Physical Chemistry of Nucleic Acids

项目摘要 核小体是真核RNA聚合酶转录的机械和能量障碍。 细胞内这一屏障的动态调控是基因表达调控的主要机制。 核小体屏障的不适当调节会导致许多病理情况，包括癌症。 在这里，我们将使用具有单分子荧光检测的高分辨率光学镊子(Fleezer)来 描述核小体对转录动力学的调控，以及人类POL II(HPOL II)如何 影响核小体的完整性。 我们将首先表征单个HPOL II的伸长动力学。具体来说，我们将遵循 HPOL II在单碱基对(BP)分辨率和位置精度(±3BP)下的研究进展我们将测量 HPOL II的无暂停速度、暂停概率、暂停持续时间和回溯动态，以及测试 这些动态是如何受力、伸长系数、磷酸化状态等因素调节的 RPB1的C-末端结构域，以及模板DNA上存在的扭转约束。这 分析将导致详细描述HPOL II的机械力化学循环以及它是如何被调节的。 同时，我们将用两个例子描述核小体屏障的能量学和动力学。 方法：1)机械地从组蛋白八聚体表面解开DNA；2) 机械地解开围绕八聚体的DNA链。我们将调查 屏障由+1核小体中出现的组蛋白变体和表观遗传修饰(H_2A.Z， H3K9ac和泛素化的H2B)或在基因体内(H3K36me3和H3K79me3)。重要的是，我们还将 结合这些力量伸展测量和荧光标记的组蛋白成分的检测 八聚体(使用一个新建立的“Fleezer”系统)来建立发生在 核小体在DNA的机械解缠和解链过程中。我们寻求获得一份详细的 对屏障的高度、深度和对称性的描述，以及通过表观遗传修饰改变屏障的情况。 接下来，我们将确定核小体屏障如何改变HPOL II的动力学，进而， 利用Fleezers系统，转录酶对核小体的完整性有什么影响？我们会 研究屏障的表观遗传修饰、模板的拓扑约束以及 伸长因子改变HPOL II的动力学以及它们如何影响屏障通过的稳定性 这种酶。与泽维尔·达扎克教授合作，我们将比较在以下方面获得的HPOL II的动力学- 在体外与体内观察到的那些在核小体的背景下，通过跟踪串联重复序列的荧光 U2OS细胞中MS2噬菌体RNA结合区的研究。我们还将进行体外实验， 核萃取物。我们希望获得前所未有的对物理/生理的定量描述 控制基因表达的机制。 1

Project Summary Nucleosomes represent a mechanical and energetic barrier to transcription by eukaryotic RNA polymerases. The dynamics modulation of this barrier in the cell is a major mechanism of gene expression regulation. Improper regulation of the nucleosomal barrier results in numerous pathological conditions, including cancer. Here, we will use high resolution optical tweezers with single molecule fluorescence detection (“fleezers”) to characterize the modulation of transcriptional dynamics by nucleosomes, and how the human Pol II (hPol II) affects nucleosome integrity. We will first characterize the elongation dynamics of single hPol II. Specifically, we will follow the progress of hPol II at single base pair (bp) resolution and at a position accuracy of ±3 bp. We will measure the pause-free velocity, the pausing probability, pause duration, and backtracking dynamics of hPol II, and test how these dynamics are modulated by factors such as force, elongation factors, the phosphorylation state of the C-terminal domain of RPB1, as well as the presence of torsional constrains on the template DNA. This analysis will results in a detailed description of the mechanochemical cycle of hPol II and how it is regulated. In parallel, we will characterize the energetics and dynamics of the nucleosomal barrier using two approaches: 1) mechanically unwrapping the DNA from the surface of the histone octamer and 2) mechanically unzipping the strands of the DNA sequentially around the octamer. We will investigate how the barrier is modulated by histone variants and epigenetic modifications that appear in +1 nucleosomes (H2A.Z, H3K9ac and ubiquitinated H2B) or inside gene bodies (H3K36me3 and H3K79me3). Importantly, we will also combine these force-extension measurements with detection of fluorescently labelled histone components of the octamer (using a newly built “fleezers” system) to establish the structural changes that occur in the nucleosome during mechanical unwrapping and unzipping of the DNA. We seek to obtain a detailed description of the height, depth, and symmetry of the barrier and its alteration by epigenetic modifications. Next, we will establish how the nucleosomal barrier modifies the dynamics of hPol II and, in turn, what is the effect of the transcribing enzyme on the integrity of nucleosomes using the fleezers system. We will investigate how epigenetic modifications of the barrier, the topological constraint of the template, and elongation factors alter the dynamics of hPol II and how they affect the stability of the barrier to the passage of the enzyme. In collaboration with Prof. Xavier Darzacq, we will compare the dynamics of hPol II obtained in- vitro with those observed in-vivo in the context of nucleosomes, by tracking the fluorescence of tandem repeats of MS2 bacteriophage RNA binding domains in U2OS cells. We will also perform ex-vivo experiments using nuclear extracts. We hope to obtain an unprecedented quantitative description of the physical/physiological mechanisms that control gene expression. 1