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An insulator element 3' to the CFTR gene binds CTCF and reveals an active chromatin hub in primary cells.

Blackledge NP, Ott CJ, Gillen AE, Harris A - Nucleic Acids Res. (2009)

Bottom Line: Elements within the basal promoter of the gene do not fully explain CFTR expression patterns, suggesting that cis-regulatory elements are located elsewhere, either within the locus or in adjacent chromatin.We further demonstrate that the element functions as an enhancer blocker in a well-established in vivo assay, and by using chromatin immunoprecipitation that it recruits CTCF in vivo.Moreover, we reveal that in primary epididymis cells, the +6.8 kb DHS interacts closely with the CFTR promoter, suggesting that the CFTR locus exists in a looped conformation, characteristic of an active chromatin hub.

View Article: PubMed Central - PubMed

Affiliation: Human Molecular Genetics Program, Children's Memorial Research Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60614, USA.

ABSTRACT
Regulation of expression of the CFTR gene is poorly understood. Elements within the basal promoter of the gene do not fully explain CFTR expression patterns, suggesting that cis-regulatory elements are located elsewhere, either within the locus or in adjacent chromatin. We previously mapped DNase I hypersensitive sites (DHS) in 400 kb spanning the CFTR locus including a cluster of sites close to the 3'-end of the gene. Here we focus on a DHS at +6.8 kb from the CFTR translation end-point to evaluate its potential role in regulating expression of the gene. This DHS, which encompasses a consensus CTCF-binding site, was evident in primary human epididymis cells that express abundant CFTR mRNA. We show by DNase I footprinting and electophoretic mobility shift assays that the cis-regulatory element within this DHS binds CTCF in vitro. We further demonstrate that the element functions as an enhancer blocker in a well-established in vivo assay, and by using chromatin immunoprecipitation that it recruits CTCF in vivo. Moreover, we reveal that in primary epididymis cells, the +6.8 kb DHS interacts closely with the CFTR promoter, suggesting that the CFTR locus exists in a looped conformation, characteristic of an active chromatin hub.

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Enhancer-blocking activity at the +6.8 kb DHS region. Each construct used in the enhancer-blocking assay is depicted as follows: pNI is the empty pNI plasmid and pNI-FII (FII) contains a known insulator from the chicken β-globin locus (represented by a triangle). pNI-6.8 (6.8) contains the wild-type DHS6.8oligo (represented by a rectangle) and pNI-6.8mut (6.8mut) contains the mutant version DHS6.8mutoligo (represented by a rectangle with a cross in it). pNI-6.8NdeI (6.8NdeI) contains the wild-type DHS6.8oligo sequence cloned upstream of the HS2 enhancer (as opposed to between HS2 and the γ-neo reporter). The number of NeoR colonies obtained for empty pNI was given a value of 1, and the number of colonies obtained with all other constructs was expressed relative to this value. Error bars denote S.E.M. of triplicate experiments carried out on three separate occasions, except for 6.8NdeI which was performed in triplicate on one occasion. Different plasmid DNA preparations were used for each experiment within the triplicates. **P < 0.01.
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Figure 4: Enhancer-blocking activity at the +6.8 kb DHS region. Each construct used in the enhancer-blocking assay is depicted as follows: pNI is the empty pNI plasmid and pNI-FII (FII) contains a known insulator from the chicken β-globin locus (represented by a triangle). pNI-6.8 (6.8) contains the wild-type DHS6.8oligo (represented by a rectangle) and pNI-6.8mut (6.8mut) contains the mutant version DHS6.8mutoligo (represented by a rectangle with a cross in it). pNI-6.8NdeI (6.8NdeI) contains the wild-type DHS6.8oligo sequence cloned upstream of the HS2 enhancer (as opposed to between HS2 and the γ-neo reporter). The number of NeoR colonies obtained for empty pNI was given a value of 1, and the number of colonies obtained with all other constructs was expressed relative to this value. Error bars denote S.E.M. of triplicate experiments carried out on three separate occasions, except for 6.8NdeI which was performed in triplicate on one occasion. Different plasmid DNA preparations were used for each experiment within the triplicates. **P < 0.01.

Mentions: The DHS6.8oligo and DHS6.8mutoligo oligonucleotides were designed with BssHII sticky ends (Figure 2A), facilitating their direct cloning into the AscI site of pNI. An enhancer-blocking activity assay was performed, with the number of neomycin-resistant colonies obtained for each construct normalized to the empty pNI plasmid. Results were subjected to statistical analysis by one-way ANOVA followed by Dunnett's multiple comparison test. When inserted into the enhancer-blocking position of pNI, FII, the CTCF-binding core from the known 5′HS4 chicken β-globin insulator (28,37), significantly reduced the number of colonies (2- to 3-fold; P < 0.01) (Figure 4). The pNI-DHS6.8oligo construct also gave a significantly lower number of colonies compared to pNI (2- to 3-fold, P < 0.01). In contrast, although the pNI-DHS6.8mutoligo construct gave a slightly lower number of colonies than pNI, this was not significant (P > 0.05). This suggests that CTCF binding at the +6.8 kb DHS is responsible, at least in part, for the enhancer-blocking activity of this element.Figure 4.


An insulator element 3' to the CFTR gene binds CTCF and reveals an active chromatin hub in primary cells.

Blackledge NP, Ott CJ, Gillen AE, Harris A - Nucleic Acids Res. (2009)

Enhancer-blocking activity at the +6.8 kb DHS region. Each construct used in the enhancer-blocking assay is depicted as follows: pNI is the empty pNI plasmid and pNI-FII (FII) contains a known insulator from the chicken β-globin locus (represented by a triangle). pNI-6.8 (6.8) contains the wild-type DHS6.8oligo (represented by a rectangle) and pNI-6.8mut (6.8mut) contains the mutant version DHS6.8mutoligo (represented by a rectangle with a cross in it). pNI-6.8NdeI (6.8NdeI) contains the wild-type DHS6.8oligo sequence cloned upstream of the HS2 enhancer (as opposed to between HS2 and the γ-neo reporter). The number of NeoR colonies obtained for empty pNI was given a value of 1, and the number of colonies obtained with all other constructs was expressed relative to this value. Error bars denote S.E.M. of triplicate experiments carried out on three separate occasions, except for 6.8NdeI which was performed in triplicate on one occasion. Different plasmid DNA preparations were used for each experiment within the triplicates. **P < 0.01.
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Related In: Results  -  Collection

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Figure 4: Enhancer-blocking activity at the +6.8 kb DHS region. Each construct used in the enhancer-blocking assay is depicted as follows: pNI is the empty pNI plasmid and pNI-FII (FII) contains a known insulator from the chicken β-globin locus (represented by a triangle). pNI-6.8 (6.8) contains the wild-type DHS6.8oligo (represented by a rectangle) and pNI-6.8mut (6.8mut) contains the mutant version DHS6.8mutoligo (represented by a rectangle with a cross in it). pNI-6.8NdeI (6.8NdeI) contains the wild-type DHS6.8oligo sequence cloned upstream of the HS2 enhancer (as opposed to between HS2 and the γ-neo reporter). The number of NeoR colonies obtained for empty pNI was given a value of 1, and the number of colonies obtained with all other constructs was expressed relative to this value. Error bars denote S.E.M. of triplicate experiments carried out on three separate occasions, except for 6.8NdeI which was performed in triplicate on one occasion. Different plasmid DNA preparations were used for each experiment within the triplicates. **P < 0.01.
Mentions: The DHS6.8oligo and DHS6.8mutoligo oligonucleotides were designed with BssHII sticky ends (Figure 2A), facilitating their direct cloning into the AscI site of pNI. An enhancer-blocking activity assay was performed, with the number of neomycin-resistant colonies obtained for each construct normalized to the empty pNI plasmid. Results were subjected to statistical analysis by one-way ANOVA followed by Dunnett's multiple comparison test. When inserted into the enhancer-blocking position of pNI, FII, the CTCF-binding core from the known 5′HS4 chicken β-globin insulator (28,37), significantly reduced the number of colonies (2- to 3-fold; P < 0.01) (Figure 4). The pNI-DHS6.8oligo construct also gave a significantly lower number of colonies compared to pNI (2- to 3-fold, P < 0.01). In contrast, although the pNI-DHS6.8mutoligo construct gave a slightly lower number of colonies than pNI, this was not significant (P > 0.05). This suggests that CTCF binding at the +6.8 kb DHS is responsible, at least in part, for the enhancer-blocking activity of this element.Figure 4.

Bottom Line: Elements within the basal promoter of the gene do not fully explain CFTR expression patterns, suggesting that cis-regulatory elements are located elsewhere, either within the locus or in adjacent chromatin.We further demonstrate that the element functions as an enhancer blocker in a well-established in vivo assay, and by using chromatin immunoprecipitation that it recruits CTCF in vivo.Moreover, we reveal that in primary epididymis cells, the +6.8 kb DHS interacts closely with the CFTR promoter, suggesting that the CFTR locus exists in a looped conformation, characteristic of an active chromatin hub.

View Article: PubMed Central - PubMed

Affiliation: Human Molecular Genetics Program, Children's Memorial Research Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60614, USA.

ABSTRACT
Regulation of expression of the CFTR gene is poorly understood. Elements within the basal promoter of the gene do not fully explain CFTR expression patterns, suggesting that cis-regulatory elements are located elsewhere, either within the locus or in adjacent chromatin. We previously mapped DNase I hypersensitive sites (DHS) in 400 kb spanning the CFTR locus including a cluster of sites close to the 3'-end of the gene. Here we focus on a DHS at +6.8 kb from the CFTR translation end-point to evaluate its potential role in regulating expression of the gene. This DHS, which encompasses a consensus CTCF-binding site, was evident in primary human epididymis cells that express abundant CFTR mRNA. We show by DNase I footprinting and electophoretic mobility shift assays that the cis-regulatory element within this DHS binds CTCF in vitro. We further demonstrate that the element functions as an enhancer blocker in a well-established in vivo assay, and by using chromatin immunoprecipitation that it recruits CTCF in vivo. Moreover, we reveal that in primary epididymis cells, the +6.8 kb DHS interacts closely with the CFTR promoter, suggesting that the CFTR locus exists in a looped conformation, characteristic of an active chromatin hub.

Show MeSH
Related in: MedlinePlus