A toolkit for visualizing and manipulating chromosomal interactions in living cells.

项目总结/摘要 真核基因组结构在三维空间和时间上都有波动，必须精确地 以实现基因表达的调控。这一结构构成了监管互动的基础， 远端控制元件（即增强子）和它们靶向的基因。虽然成千上万的增强剂 现在已经被注释和他们的相互作用的基因伙伴确定，我们有一个穷人的理解， 这些相互作用的分子性质，它们的调节，以及它们在细胞中的意义。染色体 构象捕获（3C）技术加速了对染色体组织的研究，但仅提供 群体平均快照时间分辨率差，因此无法描述染色体相互作用 在其相关背景下。阐明增强子-基因在活的单细胞中相互作用的分子基础， 对理解转录控制至关重要。为了满足这一需求，我们正在构建新的可视化工具， 并操纵活细胞中的染色体相互作用。 该建议旨在通过探索模型的动力学和控制来剖析增强子功能 染色体相互作用：Sox 2基因及其远端的必需Sox 2控制区（SCR）。Sox 2编码一种 组织特异性转录因子（TF）参与多能性和重编程，使其转录 控制广泛的利益。在小鼠胚胎干细胞（ESC）中，Sox 2的表达是通过 SCR的功能，位于>100 kb以外。为了探索Sox 2与其增强子相互作用的动力学， 我们正在将标记DNA的技术与基因组编辑的进展相结合。以确定 这些动力学如何影响基因表达，我们正在开发工具， Sox 2/SCR相互作用的稳定性，并测定个体中Sox 2基因表达的结果。 活细胞这些研究应该广泛地告知我们的理解转录控制增强子 元件，并应推广到广泛的其他基因组背景。

PROJECT SUMMARY/ABSTRACT Eukaryotic genome structure fluctuates across both 3-dimensional space and time and must be precisely orchestrated to achieve regulated gene expression. This structure underlies regulatory interactions between distal control elements (i.e. enhancers) and the genes that they target. While many thousands of enhancers have now been annotated and their interacting genic partners identified, we have a poor understanding of the molecular nature of these interactions, their regulation, and their significance in the cell. Chromosome conformation capture (3C) techniques have accelerated study of chromosomal organization but provide only population-averaged snapshots with poor temporal resolution and so fail to describe chromosomal interactions in their relevant context. Elucidating the molecular basis of enhancer-gene interactions in living, single cells is paramount to understanding transcriptional control. To address this need we are building new tools to visualize and manipulate chromosomal interactions in living cells. This proposal aims to dissect enhancer function by probing the dynamics and control of a model chromosomal interaction: the Sox2 gene and its distal, essential Sox2 Control Region (SCR). Sox2 encodes a tissue-specific transcription factor (TF) involved in pluripotency and reprogramming, making its transcriptional control of broad interest. In mouse embryonic stem cells (ESCs), Sox2 expression is established through the function of the SCR, located >100 kb away. To probe the dynamics of Sox2's interaction with its enhancer in living ESCs, we are combining techniques for marking DNA with advances in genome editing. To determine how these dynamics inform gene expression, we are developing tools to acutely and specifically manipulate the stability of the Sox2/SCR interaction and assay the consequences on Sox2 gene expression in individual living cells. These studies should broadly inform our understanding of transcriptional control by enhancer elements and should be generalizable to a wide range of other genomic contexts.