Limits...
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.

Show MeSH

Related in: MedlinePlus

Depletion of CTCF or RAD21 alters long-range interactions across the CFTR locus. (A)–(D) q3C interactions in cells treated with NC siRNA (gray) or siRNA targeting CTCF (A) and (B) (black), or RAD21 (C) and (D) (black). The 3C bait (dotted line) is in a HindIII fragment at the CFTR promoter (A) and (C) or at the −20.9 kb insulator element (B) and (D). (E) ChIP for FOXA2 on cells transfected with NC (gray) or FOXA1/2 siRNA (black). Data are shown as percent recovery over input. (F) q3C interactions in NC (gray) or FOXA1/2 siRNA treated cells (black). The 3C bait (dotted line) is in a HindIII fragment at the CFTR promoter. Multiple sites were tested for interactions within distal HindIII fragments across the CFTR locus. x-axis = position relative to translational start site of CFTR, y-axis = interaction frequency relative to that between two fragments in the ubiquitously expressed ERCC3 gene. Data shown are from one representative q3C experiment, n = 3. Error bars represent the ± SEM of duplicate qPCRs for each fragment. ∧ indicate sites that are statistically different after knockdown as determined by an unpaired, two-tailed Student's t-test, n = 3, P-values listed in Supplementary Table S2. Other sites of interest marked with arrows. Inset panels show relative siRNA-mediated depletion of CTCF or RAD21.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150766&req=5

Figure 2: Depletion of CTCF or RAD21 alters long-range interactions across the CFTR locus. (A)–(D) q3C interactions in cells treated with NC siRNA (gray) or siRNA targeting CTCF (A) and (B) (black), or RAD21 (C) and (D) (black). The 3C bait (dotted line) is in a HindIII fragment at the CFTR promoter (A) and (C) or at the −20.9 kb insulator element (B) and (D). (E) ChIP for FOXA2 on cells transfected with NC (gray) or FOXA1/2 siRNA (black). Data are shown as percent recovery over input. (F) q3C interactions in NC (gray) or FOXA1/2 siRNA treated cells (black). The 3C bait (dotted line) is in a HindIII fragment at the CFTR promoter. Multiple sites were tested for interactions within distal HindIII fragments across the CFTR locus. x-axis = position relative to translational start site of CFTR, y-axis = interaction frequency relative to that between two fragments in the ubiquitously expressed ERCC3 gene. Data shown are from one representative q3C experiment, n = 3. Error bars represent the ± SEM of duplicate qPCRs for each fragment. ∧ indicate sites that are statistically different after knockdown as determined by an unpaired, two-tailed Student's t-test, n = 3, P-values listed in Supplementary Table S2. Other sites of interest marked with arrows. Inset panels show relative siRNA-mediated depletion of CTCF or RAD21.

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.


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)

Depletion of CTCF or RAD21 alters long-range interactions across the CFTR locus. (A)–(D) q3C interactions in cells treated with NC siRNA (gray) or siRNA targeting CTCF (A) and (B) (black), or RAD21 (C) and (D) (black). The 3C bait (dotted line) is in a HindIII fragment at the CFTR promoter (A) and (C) or at the −20.9 kb insulator element (B) and (D). (E) ChIP for FOXA2 on cells transfected with NC (gray) or FOXA1/2 siRNA (black). Data are shown as percent recovery over input. (F) q3C interactions in NC (gray) or FOXA1/2 siRNA treated cells (black). The 3C bait (dotted line) is in a HindIII fragment at the CFTR promoter. Multiple sites were tested for interactions within distal HindIII fragments across the CFTR locus. x-axis = position relative to translational start site of CFTR, y-axis = interaction frequency relative to that between two fragments in the ubiquitously expressed ERCC3 gene. Data shown are from one representative q3C experiment, n = 3. Error bars represent the ± SEM of duplicate qPCRs for each fragment. ∧ indicate sites that are statistically different after knockdown as determined by an unpaired, two-tailed Student's t-test, n = 3, P-values listed in Supplementary Table S2. Other sites of interest marked with arrows. Inset panels show relative siRNA-mediated depletion of CTCF or RAD21.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150766&req=5

Figure 2: Depletion of CTCF or RAD21 alters long-range interactions across the CFTR locus. (A)–(D) q3C interactions in cells treated with NC siRNA (gray) or siRNA targeting CTCF (A) and (B) (black), or RAD21 (C) and (D) (black). The 3C bait (dotted line) is in a HindIII fragment at the CFTR promoter (A) and (C) or at the −20.9 kb insulator element (B) and (D). (E) ChIP for FOXA2 on cells transfected with NC (gray) or FOXA1/2 siRNA (black). Data are shown as percent recovery over input. (F) q3C interactions in NC (gray) or FOXA1/2 siRNA treated cells (black). The 3C bait (dotted line) is in a HindIII fragment at the CFTR promoter. Multiple sites were tested for interactions within distal HindIII fragments across the CFTR locus. x-axis = position relative to translational start site of CFTR, y-axis = interaction frequency relative to that between two fragments in the ubiquitously expressed ERCC3 gene. Data shown are from one representative q3C experiment, n = 3. Error bars represent the ± SEM of duplicate qPCRs for each fragment. ∧ indicate sites that are statistically different after knockdown as determined by an unpaired, two-tailed Student's t-test, n = 3, P-values listed in Supplementary Table S2. Other sites of interest marked with arrows. Inset panels show relative siRNA-mediated depletion of CTCF or RAD21.
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.

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.

Show MeSH
Related in: MedlinePlus