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Architectural proteins CTCF and cohesin have distinct roles in modulating the higher order structure and expression of the CFTR locus.

Gosalia N, Neems D, Kerschner JL, Kosak ST, Harris A - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

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.

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CTCF and cohesin binding are interdependent at the CFTR locus. (A) UCSC Genome Browser graphic of the CFTR locus showing CTCF/cohesin binding sites (ENCODE data) marked by vertical black bars. (B) and (C) ChIP for CTCF, RAD21 and SMC1 in Caco2 cells transfected with negative control (NC) siRNA (gray) or siRNA targeting CTCF (B) or RAD21 (C) (black) for knockdown (KD). Data from one representative experiment are shown as percent recovery over input, n ≥ 2. qPCR was performed in duplicate at each site and error bars are ± SEM. Arrows denote sites of particular interest discussed in the text. Inset panels show relative siRNA-mediated depletion of CTCF or RAD21.
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Figure 1: CTCF and cohesin binding are interdependent at the CFTR locus. (A) UCSC Genome Browser graphic of the CFTR locus showing CTCF/cohesin binding sites (ENCODE data) marked by vertical black bars. (B) and (C) ChIP for CTCF, RAD21 and SMC1 in Caco2 cells transfected with negative control (NC) siRNA (gray) or siRNA targeting CTCF (B) or RAD21 (C) (black) for knockdown (KD). Data from one representative experiment are shown as percent recovery over input, n ≥ 2. qPCR was performed in duplicate at each site and error bars are ± SEM. Arrows denote sites of particular interest discussed in the text. Inset panels show relative siRNA-mediated depletion of CTCF or RAD21.

Mentions: To determine the contribution of CTCF and cohesin to the 3D structure of the CFTR locus we first identified sites occupied by these factors. Inspection of ENCODE data (23) from multiple cell types revealed binding sites for CTCF and RAD21 (SCC1) and these were confirmed by ChIP in Caco2 intestinal carcinoma cells, which express abundant CFTR. CTCF/RAD21 binding was seen at −80.1 kb (I) 5′ to the translational start site (TSS) of the CFTR locus, within an intron of ASZ1 (corresponding to the -79.5 kb DNase I hypersensitive site (DHS) reported earlier (24)) and also at sites in the CTTNBP2 gene, +48.9 kb (IV) and +83.7 kb (V) from translational stop site of CFTR (Figure 1A and Table 1). The ENCODE data also confirmed CTCF occupancy at enhancer-blocking insulator elements −20.9 kb (II) 5′ to the TSS that we described previously (3,4). CTCF does not bind to an element at +15.6 kb that we showed to be a CTCF independent enhancer-blocking insulator (4). Modest enrichment of CTCF and RAD21 at the CFTR promoter is likely a result of indirect interactions with distal CTCF binding sites, since no CTCF binding motifs are predicted in silico or observed in genome-wide CTCF ChIP-seq data (23) within the promoter region. Moreover, we saw no evidence for CTCF occupancy at the site shown in intron 2 in ENCODE data. RAD21 occupancy was also seen in Caco2 cells at the majority of the CTCF sites (I, II, IV and V). CTCF and RAD21 occupancy at each of these sites was next confirmed by ChIP in airway cell lines (Calu-3 and 16HBE14o-), which are known to exhibit different mechanisms of CFTR regulation than are seen in intestinal cells (Supplementary Figure S1, (1,25,26)). The CTCF/RAD21 binding sites at −80.1 kb (I), +48.9 kb (IV) and +83.7 kb (V) are ubiquitously occupied, while those at −20.9 kb (II) and +6.8 kb (III) show variable occupancy in the different cell lines. The most marked difference between the airway cells and Caco2 is the prominence of CTCF/RAD21 occupancy at site I and relatively low levels at site IV in the airway.


Architectural proteins CTCF and cohesin have distinct roles in modulating the higher order structure and expression of the CFTR locus.

Gosalia N, Neems D, Kerschner JL, Kosak ST, Harris A - Nucleic Acids Res. (2014)

CTCF and cohesin binding are interdependent at the CFTR locus. (A) UCSC Genome Browser graphic of the CFTR locus showing CTCF/cohesin binding sites (ENCODE data) marked by vertical black bars. (B) and (C) ChIP for CTCF, RAD21 and SMC1 in Caco2 cells transfected with negative control (NC) siRNA (gray) or siRNA targeting CTCF (B) or RAD21 (C) (black) for knockdown (KD). Data from one representative experiment are shown as percent recovery over input, n ≥ 2. qPCR was performed in duplicate at each site and error bars are ± SEM. Arrows denote sites of particular interest discussed in the text. Inset panels show relative siRNA-mediated depletion of CTCF or RAD21.
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Figure 1: CTCF and cohesin binding are interdependent at the CFTR locus. (A) UCSC Genome Browser graphic of the CFTR locus showing CTCF/cohesin binding sites (ENCODE data) marked by vertical black bars. (B) and (C) ChIP for CTCF, RAD21 and SMC1 in Caco2 cells transfected with negative control (NC) siRNA (gray) or siRNA targeting CTCF (B) or RAD21 (C) (black) for knockdown (KD). Data from one representative experiment are shown as percent recovery over input, n ≥ 2. qPCR was performed in duplicate at each site and error bars are ± SEM. Arrows denote sites of particular interest discussed in the text. Inset panels show relative siRNA-mediated depletion of CTCF or RAD21.
Mentions: To determine the contribution of CTCF and cohesin to the 3D structure of the CFTR locus we first identified sites occupied by these factors. Inspection of ENCODE data (23) from multiple cell types revealed binding sites for CTCF and RAD21 (SCC1) and these were confirmed by ChIP in Caco2 intestinal carcinoma cells, which express abundant CFTR. CTCF/RAD21 binding was seen at −80.1 kb (I) 5′ to the translational start site (TSS) of the CFTR locus, within an intron of ASZ1 (corresponding to the -79.5 kb DNase I hypersensitive site (DHS) reported earlier (24)) and also at sites in the CTTNBP2 gene, +48.9 kb (IV) and +83.7 kb (V) from translational stop site of CFTR (Figure 1A and Table 1). The ENCODE data also confirmed CTCF occupancy at enhancer-blocking insulator elements −20.9 kb (II) 5′ to the TSS that we described previously (3,4). CTCF does not bind to an element at +15.6 kb that we showed to be a CTCF independent enhancer-blocking insulator (4). Modest enrichment of CTCF and RAD21 at the CFTR promoter is likely a result of indirect interactions with distal CTCF binding sites, since no CTCF binding motifs are predicted in silico or observed in genome-wide CTCF ChIP-seq data (23) within the promoter region. Moreover, we saw no evidence for CTCF occupancy at the site shown in intron 2 in ENCODE data. RAD21 occupancy was also seen in Caco2 cells at the majority of the CTCF sites (I, II, IV and V). CTCF and RAD21 occupancy at each of these sites was next confirmed by ChIP in airway cell lines (Calu-3 and 16HBE14o-), which are known to exhibit different mechanisms of CFTR regulation than are seen in intestinal cells (Supplementary Figure S1, (1,25,26)). The CTCF/RAD21 binding sites at −80.1 kb (I), +48.9 kb (IV) and +83.7 kb (V) are ubiquitously occupied, while those at −20.9 kb (II) and +6.8 kb (III) show variable occupancy in the different cell lines. The most marked difference between the airway cells and Caco2 is the prominence of CTCF/RAD21 occupancy at site I and relatively low levels at site IV in the airway.

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.

View Article: PubMed Central - PubMed

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.

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Related in: MedlinePlus