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

 描述(由申请人提供)：染色质的高阶组织产生对高保真染色体分离、DNA损伤修复和基因表达调节至关重要的结构。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。这项建议通过分析粘附素来解决这一问题，粘附素是SMC(染色体结构维护)蛋白质复合体家族的成员。粘附素将DNA的两个区域连接在一起，介导姐妹染色单体粘附素、DNA修复、有丝分裂染色体凝聚和转录调节。粘附素的生物学功能的重要性从它在癌症进展、年龄相关性出生缺陷和干细胞多能性中的新角色中可见一斑。通过利用酵母遗传学、细胞学、新的生化和单分子成像分析，我们发现了新的粘附素活性和调节因子。这些发现导致了新的模型：1)粘附素对DNA的拓扑捕获；2)粘附素ATPase和寡聚在粘附素的DNA结合和DNA拴系活动中的作用；3)这些活动的时空调节；以及4)粘附素与其他SMC复合体的协调作用，以促进更高顺序的染色体结构。基于这些模型，我们建议进行实验，以了解粘附素活动的分子基础，它们的调节，以及这些活动如何促进染色体的组织和功能。另一种新出现但知之甚少的染色体功能介体是R-环。当转录本与同源染色体位置杂交时，就会产生R-环，产生RNA-DNA杂交体和移位的单链DNA。R环可导致严重的染色体重排(GCRs)，并与癌症和脆弱染色体部位有关。R环还可以通过调节表观遗传标记和反义RNA来调节基因的表达。我们开发了新的遗传和细胞学分析来确定许多新的R环形成的抑制和增强剂以及它们形成的新机制。我们还开发了一种新的定量和高精度技术来在全基因组范围内定位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导致DNA损伤，这种损伤是如何导致GCR的？R-环是否调节染色体过程的其他方面，如同源重组和凝聚？通过回答这些问题，我们将阐明R环形成的分子机制，并为R环调节染色体功能的不同机制提供关键的见解。

 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.