Mechanisms of chromatin regulation of transcription

染色质调控转录的机制 项目总结 在过去的十年里，波里尔实验室致力于为染色质生物学和 通过制定一项研究计划来实现转录调控，该计划的重点是 染色质和核小体结构动力学调节基因组对转录调控的可及性 复合体。我们已经通过自己和合作开发和应用了广泛的 能够进行机制研究的实验工具，包括单分子荧光、单分子作用力 光谱学、集合荧光和组蛋白化学连接。结合起来，我们在数量上 研究了染色质调节剂包括组蛋白翻译后修饰(PTM)、PTM结合 蛋白质(阅读器)、染色质重构体、连接器组蛋白和转录因子起着控制基因组的作用 染色质调节复合体的可达性。在这项工作的基础上，我们建议调查两个中心 未来5年染色质生物学和转录领域的问题：(I)先锋如何 转录因子以致密核小体和染色质中的识别位点为靶点，然后 促进染色质分解？(2)表观遗传调节器如何共同发挥作用，以协同或 拮抗地调节基因组对转录调控复合体的可及性？ 先锋因子是细胞分化的主要调节因子，与核小体耗竭有关， 并以某种方式访问它们在致密染色质中的结合部位，而这些结合部位是标准染色质无法访问的 转录因子(TF)。此外，PF功能障碍与疾病密切相关，最显著的是 癌症。通过结合单分子和系综研究，我们将直接测试PF的不同机制 功能，并确定PFS与规范TFS的区别。此外，通过协作工作 我们将在体内研究相同的PFS，以确定从我们的体外产生的PF机制 研究会影响它们在体内的功能。这第一个方向将提供对先锋因素如何获得 在致密染色质内访问它们的站点，以及PFS与规范的TF的区别。 包括组蛋白PTM在内的表观遗传调控因子及其读取器和编写器对生物体至关重要 发展、衰老和包括癌症在内的多种疾病。我们和其他人已经调查了这些 监管机构是单独的，但它们是如何结合在一起发挥作用的，在很大程度上仍是未知的。利用我们的能力 用PTM、组蛋白H3读取器和最新的连接物组蛋白的任意组合制备核心组蛋白和最近的连接物组蛋白 最近生化量的内源性人类SAGA和ATAC复合体，我们将使用单一的 基于分子、系综和ES细胞的方法来确定这些生物相关的组合如何 监管机构控制着对规范金融工具的可及性，并影响PFS。这第二个方向将提供一个 理解关键的表观遗传转录调控因子的机制和功能基础 差异控制染色质动力学、可及性和转录。

MECHANISMS OF CHROMATIN REGULATION OF TRANSCRIPTION PROJECT SUMMARY Over the past decade, the Poirier lab has worked to contribute to the fields of chromatin biology and transcription regulation by developing a research program that is focused on the mechanisms by which chromatin and nucleosome structural dynamics regulate genome accessiblity to transcription regulatory complexes. We have developed and applied on our own and through collaborations, a wide range of experimental tools that enable mechanistic studies including single molecule fluorscence, single molecule force spectroscopy, ensemble fluorescence, and histone chemical ligations. In combination, we have quantitatively investigated how chromatin regulators including histone post translational modifications (PTMs), PTM binding proteins (readers), chromatin remodelers, linker histones, and transcripton factors function to control genome accessiblity to chromatin regulatory complexes. Building off this work, we propose to investigate two central questions in the fields of chromatin biology and transcription in the coming 5 years: (i) How do pioneer transcription factors target their recognition sites within compact nucleosomes and chromatin, and then facilitate chromatin decompaction? (ii) How do epigenetic regulators function together to synergistically or antagonistically regulate genome accessibility to transcription regulatory complexes? Pioneer factors (PFs) are master regulators of cell differentiation, are correlated with nucleosome depletion, and somehow access their binding sites within compact chromatin that are inaccessible to canonical transcription factors (TFs). Furthermore, PF disfunction is strongly correlated with disease most notably cancer. By combining single molecule and ensemble studies we will directly test distinct mechanisms of PF function, and determine what differentiates PFs from canonical TFs. Furthermore, through collaborative work we will investigate the same PFs in vivo to determine how the PF mechanisms that emerge from our in vitro studies impact their functions in vivo. This first direction will provide key insights into how pioneer factors gain access to their sites within compact chromatin and what differentiates PFs from canonical TFs. Epigenetic regulators including histone PTMs, and their readers and writers are critical to organismal development, aging and numerous diseases including cancer. We and others have investigated these regulators individually, yet how they function in combination remains largely unknown. Leveraging our ability to prepare core histone and most recently linker histones with any combination of PTMs, histone H3 readers, and most recently biochemical quantities of endogenous human SAGA and ATAC complexes, we will use single molecule, ensemble and ES cell-based methods to determine how biologically relevant combinations of these regulators control accessibility to canonical TFs and influence PFs. This second direction will provide a mechanistic and functional foundation for understanding how key epigenetic transcriptional regulators differentially control chromatin dynamics, accessibility and transcription.