Elucidating chromosome structure and function through the lens of SMC complexes and R-loops

 描述（由申请人提供）：染色质的高级组织产生高保真染色体分离、DNA损伤修复和基因表达调控所必需的结构。确定这些结构中的染色质组织，形成这些结构的机制以及它们所发挥的分子功能是细胞生物学中最重要和最艰巨的任务之一。该提案通过分析粘附素来解决这一任务，粘附素是SMC（染色体结构维持）蛋白质复合物家族的成员。粘着蛋白将DNA的两个区域连接在一起以介导姐妹染色单体粘着蛋白、DNA修复、有丝分裂染色体凝聚和转录调节。从其在癌症进展、年龄依赖性出生缺陷和干细胞多能性中的新兴作用中可以明显看出粘附素的生物学功能的重要性。通过利用酵母遗传学，细胞学，新的生物化学和单分子成像分析，我们发现了新的粘蛋白的活动和监管机构。这些发现为以下方面提供了新的模型：1）DNA被粘附素的拓扑捕获; 2）粘附素ATP酶和寡聚化在粘附素的DNA结合和DNA束缚活性中的作用; 3）这些活性的时空调节;以及4）粘附素与其他SMC复合物的协调以促进更高级的染色体结构。基于这些模型，我们提出的实验，将告知分子基础上的凝聚素的活动，他们的调节，以及这些活动如何促进染色体的组织和功能。 另一个新兴但知之甚少的染色体功能介质是R环。当转录本与同源染色体位点杂交时，产生R环，产生RNA-DNA杂交体和置换的单链DNA。R环可导致染色体重排（GCR），并与癌症和脆性染色体位点有关。R环还可以通过调节表观遗传标记和反义RNA来调节基因表达。我们开发了新的遗传学和细胞学检测，以确定许多新的抑制剂和增强剂的R-环形成和新的机制，为他们的形成。我们还开发了一种新的定量和高精度的技术来映射全基因组的R环。通过这些工具，我们将解决该领域的关键问题。RNA、DNA和蛋白质的哪些特征调节R环的形成？R环和侧翼染色质的哪些特征决定了它们是否会引起DNA损伤，这种损伤是如何引起GCR的？R环是否调节染色体过程的其他方面，如同源重组和缩合？通过回答这些问题，我们将阐明R环形成的分子机制，并提供关键的见解，R环调节染色体功能的不同机制。

 DESCRIPTION (provided by applicant): Higher order organization of chromatin generates structures that are essential for high fidelity chromosome segregation, DNA damage repair, and the regulation of gene expression. Defining the chromatin organization in these structures, the mechanisms for forming these structures, and the molecular functions they play are amongst the most important and daunting tasks in cell biology. This proposal tackles this task through the analysis of cohesin, a member of the SMC (Structural Maintenance of Chromosomes) family of protein complexes. Cohesin tethers together two regions of DNA to mediate sister chromatid cohesin, DNA repair, mitotic chromosome condensation and transcription regulation. The importance of cohesin's biological functions is evident from its emerging roles in cancer progression, age-dependent birth defects, and stem cell pluripotency. By utilizing yeast genetics, cytology, new biochemical and single-molecule imaging assays, we have discovered novel cohesin activities and regulators. These discoveries lead to new models for: 1) the topological entrapment of DNA by cohesin; 2) the roles of cohesin ATPases and oligomerization in cohesin's DNA binding and DNA tethering activities; 3) the spatial-temporal regulation of these activities; and 4) cohesin's coordination with other SMC complexes to promote higher order chromosome structure. Based on these models we propose experiments that will inform on the molecular basis for cohesin's activities, their regulation, and how these activities facilitte chromosome organization and function. Another emerging but poorly understood mediators of chromosome function are R-loops. R-loops result when transcripts hybridize to homologous chromosomal sites, generating an RNA-DNA hybrid and a displaced single-stranded DNA. R-loops can lead to gross chromosomal rearrangements (GCRs) and have been linked to cancer and fragile chromosome sites. R-loops can also regulate gene expression by modulating epigenetic marks and antisense RNA. We developed novel genetic and cytological assays to identify many new inhibitors and enhancers of R-loop formation and new mechanisms for their formation. We also have developed a novel quantitative and high-precision technique to map R-loops genome-wide. With these tools we will address key questions in the field. Which features of RNA, DNA and proteins regulate R-loop formation? What characteristics of R-loops and flanking chromatin determine whether they induce DNA damage, and how does this damage cause GCRs? Do R-loops regulate other aspects of chromosomal processes like homologous recombination and condensation? By answering these questions we will elucidate the molecular mechanism of R-loop formation and provide critical insights into the diverse mechanisms by which R-loops modulate chromosome function.