Constitutively bound CTCF sites maintain 3D chromatin architecture and long-range epigenetically regulated domains

Abstract

The architectural protein CTCF is a mediator of chromatin conformation, but how CTCF binding to DNA is orchestrated to maintain long-range gene expression is poorly understood. Here we perform RNAi knockdown to reduce CTCF levels and reveal a shared subset of CTCF-bound sites are robustly resistant to protein depletion. The 'persistent' CTCF sites are enriched at domain boundaries and chromatin loops constitutive to all cell types. CRISPR-Cas9 deletion of 2 persistent CTCF sites at the boundary between a long-range epigenetically active (LREA) and silenced (LRES) region, within the Kallikrein (KLK) locus, results in concordant activation of all 8 KLK genes within the LRES region. CTCF genome-wide depletion results in alteration in Topologically Associating Domain (TAD) structure, including the merging of TADs, whereas TAD boundaries are not altered where persistent sites are maintained. We propose that the subset of essential CTCF sites are involved in cell-type constitutive, higher order chromatin architecture.

A comparative analysis of chromatin accessibility in cattle, pig, and mouse tissues

“…Indeed, regions that were globally accessible in all pig and cattle tissues were particularly enriched for CTCF recognition motifs, suggesting that these regions may delineate TAD boundaries, although direct profiling of chromatin interactions will be necessary to provide more definitive annotations of 3D chromatin structure. Interestingly, out of all accessible CTCF motifs, almost a third were only accessible in a single tissue, which is consistent with CTCF binding being highly variable in different cell types [37,38], even though TAD structure is largely consistent [32][33][34]. Only a fraction of CTCF binding sites (15%) actually localize to TAD boundaries [39], and most CTCF binding sites are interspersed with enhancers, stabilizing enhancer-promoter interactions [40], and forming cell type-specific chromatin loops linked to differential gene expression [40][41][42].…”

“…Although efforts are currently underway to further characterize these regulatory elements as enhancers, silencers, insulators, promoters, etc. [29,30], motif enrichment analysis highlighted some of their potential regulatory [31], which are largely invariable across cell types [32][33][34] and even across species [32,33,35,36]. Indeed, regions that were globally accessible in all pig and cattle tissues were particularly enriched for CTCF recognition motifs, suggesting that these regions may delineate TAD boundaries, although direct profiling of chromatin interactions will be necessary to provide more definitive annotations of 3D chromatin structure.…”

Inhibitor of Differentiation 4 (ID4) represses myoepithelial differentiation of mammary stem cells through its interaction with HEB

“…Our ChIP-seq protocol was adapted from (Khoury, Achinger-Kawecka et al, 2020). Comma-Dβ cells were grown in 150 mm or 100 mm dishes (Corning) until 80-90% confluent.…”

Genes on Different Strands Mark Boundaries Associated with Co-regulation Domains

“…There is thus a distance bias to consider when comparing the elements separating different types of pairs. TAD boundaries are known to have insulator properties, limiting the effects of enhancers to nearby genes on the other side of the boundary (9,12,14,45). If true, the prevalence of TAD boundaries between genes having opposite behaviours following DEX induction should be higher.…”