Regulation of chromatin folding in space and time

摘要 真核基因组以其线性序列编码遗传信息，但适当地表达 他们的基因需要染色体折叠成复杂的、空间上不同的三维结构。 基因组方法的最新进展揭示了DNA相互作用的层次结构，从小到小 将基因和增强子连接到更大的染色体区域和核的染色质环 车厢。然而，尽管这些组织特征和它们的 对基因功能的影响，我们对染色体是如何在空间上分割的了解非常有限， 功能包装，并相对定位在细胞核内。技术限制也阻碍了我们的 能够提出关于细胞间的可变性和染色质折叠之间的关系的问题， 定位，并在单个单元分辨率下工作。 我们之前的研究包括开发两种使用原位荧光的技术 杂交(FISH)在单细胞分辨率下询问染色体定位。我们的目标是在此基础上再接再厉 研究并使用这些工具来阐明染色体片段是如何找到彼此并形成稳定的 细胞内的相互作用。为此，我们开发了一种快速而准确的方法来确定候选人 参与染色体的相互作用。我们现在建议使用这项技术来隔离新的架构 蛋白质，随后将进行一系列基因组和原位分析，以表征 候选人。总的来说，这里提出的研究将揭示潜在的新的分子机制 核组织，为研究染色质折叠和定位是如何建立的提供了新的途径 和遗传，以及失调的组织如何导致疾病。

Abstract Eukaryotic genomes encode genetic information in their linear sequence, but appropriate expression of their genes requires chromosomes to fold into complex and spatially distinct three-dimensional structures. Recent advances in genomic-based approaches have uncovered a hierarchy of DNA interactions, from small chromatin loops that connect genes and enhancers to larger chromosomal domains and nuclear compartments. However, despite the remarkable conservation of these organizational features and their impact on gene function, we have a very limited understanding of how chromosomes are spatially partitioned, functionally packaged, and relatively positioned in the nucleus. Technical limitations have also hindered our ability to ask questions regarding cell-to-cell variability and the relationship between chromatin folding, positioning, and function at single cell resolution. Our previous studies involved the development of two technologies that use fluorescent in situ hybridization (FISH) to interrogate chromosome positioning at single-cell resolution. Our goal is to build on this work and use these tools to elucidate how chromosomal segments find each other and then form stable interactions within cells. To this end, we have developed a rapid and precise method for identifying candidates involved in chromosome interactions. We now propose to employ this technology to isolate novel architectural proteins, which will be followed by a battery of genomic and in situ-based assays to characterize the candidates. Collectively, the studies proposed here will uncover novel molecular mechanisms underlying nuclear organization, providing a new avenue to study how chromatin folding and positioning is established and inherited, and how dysfunctional organization contributes to disease.