Two unrelated distal genes activated by a shared enhancer benefit from localizing inside the same small topological domain (4C).. Two unrelated distal genes activated by a shared enhancer benefit from localizing inside the same small topological domain (4C).

Enhancers are tissue-specific regulatory DNA elements that can activate transcription of genes over distance. Their target genes most often locate in the same topologically associating domain (TAD). TADs are structural entities within chromosomes in which cohesin DNA loop extrusion supports intra-domain DNA contacts and CTCF-bound boundaries serve to halt and concentrate paralogous genes. Enhancers shared by multiple unrelated genes may be more common than those acting on paralogous gene clusters, but are underexplored. Here, we analyzed the interplay between an enhancer and two distal, functionally unrelated, genes residing at opposite domain boundaries. The enhancer stimulated boundary and gene-gene contacts and required cohesin as well as two intact domain boundaries to support gene activation. The two genes preferentially transcribed when spatially together, but did not rely on each other’s transcription, nor showed gene competition. Placing them and the enhancer in smaller domains caused their upregulation. Domain boundaries, therefore, can support long-range enhancer-promoter communication inside domains. Boundary proximity thereby matters as the distal enhancer functions more effectively inside smaller domains. We propose that smaller domains have more concentrated cohesin loop extrusion activity to facilitate enhancer action over distance. Overall design: 4C-seq data with viewpoints located in the MYEOV-CCND1 locus of WT, enhancer deleted, MYEOV deleted, CCND1 deleted and CTCF2 deleted K562 cells.

增强子（Enhancer）是一类组织特异性的调控DNA元件，可远距离激活基因的转录过程。其靶基因大多位于同一拓扑关联结构域（topologically associating domain, TAD）内。TAD是染色体内部的结构实体，其中黏连蛋白（cohesin）介导的DNA环挤出过程支持结构域内部的DNA相互作用，而结合CTCF的结构域边界可阻断跨边界的DNA相互作用，并使旁系同源基因富集于边界区域。相较于作用于旁系同源基因簇的增强子，多份不相关基因共享的增强子或许更为常见，但相关研究仍有待深入挖掘。
本研究针对位于相反TAD边界的某一增强子与两个远端、功能上无关联的基因之间的相互作用展开分析。该增强子可促进边界区域及基因间的染色质相互作用，其介导的基因激活过程依赖于黏连蛋白与两个完整的TAD边界。两个基因在空间上彼此靠近时转录水平更高，但二者并不依赖对方的转录过程，亦未出现基因竞争现象。将这两个基因与该增强子置于更小的TAD结构域内时，它们的转录水平会上调。由此可见，TAD边界可支持结构域内部的远距离增强子-启动子通讯，而边界的邻近性同样至关重要：远端增强子在更小的TAD结构域内可更高效地发挥调控功能。我们推测，更小的TAD结构域内部黏连蛋白环挤出活动更为集中，从而助力增强子实现远距离基因调控。
实验整体设计：针对野生型（WT）、增强子敲除、MYEOV敲除、CCND1敲除及CTCF2敲除的K562细胞系，选取位于MYEOV-CCND1基因座的测序视点（viewpoints）获取4C-seq数据。