Outward-oriented sites within clustered CTCF boundaries are key for intraTAD chromatin interactions and gene regulation

Abstract

AbstractCTCF plays an important role in adjusting insulation at TAD boundaries, where clustered CBS (CTCF-binding site) elements are often arranged in a somewhat tandem array with a complex divergent or convergent orientation. Here usingcPcdhandHOXDloci 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 elementsin vivoor CBS elementsin vitro, in conjunction with chromosome conformation capture and RNA-seq analyses, we show that deletions of outward-oriented CBS elements weaken the strength of 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.