Mechanisms and functions of repressive chromatin structure in quiescent cells.

项目摘要 染色质的构象是细胞调节DNA模板化的主要机制。 流程.与这种广泛的作用相一致，酶和结构成分的畸变 控制染色质动力学与广泛的人类疾病有关，包括 大多数癌症和越来越多的遗传和发育障碍。最近的技术 这些进展增加了我们对核小体在DNA上的位置是如何被调节的知识， 染色质如何形成称为染色质结构域的大型三维（3D）环。3D染色质结构是 假设能够根据上下文促进和抑制转录。然而，在这方面， 理解这些类型的染色质结构的机制和功能是困难的， 目前的方法分辨率低，几乎不可能确定染色质结构 在细胞内以必要的尺度确定其与单基因表达的关系。结果导致 一个长期存在的假设，即3D染色质结构在这个水平上调节转录，在很大程度上尚未得到验证 in a physiological生理context上下文.检查预期发挥功能的细胞中的3D染色质结构 广泛地说，候选人已经实施了一种能够绘制3D染色质结构的基因组学方法 全基因组在前所未有的单核小体150碱基对分辨率在静止S。啤酒。 静止酵母具有静止细胞的保守特征，特别是广泛的转录 抑制和染色质凝聚，这使他们成为一个很好的模型，以确定机制 3D染色质结构通过其抑制转录。初步结果导致了这样的假设， 在静止细胞中，凝聚素复合物通过诱导静止特异性3D染色质来抑制转录 结构.本提案的目的1将确定如何凝聚蛋白的目标，以形成染色质结构域， 使用基因组学、显微镜和单分子磁性镊子测定来观察静止。目标2将检查 染色质在结构域内的构象，以确定其是否在较小尺度上折叠成3D结构 并研究这种尺度的染色质结构是否是抑制转录的机制 在静止期。这项工作的指导部分将在博士的赞助下完成。 Toshio Tsukiyama是一位染色质领域的专家，他在一个主要的基础科学研究所工作， 弗雷德哈钦森癌症研究中心。候选人还将接受单一培训- 在苏·比金斯博士的监督下进行分子生物化学测定，并将扩大她在以下方面的熟练程度： 生物信息学通过课程和独立研究。这项研究和培训将提供 候选人具有令人兴奋的模型系统和成功的独立职业所需的技能。

Project Summary The conformation of chromatin is a primary mechanism by which the cell regulates DNA-templated processes. Consistent with this broad role, aberrations in the enzymes and structural components responsible for controlling chromatin dynamics have been linked to an extensive range of human diseases, including the majority of cancers and an increasing number of genetic and developmental disorders. Recent technological advancements have increased our knowledge of how the positions of nucleosomes on DNA are regulated and how chromatin forms large three-dimensional (3D) loops called chromatin domains. 3D chromatin structure is hypothesized to be capable of both promoting and inhibiting transcription depending on context. However, understanding the mechanisms and functions of these types of chromatin structures has been difficult due to the low resolution of current methods, which have made it almost impossible to determine chromatin structure within cells at scales necessary to determine its relationship to the expression of single-genes. As a result, the long-held hypothesis that 3D chromatin structure at this level regulates transcription has been largely untested in a physiological context. To examine 3D chromatin structure in cells in which it is expected to function extensively, the candidate has implemented a genomics method capable of mapping 3D chromatin structure genome-wide at unprecedented single-nucleosome 150 base pair resolution in quiescent S. cerevisiae. Quiescent yeast bear conserved hallmarks of quiescent cells, in particular widespread transcriptional repression and chromatin condensation, which make them an excellent model for determining the mechanisms by which 3D chromatin structure represses transcription. Preliminary results have led to the hypothesis that in quiescent cells, the condensin complex represses transcription by inducing quiescence-specific 3D chromatin structures. Aim 1 of this proposal will determine how condensin is targeted to form chromatin domains during quiescence using genomics, microscopy, and a single-molecule magnetic tweezer assay. Aim 2 will examine the conformation of chromatin within domains to determine if it is folded into 3D structure at a smaller scale and investigate whether chromatin structure at this scale is the mechanism by which transcription is repressed during quiescence. The mentored component of this work will be completed under the sponsorship of Dr. Toshio Tsukiyama, an expert in the chromatin field, at one of the premier institutes for basic science and cancer research, the Fred Hutchinson Cancer Research Center. The candidate will also be trained in single- molecule biochemical assays under the supervision of Dr. Sue Biggins, and will expand her proficiency in bioinformatics through coursework and independent study. This research and training will provide the candidate with an exciting model system and the skills necessary for a successful independent career.