Molecular dissection of Topologically Associating Domains (TADs)

三维折叠是脊椎动物基因组的一种固有特性，它是为了在细胞核这样的小空间里容纳一个单细胞大约2米长的DNA。这种折叠，远不是一个随机的过程，似乎是高度预定的，并强烈影响基因表达。在亚染色体尺度上，我们的基因组被划分为称为拓扑关联域（TADs）的自相互作用单元，范围从几百个碱基对到几个兆碱基。TADs由所谓的边界区域划分，将它们与基因组的其余部分隔离开来，从而促进了其中包含的位点之间的接触。有人提出，tad代表了基因组的一个基本结构单元，它被认为是引导调控元件到它们的同源基因。最近，TADs及其边界的破坏被证明与先天性畸形或癌症等人类疾病有关。尽管TADs具有生物学意义，但构建这种基本结构所需的基本成分仍然难以捉摸。在本提案中，我的目标是分子解剖TAD结构域及其边界区域，以了解这些结构如何形成并受到其基因组背景的影响。通过结合Capture-HiC或CRISPR/Cas等尖端技术来产生携带特定突变的小鼠，我将：1)评估TAD边界在结构域组织中的贡献；2)确定赋予边界函数的最小区域；3)评估基因组背景对TAD边界函数的影响。从这一建议中得出的潜在结果将促进我们对染色质组织的一般原理以及它们的改变如何引起异常表型的理解。

3D folding is an inherent property of the vertebrate genome in order to accommodate the roughly 2m of DNA that a single cell contains in such a tiny space as the nucleus. This folding, far from being a random process, appears to be highly predetermined and strongly influences gene expression. At the subchromosomal scale, our genome is partitioned in self-interacting units called Topologically Associating Domains (TADs), ranging from a few hundreds of base pairs to several megabases. TADs are delimited by so-called boundary regions that isolate them from the rest of the genome, thus promoting contacts between the loci contained within them. It has been proposed that TADs represent a fundamental structural unit of the genome, which is thought to guide regulatory elements to their cognate genes. Recently, the disruption of TADs and their boundaries was shown to be associated to human disorders such as congenital malformations or cancer. Despite the biological relevance of TADs, the essential components required to build such a fundamental structure remain elusive. In this proposal, I aim to molecularly dissect TAD domains and their boundary regions, in order to understand how these structures can be formed and influenced by their genomic context. By combining cutting-edge technologies such as Capture-HiC or CRISPR/Cas to generate mice carrying specific mutations, I am to: 1) evaluate the contribution of TAD boundaries in domain organization; 2) determine the minimal region to confer boundary function and 3) evaluate the impact of genomic context on TAD boundary function. The potential results derived from this proposal would advance in our understanding of the general principles of chromatin organization and how their alteration can originate abnormal phenotypes.