better defines the binding site for TFIID and is surprisingly invariant over a wide range of promoter strength. We identified a sequence motif associated with promoter-proximal pausing and demonstrated that cap methylation only begins once transcripts are about 30 nt long. Mapping also revealed a ∼150 bp periodic downstream sequence element (PDE) following the typical pause location, strongly suggestive of a +1 nucleosome positioning element. A nuclear run-off assay utilizing the unique properties of the DNA fragmentation factor (DFF) coupled with sequencing of DFF protected fragments demonstrated that a +1 nucleosome is present downstream of paused Pol II. Our data more clearly define the human Pol II promoter: a TFIID binding site with built-in downstream information directing ubiquitous promoter-proximal pausing and downstream nucleosome location.
为了更好地理解人类 RNA 聚合酶 II（Pol II）启动子在启动子近端暂停和局部染色质组织中的作用，通过深度测序准确识别了新生加帽转录本的 5'和 3'末端以及附近核小体的位置。高质量的可视化工具揭示了一个优选序列，该序列定义了超过 177000 个核心启动子，其强度差异超过 10000 倍。该序列特征包含并更好地定义了 TFIID 的结合位点，并且在广泛的启动子强度范围内惊人地不变。我们确定了与启动子近端暂停相关的序列基序，并证明只有当转录物大约 30nt 长时，帽甲基化才开始。映射还揭示了在典型暂停位置之后存在一个约 150bp 的下游序列元件（PDE），强烈暗示存在一个 +1 核小体定位元件。利用 DNA 片段化因子（DFF）的独特性质进行的核流出测定，以及对 DFF 保护片段的测序表明，暂停的 Pol II 下游存在一个 +1 核小体。我们的数据更清楚地定义了人类 Pol II 启动子：一个 TFIID 结合位点，内置下游信息，指导普遍存在的启动子近端暂停和下游核小体位置。
