Architectural proteins CTCF and cohesin have distinct roles in modulating the higher order structure and expression of the CFTR locus.
Bottom Line: CTCF mediates the interactions between CTCF/cohesin binding sites, some of which have enhancer-blocking insulator activity.Cohesin shares this tethering role, but in addition stabilizes interactions between the promoter and cis-acting intronic elements including enhancers, which are also dependent on the forkhead box A1/A2 (FOXA1/A2) transcription factors (TFs).Disruption of the three-dimensional structure of the CFTR gene by depletion of CTCF or RAD21 increases gene expression, which is accompanied by alterations in histone modifications and TF occupancy across the locus, and causes internalization of the gene from the nuclear periphery.
Affiliation: Human Molecular Genetics Program, Lurie Children's Research Center, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA.Show MeSH
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Mentions: CTCF and cohesin have important roles in the establishment and maintenance of long-range chromatin interactions, which are required for normal gene expression. To determine if CTCF and RAD21 are critical for chromatin looping across the CFTR locus, q3C was performed after siRNA-mediated depletion of CTCF or RAD21 in Caco2 cells. No marked changes in interactions between the CFTR promoter (bait marked as a dotted line) and the cis-regulatory elements within and flanking the gene were seen after CTCF knockdown (Figure 2A). Minor reductions were evident in promoter interactions with the −20.9 kb (II), +6.8 kb/+15.6 kb (III) enhancer-blocking insulators closest to the locus (marked by arrows). However, with a 3C bait at the −20.9 kb insulator (dotted line, Figure 2B), depletion of CTCF was seen to decrease interactions with a site 3′+20 kb (marked by arrow) and significantly with the +48.9 kb (IV) site (Figure 2B, marked ∧). In contrast, RAD21 depletion caused a partial loss of interactions between the CFTR promoter and all of the cis-acting elements across the locus (Figure 2C). These included both insulator elements flanking the locus (II and III) and intronic enhancers, such as the one at DHS11. A similar decrease in interactions across CFTR was evident using the 3C bait at −20.9 kb, including between this insulator element and the promoter, and also the enhancer at DHS11 (Figure 2D, significant sites marked ∧). However, the reduction in RAD21 did not alter the interactions between the −20.9 kb bait and elements 3′ to CFTR, particularly the +48.9 kb site (IV) which is impacted by CTCF knockdown (Figure 2D). In combination, these q3C data after CTCF or RAD21 depletion suggest that CTCF has a dominant role in the chromatin looping that draws together insulator elements 5′ and 3′ to the CFTR locus and brings them close to the promoter. In contrast, though the cohesin complex, evaluated by RAD21, also contributes to the maintenance of CTCF-mediated interactions across the locus, it appears to have an additional role in stabilizing the association between intronic enhancers and the gene promoter.
Affiliation: Human Molecular Genetics Program, Lurie Children's Research Center, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA.