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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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Depletion of architectural proteins alters histone modifications and transcription factor occupancy. (A)–(C) ChIP for H3K9me3 (A), H3K9ac (B) and H3K27ac (C) in Caco2 cells transfected with NC siRNA (gray) or a siRNA targeting CTCF (black). Data are shown as percent recovery over input and each histone modification is normalized to recovery over input of unmodified histone H3 at each site, n = 3. ∧ indicate sites that are statistically different after knockdown as determined by an unpaired, two-tailed Student's t-test, P-values are listed in Supplementary Table S2. Other sites of interest are marked with arrows. (D)–(F) ChIP for FOXA1 (D), FOXA2 (E) and CDX2 (F) in Caco2 cells transfected with NC siRNA (gray) or siRNAs targeting CTCF and RAD21 (black). Data are shown as percent recovery over input, n = 3. *P < 0.05; **P < 0.01 as determined by an unpaired, two-tailed Student's t-test.
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Figure 5: Depletion of architectural proteins alters histone modifications and transcription factor occupancy. (A)–(C) ChIP for H3K9me3 (A), H3K9ac (B) and H3K27ac (C) in Caco2 cells transfected with NC siRNA (gray) or a siRNA targeting CTCF (black). Data are shown as percent recovery over input and each histone modification is normalized to recovery over input of unmodified histone H3 at each site, n = 3. ∧ indicate sites that are statistically different after knockdown as determined by an unpaired, two-tailed Student's t-test, P-values are listed in Supplementary Table S2. Other sites of interest are marked with arrows. (D)–(F) ChIP for FOXA1 (D), FOXA2 (E) and CDX2 (F) in Caco2 cells transfected with NC siRNA (gray) or siRNAs targeting CTCF and RAD21 (black). Data are shown as percent recovery over input, n = 3. *P < 0.05; **P < 0.01 as determined by an unpaired, two-tailed Student's t-test.

Mentions: Since looping of the CFTR locus is associated with active gene expression, perturbation of the 3D structure by depletion of the architectural proteins was predicted to repress, not increase, CFTR expression. To investigate the mechanism underlying the increase in CFTR expression after depletion of CTCF and RAD21, we evaluated changes in the chromatin landscape of the locus. ChIP for three histone modifications, H3K9me3, H3K9ac and H3K27ac, was performed after depletion of CTCF in Caco2 cells (Figure 5A–C). H3K9me3 is found at repressed or inactive regions of chromatin including at promoters, silenced enhancers, and in the gene body (30). H3K9ac is generally associated with active or transcribed regions of the genome, specifically at promoters, transcription start sites and regulatory elements (31,32) and H3K27ac is a mark of active enhancers (30). Enrichment of each histone mark was calculated as recovery over percent input and normalized to total histone H3 at each site. Loss of CTCF decreased enrichment of the H3K9me3 mark at −20.9 kb (site II), the promoter (-0.5 kb), and within the gene body at a site in intron 14a that is not associated with any known cis-regulatory element (Figure 5A, marked by arrows). Correspondingly, H3K9ac increased at the −20.9 kb site, though the modification remained unchanged at all other regions tested (Figure 5B). The greatest changes in histone marks after CTCF depletion were in H3K27ac enrichment, which increased significantly at the known, active enhancers in introns 1 and 11 and a cis-element in intron 10 (Figure 5C, marked by arrows and ∧).


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 architectural proteins alters histone modifications and transcription factor occupancy. (A)–(C) ChIP for H3K9me3 (A), H3K9ac (B) and H3K27ac (C) in Caco2 cells transfected with NC siRNA (gray) or a siRNA targeting CTCF (black). Data are shown as percent recovery over input and each histone modification is normalized to recovery over input of unmodified histone H3 at each site, n = 3. ∧ indicate sites that are statistically different after knockdown as determined by an unpaired, two-tailed Student's t-test, P-values are listed in Supplementary Table S2. Other sites of interest are marked with arrows. (D)–(F) ChIP for FOXA1 (D), FOXA2 (E) and CDX2 (F) in Caco2 cells transfected with NC siRNA (gray) or siRNAs targeting CTCF and RAD21 (black). Data are shown as percent recovery over input, n = 3. *P < 0.05; **P < 0.01 as determined by an unpaired, two-tailed Student's t-test.
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Figure 5: Depletion of architectural proteins alters histone modifications and transcription factor occupancy. (A)–(C) ChIP for H3K9me3 (A), H3K9ac (B) and H3K27ac (C) in Caco2 cells transfected with NC siRNA (gray) or a siRNA targeting CTCF (black). Data are shown as percent recovery over input and each histone modification is normalized to recovery over input of unmodified histone H3 at each site, n = 3. ∧ indicate sites that are statistically different after knockdown as determined by an unpaired, two-tailed Student's t-test, P-values are listed in Supplementary Table S2. Other sites of interest are marked with arrows. (D)–(F) ChIP for FOXA1 (D), FOXA2 (E) and CDX2 (F) in Caco2 cells transfected with NC siRNA (gray) or siRNAs targeting CTCF and RAD21 (black). Data are shown as percent recovery over input, n = 3. *P < 0.05; **P < 0.01 as determined by an unpaired, two-tailed Student's t-test.
Mentions: Since looping of the CFTR locus is associated with active gene expression, perturbation of the 3D structure by depletion of the architectural proteins was predicted to repress, not increase, CFTR expression. To investigate the mechanism underlying the increase in CFTR expression after depletion of CTCF and RAD21, we evaluated changes in the chromatin landscape of the locus. ChIP for three histone modifications, H3K9me3, H3K9ac and H3K27ac, was performed after depletion of CTCF in Caco2 cells (Figure 5A–C). H3K9me3 is found at repressed or inactive regions of chromatin including at promoters, silenced enhancers, and in the gene body (30). H3K9ac is generally associated with active or transcribed regions of the genome, specifically at promoters, transcription start sites and regulatory elements (31,32) and H3K27ac is a mark of active enhancers (30). Enrichment of each histone mark was calculated as recovery over percent input and normalized to total histone H3 at each site. Loss of CTCF decreased enrichment of the H3K9me3 mark at −20.9 kb (site II), the promoter (-0.5 kb), and within the gene body at a site in intron 14a that is not associated with any known cis-regulatory element (Figure 5A, marked by arrows). Correspondingly, H3K9ac increased at the −20.9 kb site, though the modification remained unchanged at all other regions tested (Figure 5B). The greatest changes in histone marks after CTCF depletion were in H3K27ac enrichment, which increased significantly at the known, active enhancers in introns 1 and 11 and a cis-element in intron 10 (Figure 5C, marked by arrows and ∧).

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