Tissue-specific CTCF/Cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo (RNA-Seq)

The genome is organized via CTCF/cohesin binding sites, which partition chromosomes into 1-5Mb topologically associated domains (TADs), and further into smaller contact sub-domains within TADs (sub-TADs; 40-1000kb). Here we examined in vivo an ~80kb sub-TAD, containing the mouse a-globin gene cluster, lying within a ~1Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF/cohesin sites which are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the a-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF/cohesin boundary extends the sub-TAD to the adjacent upstream CTCF/cohesin binding site. The a-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF/cohesin boundaries in this sub-TAD regulate both directionality and specificity of enhancer interactions with surrounding promoters. Overall design: RNA-seq of globin-depleted polyA-isolated RNA libraries isolated from primary erythroid ter119+ cells obtained from the spleens of acetylphenylhydrazine treated mice. Wild-type C57BL/6 mice were comparied with mice containing deletions for HS-29 (D29) or HS-38 and HS-39 (D3839) CTCF binding sites. Biological triplicates are included for each mouse and are analysed compared to WT (C57)基因组通过CTCF/黏连蛋白结合位点进行组织，此类位点将染色体划分为1~5Mb的拓扑关联结构域（topologically associated domains, TADs），并在TAD内部进一步拆分为更小的接触亚结构域（sub-TADs，即亚TAD；40~1000kb）。本研究针对一个约80kb的亚TAD开展了体内实验，该亚TAD包含小鼠α-珠蛋白基因簇，位于一个约1Mb的TAD内部。我们发现，该亚TAD两侧主要分布有同向排列的CTCF/黏连蛋白结合位点，此类位点可被CTCF普遍结合，但仅在红细胞生成过程中发生相互作用，从而构成一个自我相互作用的红系特异性区域。正常情况下，α-珠蛋白调控元件仅作用于增强子下游的启动子；而删除保守的上游CTCF/黏连蛋白边界后，该亚TAD会延伸至相邻的上游CTCF/黏连蛋白结合位点。此时α-珠蛋白增强子可与侧翼染色质发生相互作用，上调该延伸后亚TAD内部基因的表达水平。该亚TAD内的CTCF/黏连蛋白边界并非仅作为染色质修饰的屏障，同时还可调控增强子与周围启动子相互作用的方向性与特异性。实验整体设计：对经乙酰苯肼处理的小鼠脾脏来源的原代红系ter119阳性细胞中提取的、去珠蛋白的聚腺苷酸化RNA文库开展RNA测序。以野生型C57BL/6小鼠为对照，对比分析了携带HS-29（D29）或HS-38与HS-39（D3839）CTCF结合位点缺失的小鼠。每组小鼠均设置3次生物学重复，并与野生型（C57）小鼠样本进行对比分析。