Outward-oriented sites within clustered CTCF boundaries are key for intraTAD chromatin interactions and gene regulation [4C-seq]. Outward-oriented sites within clustered CTCF boundaries are key for intraTAD chromatin interactions and gene regulation [4C-seq]

CTCF plays an important role in 3D genome organization by adjusting insulation at TAD boundaries, where clustered CBS (CTCF-binding site) elements are often arranged in tandem array with a complex divergent or convergent orientation. Here using cPcdh and HOXD loci as a paradigm, we look into the clustered CTCF TAD boundaries and find that, counterintuitively, outward-oriented CBS elements are crucial for inward enhancer-promoter interactions as well as for gene regulation. Specifically, by combinatorial deletions of a series of putative enhancer elements in vivo or CBS elements in vitro, in conjunction with chromosome conformation capture and RNA-seq analyses, we show that deletions of outward-oriented CBS elements weaken the strength of long-distance intraTAD promoter-enhancer interactions and enhancer activation of target genes. Our data highlight the crucial role of outward-oriented CBS elements within the clustered CTCF TAD boundaries and have interesting implications on the organization principles of clustered CTCF sites within TAD boundaries. Overall design: QHR4C-seq of P0 old wild type (WT) and several mouse line mice and HEK293T cells were seperately generated by deep sequencing using Illumina Novaseq 6000

CTCF（CCCTC结合因子）通过调控拓扑关联结构域（Topologically Associating Domain, TAD）边界的绝缘作用，在三维基因组组织中发挥关键功能。簇状的CBS（CTCF结合位点，CTCF-binding site）元件通常以串联阵列形式排布，并呈现复杂的发散或收敛取向。本研究以cPcdh（簇状原钙粘蛋白）基因座与HOXD基因座为研究模型，针对簇状CTCF结合的TAD边界展开探究，结果与直觉相悖地发现，向外取向的CBS元件对于向内的增强子-启动子相互作用以及基因调控均至关重要。具体而言，通过在体内对一系列推定增强子元件或在体外对CBS元件进行组合缺失，并结合染色体构象捕获与RNA测序（RNA-seq）分析，本研究证实：向外取向的CBS元件缺失会减弱长距离TAD内部启动子-增强子相互作用的强度，并削弱靶基因的增强子激活效应。本研究数据凸显了簇状CTCF结合TAD边界内向外取向CBS元件的关键作用，同时为TAD边界内簇状CTCF位点的组织原则提供了新的研究启示。实验设计概况：本研究采用Illumina NovaSeq 6000测序平台开展深度测序，分别构建了出生后0日龄（P0）野生型（wild type, WT）小鼠、多款基因工程小鼠品系以及HEK293T细胞的QHR4C-seq文库。