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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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Altered nuclear positioning of CFTR alleles after loss of CTCF or RAD21. (A)–(C) Representative FISH images for CFTR in Caco2 cells treated with NC siRNA (A), CTCF siRNA (B), or RAD21 siRNA (C). Nuclei are stained with DAPI and the CFTR alleles are white dots. (D) Quantification of the minimum distance to the nuclear periphery of the CFTR alleles in all three conditions. **P < 0.01 as determined by an unpaired, two-tailed Student's t-test.
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Figure 6: Altered nuclear positioning of CFTR alleles after loss of CTCF or RAD21. (A)–(C) Representative FISH images for CFTR in Caco2 cells treated with NC siRNA (A), CTCF siRNA (B), or RAD21 siRNA (C). Nuclei are stained with DAPI and the CFTR alleles are white dots. (D) Quantification of the minimum distance to the nuclear periphery of the CFTR alleles in all three conditions. **P < 0.01 as determined by an unpaired, two-tailed Student's t-test.

Mentions: The increase in CFTR mRNA and CFTR protein, coupled with the changes in chromatin structure and TF occupancy upon CTCF/RAD21 depletion, led us to evaluate the positioning of the CFTR alleles within the nucleus. A previous study demonstrated that the perinuclear positioning of the inactive CFTR locus was dependent on type A lamins, CTCF and a histone deacetylase (HDAC) (34). FISH followed by 2D analysis revealed that treatment of cells with a CTCF siRNA significantly decreased the percentage of alleles at the nuclear periphery in two cell types (34). To determine the effect of CTCF and RAD21 depletion on nuclear positioning of the CFTR alleles we performed 3D FISH with cells grown on glass coverslips. Caco2 cells, which are highly aneuploid, generally have three copies of chromosome 7 per cell, as evidenced by the three distinct FISH signals in each nucleus in negative control, CTCF, or RAD21 siRNA-treated cells (Figure 6A–C). Quantification of CFTR nuclear positioning after CTCF or RAD21 depletion showed a significant increase in the average minimum distance to the nuclear periphery relative to non-targeting siRNA treated cells (Figure 6D). Hence, depletion of both architectural proteins is associated with internalization of the CFTR locus within the nucleus.


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)

Altered nuclear positioning of CFTR alleles after loss of CTCF or RAD21. (A)–(C) Representative FISH images for CFTR in Caco2 cells treated with NC siRNA (A), CTCF siRNA (B), or RAD21 siRNA (C). Nuclei are stained with DAPI and the CFTR alleles are white dots. (D) Quantification of the minimum distance to the nuclear periphery of the CFTR alleles in all three conditions. **P < 0.01 as determined by an unpaired, two-tailed Student's t-test.
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Related In: Results  -  Collection

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Figure 6: Altered nuclear positioning of CFTR alleles after loss of CTCF or RAD21. (A)–(C) Representative FISH images for CFTR in Caco2 cells treated with NC siRNA (A), CTCF siRNA (B), or RAD21 siRNA (C). Nuclei are stained with DAPI and the CFTR alleles are white dots. (D) Quantification of the minimum distance to the nuclear periphery of the CFTR alleles in all three conditions. **P < 0.01 as determined by an unpaired, two-tailed Student's t-test.
Mentions: The increase in CFTR mRNA and CFTR protein, coupled with the changes in chromatin structure and TF occupancy upon CTCF/RAD21 depletion, led us to evaluate the positioning of the CFTR alleles within the nucleus. A previous study demonstrated that the perinuclear positioning of the inactive CFTR locus was dependent on type A lamins, CTCF and a histone deacetylase (HDAC) (34). FISH followed by 2D analysis revealed that treatment of cells with a CTCF siRNA significantly decreased the percentage of alleles at the nuclear periphery in two cell types (34). To determine the effect of CTCF and RAD21 depletion on nuclear positioning of the CFTR alleles we performed 3D FISH with cells grown on glass coverslips. Caco2 cells, which are highly aneuploid, generally have three copies of chromosome 7 per cell, as evidenced by the three distinct FISH signals in each nucleus in negative control, CTCF, or RAD21 siRNA-treated cells (Figure 6A–C). Quantification of CFTR nuclear positioning after CTCF or RAD21 depletion showed a significant increase in the average minimum distance to the nuclear periphery relative to non-targeting siRNA treated cells (Figure 6D). Hence, depletion of both architectural proteins is associated with internalization of the CFTR locus within the nucleus.

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