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

siRNA-mediated depletion of architectural proteins augments CFTR expression. (A), (D) and (G) Western blots show siRNA-mediated depletion of CTCF (A), RAD21 (D), or both (G) in Caco2 cells with β-tubulin as the loading control. (B) and (E) ImageJ quantification of western blots shown in (A) and (D), respectively. (C), (F) and (H) RT-qPCR for CFTR expression in Caco2 cells after knockdown of CTCF (C), RAD21 (F) or both (H) (black) compared to NC siRNA treated cells (gray). (I)–(K) Western blots show CFTR levels after loss of CTCF (I), RAD21 (J) or both factors (K) in Caco2 cells with β-tubulin as the loading control. (L) ImageJ quantification of the CFTR western blots, n ≥ 3. *P < 0.05; **P < 0.01 as determined by an unpaired, two-tailed Student's t-test. CT/RA KD is short for CTCF and RAD21 knockdown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: siRNA-mediated depletion of architectural proteins augments CFTR expression. (A), (D) and (G) Western blots show siRNA-mediated depletion of CTCF (A), RAD21 (D), or both (G) in Caco2 cells with β-tubulin as the loading control. (B) and (E) ImageJ quantification of western blots shown in (A) and (D), respectively. (C), (F) and (H) RT-qPCR for CFTR expression in Caco2 cells after knockdown of CTCF (C), RAD21 (F) or both (H) (black) compared to NC siRNA treated cells (gray). (I)–(K) Western blots show CFTR levels after loss of CTCF (I), RAD21 (J) or both factors (K) in Caco2 cells with β-tubulin as the loading control. (L) ImageJ quantification of the CFTR western blots, n ≥ 3. *P < 0.05; **P < 0.01 as determined by an unpaired, two-tailed Student's t-test. CT/RA KD is short for CTCF and RAD21 knockdown.

Mentions: To determine the effect of disruption of looping across the CFTR locus on gene expression, CFTR mRNA levels were measured after siRNA-mediated depletion of CTCF (Figure 4A and B) or RAD21 (Figure 4D and E) in Caco2 cells. A greater than 80% reduction of both architectural proteins significantly increased CFTR mRNA levels 1.4 - (CTCF, Figure 4C) and 1.6- (RAD21, Figure 4F) fold in comparison to a non-targeting control siRNA, when measured by reverse transcription and qPCR (RT-qPCR). Depletion of both CTCF and RAD21 together (Figure 4G) led to 2.8-fold increase in CFTR expression (Figure 4H), showing an additive effect, which is consistent with data showing distinct roles of CTCF and cohesin at the CFTR locus (Figure 2). To ensure the increase in CFTR expression was gene-specific and not simply an artifact of generalized disruption of chromatin architecture, we analyzed the expression of several other genes that are regulated by CTCF and cohesin. Depletion of RAD21 in HeLa cells repressed the solute carrier family 35, member C2 (SLC35C2) gene (5) and we observed the same effect in Caco2 cells after RAD21 knockdown (Supplementary Figure S2). In previous work, we also identified CTCF as a regulator of the gel-forming mucin genes on 11p15.5 (29) and after siRNA-mediated depletion of CTCF we observed a decrease in MUC2 and an increase in MUC6 mRNA (Supplementary Figure S2). These data confirm that the increased expression of CFTR that is seen after CTCF and RAD21 knockdown is likely a gene-specific effect. Next, we sought to determine if the observed increase in CFTR mRNA levels also occurred at the protein level. Western blots for CFTR after depletion of CTCF, RAD21, or both factors led to a marked increase in CFTR protein expression (Figure 4I–K). This was confirmed by quantification of the scanned western blots, showing a significant, ∼ 3-fold increase in CFTR protein levels after combined CTCF and RAD21 depletion, a 2-fold increase after RAD21 reduction alone, and a 1.5-fold increase after CTCF depletion alone (Figure 4L).


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)

siRNA-mediated depletion of architectural proteins augments CFTR expression. (A), (D) and (G) Western blots show siRNA-mediated depletion of CTCF (A), RAD21 (D), or both (G) in Caco2 cells with β-tubulin as the loading control. (B) and (E) ImageJ quantification of western blots shown in (A) and (D), respectively. (C), (F) and (H) RT-qPCR for CFTR expression in Caco2 cells after knockdown of CTCF (C), RAD21 (F) or both (H) (black) compared to NC siRNA treated cells (gray). (I)–(K) Western blots show CFTR levels after loss of CTCF (I), RAD21 (J) or both factors (K) in Caco2 cells with β-tubulin as the loading control. (L) ImageJ quantification of the CFTR western blots, n ≥ 3. *P < 0.05; **P < 0.01 as determined by an unpaired, two-tailed Student's t-test. CT/RA KD is short for CTCF and RAD21 knockdown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: siRNA-mediated depletion of architectural proteins augments CFTR expression. (A), (D) and (G) Western blots show siRNA-mediated depletion of CTCF (A), RAD21 (D), or both (G) in Caco2 cells with β-tubulin as the loading control. (B) and (E) ImageJ quantification of western blots shown in (A) and (D), respectively. (C), (F) and (H) RT-qPCR for CFTR expression in Caco2 cells after knockdown of CTCF (C), RAD21 (F) or both (H) (black) compared to NC siRNA treated cells (gray). (I)–(K) Western blots show CFTR levels after loss of CTCF (I), RAD21 (J) or both factors (K) in Caco2 cells with β-tubulin as the loading control. (L) ImageJ quantification of the CFTR western blots, n ≥ 3. *P < 0.05; **P < 0.01 as determined by an unpaired, two-tailed Student's t-test. CT/RA KD is short for CTCF and RAD21 knockdown.
Mentions: To determine the effect of disruption of looping across the CFTR locus on gene expression, CFTR mRNA levels were measured after siRNA-mediated depletion of CTCF (Figure 4A and B) or RAD21 (Figure 4D and E) in Caco2 cells. A greater than 80% reduction of both architectural proteins significantly increased CFTR mRNA levels 1.4 - (CTCF, Figure 4C) and 1.6- (RAD21, Figure 4F) fold in comparison to a non-targeting control siRNA, when measured by reverse transcription and qPCR (RT-qPCR). Depletion of both CTCF and RAD21 together (Figure 4G) led to 2.8-fold increase in CFTR expression (Figure 4H), showing an additive effect, which is consistent with data showing distinct roles of CTCF and cohesin at the CFTR locus (Figure 2). To ensure the increase in CFTR expression was gene-specific and not simply an artifact of generalized disruption of chromatin architecture, we analyzed the expression of several other genes that are regulated by CTCF and cohesin. Depletion of RAD21 in HeLa cells repressed the solute carrier family 35, member C2 (SLC35C2) gene (5) and we observed the same effect in Caco2 cells after RAD21 knockdown (Supplementary Figure S2). In previous work, we also identified CTCF as a regulator of the gel-forming mucin genes on 11p15.5 (29) and after siRNA-mediated depletion of CTCF we observed a decrease in MUC2 and an increase in MUC6 mRNA (Supplementary Figure S2). These data confirm that the increased expression of CFTR that is seen after CTCF and RAD21 knockdown is likely a gene-specific effect. Next, we sought to determine if the observed increase in CFTR mRNA levels also occurred at the protein level. Western blots for CFTR after depletion of CTCF, RAD21, or both factors led to a marked increase in CFTR protein expression (Figure 4I–K). This was confirmed by quantification of the scanned western blots, showing a significant, ∼ 3-fold increase in CFTR protein levels after combined CTCF and RAD21 depletion, a 2-fold increase after RAD21 reduction alone, and a 1.5-fold increase after CTCF depletion alone (Figure 4L).

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