Functional dissection of regulatory landscapes reveals non-essential and instructive roles of TADs in regulating gene expression [ATAC-Seq]

The genome is organized in megabase­sized three-dimensional units, called Topologically Associated Domains (TADs), that are separated by boundaries. TADs bring distant cis-regulatory elements into proximity, facilitated by the cooperative action of cohesin and the DNA binding factor CTCF. However, how TADs and their boundaries impinge on enhancer function remains an open question. Here, we investigate TAD function in vivo in mice at the Sox9/Kcnj locus. We find that TADs are formed by a redundant system of CTCF sites requiring the removal of all major sites within the TAD and at the boundary for two neighboring TADs to fuse. TAD fusion resulted in a low degree of regulatory spread from the Sox9 to the Kcnj TAD, but no major changes in gene expression, indicating that TAD structures provide robustness and precision, but are not essential for developmental gene regulation. Gene misexpression and consecutive disease phenotypes, however, were attained by re-directing regulatory activity through inversions and/or the re-positioning of boundaries. Thus, efficient re-wiring of enhancer promoter interaction and aberrant disease causing gene activation is not induced by a mere loss of insulation but requires the re-direction of contacts. We used ATAC-seq to compare chromatin accessibility in 3 different tissues and to compare the accessibility of the enhancer in WT and mutant animals.

基因组以兆碱基级的三维结构单元进行组织，这类单元被称为拓扑关联结构域（Topologically Associated Domains，TADs），彼此之间由边界区域分隔开。TADs可使远距离的顺式调控元件相互靠近，这一过程由黏连蛋白（cohesin）与DNA结合因子CTCF的协同作用所介导。然而，TADs及其边界区域如何影响增强子功能，仍是一个悬而未决的科学问题。本研究以小鼠Sox9/Kcnj基因座为研究对象，在活体水平探究TADs的功能。研究发现，TADs由一套冗余的CTCF结合位点系统构成，若要使两个相邻的TAD发生融合，需要去除该TAD内部以及边界区域内的所有主要CTCF结合位点。TAD融合会导致调控活性从Sox9 TAD向Kcnj TAD发生低程度的扩散，但并未引起基因表达的显著变化，这表明TAD结构可为基因调控提供稳定性与精准性，但并非发育过程中基因调控所必需的要素。不过，通过染色体倒位或边界区域的重新定位来改变调控活性的方向，可引发基因异常表达以及后续的疾病表型。由此可见，仅丧失绝缘作用并不会引发增强子-启动子相互作用的有效重连以及导致疾病的异常基因激活，这一过程需要通过重新定向染色质接触来实现。本研究采用ATAC-seq技术，对3种不同组织的染色质开放状态进行比较，并对比野生型（wild type，WT）与突变体动物中增强子的开放状态。