CTCF Binding Polarity Determines Chromatin Looping

Abstract

CCCTC-binding factor (CTCF) is an architectural protein involved in the three-dimensional (3D) organization of chromatin. In this study, we assayed the 3D genomic contact profiles of a large number of CTCF binding sites with high-resolution 4C-seq. As recently reported, our data also suggest that chromatin loops preferentially form between CTCF binding sites oriented in a convergent manner. To directly test this, we used CRISPR/Cas9 genome editing to delete core CTCF binding sites in three loci, including the CTCF site in the Sox2 super-enhancer. In all instances, CTCF and cohesin recruitment were lost, and chromatin loops with distal, convergent CTCF sites were disrupted or destabilized. Re-insertion of oppositely oriented CTCF recognition sequences restored CTCF and cohesin recruitment, but did not re-establish chromatin loops. We conclude that CTCF binding polarity plays a functional role in the formation of higher-order chromatin structure.

Identification of a regulatory domain controlling the Nppa-Nppb gene cluster during heart development and stress

“…1A,B). The convergent orientation of these CTCF sites supports potential loop anchoring at the domain boundaries (Rao et al, 2014;de Wit et al, 2015). Moreover, both CTCF-associated sites interact with the Nppa promoter and another region within the locus (black arrowheads), showing possible involvement in the regulation of Nppa expression.…”

“…TADs are separated by boundary regions enriched for convergent CTCFassociated sites (Ong and Corces, 2014;Rao et al, 2014;de Wit et al, 2015). Our 4C-seq analysis indicates that the CTCFassociated sites flanking Nppa/Nppb contact each other, encircling the gene promoters and most of their interaction partners.…”

Cis-regulatory landscapes in development and evolution

“…In this regard, the generation of chromatin interaction data from a much broader taxonomic sampling will be necessary in combination with careful examination and detailed functional dissection of many more examples of RLs. The use of novel genomic editing tools, such as the CRISPR/CAS9 system, will undoubtedly contribute to this endeavor, as highlighted by the increasing number of works that have benefited from these approaches to shed light into the molecular mechanisms underlying TAD boundaries and the establishment of chromatin loops [19,[47][48][49]. …”

Two independent modes of chromatin organization revealed by cohesin removal

“…CTCF sites). Recent experimental manipulation of CTCF motifs 15–17 and CTCF depletion 18 support the proposed role of CTCFs as boundary elements, but cohesin’s role in interphase chromatin organization and the process of loop extrusion remains ambiguous, as experimental depletions of cohesin have shown limited impact on chromatin organisation 19–21 …”