Regulation of chromatin folding in space and time

原始项目摘要 真核生物基因组以线性序列编码遗传信息，但适当表达 它们的基因需要染色体折叠成复杂且空间上不同的三维结构。 基于基因组学方法的最新进展揭示了DNA相互作用的层次结构，从小到大， 染色质环，将基因和增强子连接到更大的染色体结构域和核 隔间然而，尽管这些组织特征及其 基因功能的影响，我们对染色体如何在空间上划分的理解非常有限， 功能性包装，并相对定位在核中。技术限制也阻碍了我们的 能够提出关于细胞间变异性和染色质折叠之间关系的问题， 定位，并在单细胞分辨率下发挥作用。 我们以前的研究涉及两种技术的发展，使用荧光原位 杂交（FISH）以单细胞分辨率询问染色体定位。我们的目标是在此基础上 工作和使用这些工具来阐明染色体片段如何找到对方，然后形成稳定的 细胞内的相互作用。我可以设想我们工作的三个直接阶段。第一，快速发展， 用于鉴定参与染色体相互作用的候选物的精确方法。第二，建立一个 一组基于原位的测定，可用于表征候选物，第三个是翻译我们的 从模式生物到人类的发现。总的来说，这里提出的研究将揭示新的分子 核组织的潜在机制，提供了一个新的途径来研究染色质如何折叠， 定位是建立和继承的，以及功能失调的组织如何导致疾病。

Original Project 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. I can envision three immediate stages for our work. The first is developing a rapid and precise method for identifying candidates involved in chromosome interactions. The second is establishing a battery of in situ-based assays that can be used to characterize the candidates, and the third is translating our findings from model organisms to humans. 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.