Mammalian genomes are folded into topologically associating domains (TADs), consisting of chromatin loops anchored by CTCF and cohesin. Some loops are cell-type specific. Here we asked whether CTCF loops are established by a universal or locus-specific mechanism. Investigating the molecular determinants of CTCF clustering, we found that CTCF self-association in vitro is RNase sensitive and that an internal RNA-binding region (RBR_i) mediates CTCF clustering and RNA interaction in vivo. Strikingly, deleting the RBR_i impairs about half of all chromatin loops in mESCs and causes deregulation of gene expression. Disrupted loop formation correlates with diminished clustering and chromatin binding of RBR_i mutant CTCF, which in turn results in a failure to halt cohesin-mediated extrusion. Thus, CTCF loops fall into at least two classes: RBR_i-independent and RBR_i-dependent loops. We speculate that evidence for RBR_i-dependent loops may provide a molecular mechanism for establishing cell-specific CTCF loops, potentially regulated by RNA(s) or other RBR_i-interacting partners.

The function of the CCCTC-binding factor (CTCF) in the organization of the genome has become an important area of investigation, but the mechanisms by which CTCF dynamically contributes to genome organization are not clear. We previously discovered that CTCF binds to large numbers of endogenous RNAs, promoting its self-association. In this regard, we now report two independent features that disrupt CTCF association with chromatin: inhibition of transcription and disruption of CTCF-RNA interactions through mutations of 2 of its 11 zinc fingers that are not required for CTCF binding to its cognate DNA site: zinc finger 1 (ZF1) or zinc finger 10 (ZF10). These mutations alter gene expression profiles as CTCF mutants lose their ability to form chromatin loops and thus the ability to insulate chromatin domains and to mediate CTCF long-range genomic interactions. Our results point to the importance of CTCF-mediated RNA interactions as a structural component of genome organization.