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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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In vivo binding of CTCF at the +6.8 kb DHS region. Immunoprecipitations were performed with a CTCF-specific antibody and chromatin from (A) Caco2 cells and (B) Primary epididymis cells. No antibody control samples were also prepared, in which chromatin was incubated with Protein A agarose beads alone (data not shown). Samples were subjected to Taqman quantitative PCR analysis using probes specific for intron 17a and regions of interest 3′ to CFTR. CTCF-specific enrichment of each of these regions is shown relative to levels at intron 17a (which contains no predicted CTCF-binding sites). Vertical dashed line represents location of CFTR translation end-point, and x-axis on the right of this is drawn to scale (i.e. each data point accurately reflects the relative positions of Taqman amplicons). Immunoprecipitations were repeated at least twice. PCRs were performed in triplicate and Ct values averaged. Error bars denote S.E.M.
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Figure 3: In vivo binding of CTCF at the +6.8 kb DHS region. Immunoprecipitations were performed with a CTCF-specific antibody and chromatin from (A) Caco2 cells and (B) Primary epididymis cells. No antibody control samples were also prepared, in which chromatin was incubated with Protein A agarose beads alone (data not shown). Samples were subjected to Taqman quantitative PCR analysis using probes specific for intron 17a and regions of interest 3′ to CFTR. CTCF-specific enrichment of each of these regions is shown relative to levels at intron 17a (which contains no predicted CTCF-binding sites). Vertical dashed line represents location of CFTR translation end-point, and x-axis on the right of this is drawn to scale (i.e. each data point accurately reflects the relative positions of Taqman amplicons). Immunoprecipitations were repeated at least twice. PCRs were performed in triplicate and Ct values averaged. Error bars denote S.E.M.

Mentions: Since CTCF showed a strong interaction with DHS6.8oligoin vitro, ChIP with an antibody specific for CTCF followed by Taqman quantitative PCR analysis was used to investigate in vivo binding at this site. Chromatin from two cell types was evaluated: Caco2 colon carcinoma cells and fetal male primary epididymis cells, both of which express abundant CFTR. For Caco2 chromatin, ChIP with an antibody specific to CTCF gave an ∼5-fold enrichment of the +6.8 kb DHS relative to a region within CFTR intron 17a where there is no predicted CTCF-binding site (Figure 3A). The +15.6 kb DHS of CFTR (located ∼9 kb 3′ to the +6.8 kb DHS), that was previously demonstrated to possess CTCF-independent enhancer-blocking activity, showed no CTCF-specific enrichment, consistent with our earlier work (6). In contrast, using primary epididymis chromatin, the CTCF-specific antibody enriched the +6.8 kb DHS region by about 15-fold relative to CFTR intron 17a (Figure 3B). Approximately 2.5 kb either side of the +6.8 kb DHS, at +4.4 kb and +9.3 kb relative to the CFTR translation end-point, CTCF-specific enrichment in primary epididymis cells returned to baseline levels. The +15.6 kb DHS again showed no CTCF-specific enrichment in epididymis chromatin. For both Caco2 and primary epididymis chromatin, negative control ChIP experiments in which chromatin was immunoprecipitated with protein A beads alone (no antibody) resulted in baseline levels of enrichment at all regions (data not shown). Taken together, these results demonstrate strong in vivo binding of CTCF at the +6.8 kb DHS in primary epididymis cells, with a much lesser interaction between CTCF and the +6.8 kb DHS in the Caco2 cell line. It is noteworthy that CTCF-specific enrichment of the +6.8 kb DHS in primary epididymis cells correlates with the presence of the +6.8 kb DHS in this cell type (13).Figure 3.


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)

In vivo binding of CTCF at the +6.8 kb DHS region. Immunoprecipitations were performed with a CTCF-specific antibody and chromatin from (A) Caco2 cells and (B) Primary epididymis cells. No antibody control samples were also prepared, in which chromatin was incubated with Protein A agarose beads alone (data not shown). Samples were subjected to Taqman quantitative PCR analysis using probes specific for intron 17a and regions of interest 3′ to CFTR. CTCF-specific enrichment of each of these regions is shown relative to levels at intron 17a (which contains no predicted CTCF-binding sites). Vertical dashed line represents location of CFTR translation end-point, and x-axis on the right of this is drawn to scale (i.e. each data point accurately reflects the relative positions of Taqman amplicons). Immunoprecipitations were repeated at least twice. PCRs were performed in triplicate and Ct values averaged. Error bars denote S.E.M.
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Figure 3: In vivo binding of CTCF at the +6.8 kb DHS region. Immunoprecipitations were performed with a CTCF-specific antibody and chromatin from (A) Caco2 cells and (B) Primary epididymis cells. No antibody control samples were also prepared, in which chromatin was incubated with Protein A agarose beads alone (data not shown). Samples were subjected to Taqman quantitative PCR analysis using probes specific for intron 17a and regions of interest 3′ to CFTR. CTCF-specific enrichment of each of these regions is shown relative to levels at intron 17a (which contains no predicted CTCF-binding sites). Vertical dashed line represents location of CFTR translation end-point, and x-axis on the right of this is drawn to scale (i.e. each data point accurately reflects the relative positions of Taqman amplicons). Immunoprecipitations were repeated at least twice. PCRs were performed in triplicate and Ct values averaged. Error bars denote S.E.M.
Mentions: Since CTCF showed a strong interaction with DHS6.8oligoin vitro, ChIP with an antibody specific for CTCF followed by Taqman quantitative PCR analysis was used to investigate in vivo binding at this site. Chromatin from two cell types was evaluated: Caco2 colon carcinoma cells and fetal male primary epididymis cells, both of which express abundant CFTR. For Caco2 chromatin, ChIP with an antibody specific to CTCF gave an ∼5-fold enrichment of the +6.8 kb DHS relative to a region within CFTR intron 17a where there is no predicted CTCF-binding site (Figure 3A). The +15.6 kb DHS of CFTR (located ∼9 kb 3′ to the +6.8 kb DHS), that was previously demonstrated to possess CTCF-independent enhancer-blocking activity, showed no CTCF-specific enrichment, consistent with our earlier work (6). In contrast, using primary epididymis chromatin, the CTCF-specific antibody enriched the +6.8 kb DHS region by about 15-fold relative to CFTR intron 17a (Figure 3B). Approximately 2.5 kb either side of the +6.8 kb DHS, at +4.4 kb and +9.3 kb relative to the CFTR translation end-point, CTCF-specific enrichment in primary epididymis cells returned to baseline levels. The +15.6 kb DHS again showed no CTCF-specific enrichment in epididymis chromatin. For both Caco2 and primary epididymis chromatin, negative control ChIP experiments in which chromatin was immunoprecipitated with protein A beads alone (no antibody) resulted in baseline levels of enrichment at all regions (data not shown). Taken together, these results demonstrate strong in vivo binding of CTCF at the +6.8 kb DHS in primary epididymis cells, with a much lesser interaction between CTCF and the +6.8 kb DHS in the Caco2 cell line. It is noteworthy that CTCF-specific enrichment of the +6.8 kb DHS in primary epididymis cells correlates with the presence of the +6.8 kb DHS in this cell type (13).Figure 3.

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