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

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.

Figure 1.

In vitro DNase I footprinting of the +6.8 kb DHS probe. Experiments using (A) sense and (B) anti-sense strands are shown. Both gel images are labelled as follows: 1, AG ladder; 2, No DNase I; 3, No Caco2 nuclear extract; 4 and 5, 40 μg and 80 μg Caco2 cell nuclear extract, respectively. For both panels, protected region 1 (PR1) is highlighted by a bold vertical line. Narrow vertical line marked with an asterisk shows position of putative CTCF-binding site.

Figure 2.

In vitro binding of CTCF at the +6.8 kb DHS region. (A) The DHS6.8^oligo and DHS6.8mut^oligo probes. The putative CTCF-binding site core is highlighted. Oligonucleotides were designed to form BssHII sticky ends when annealed (shown in italics), facilitating cloning into the AscI site of pNI. Mutated bases in DHS6.8mut^oligo are underlined. (B) IVT CTCF; (C, D), Caco2 nuclear extracts. (B) EMSA using DHS6.8^oligo. Supershift was performed with an anti-CTCF antibody and anti-RAR β was used as isotype control. (C) EMSA with DHS6.8^oligo. Competition reactions were performed with 100× excess of unlabelled DHS6.8^oligo (self), DHS6.8mut^oligo (self mut), FII (known CTCF-binding site from chicken β-globin locus) and mutant (FIImut). (D) EMSA with DHS6.8^oligo. Supershift reactions were performed as in (B). (E) EMSA with DHS6.8mut^oligo. Complex marked i represents CTCF in complex with DHS6.8^oligo and ii the antibody-supershifted CTCF complex. Undefined interactions formed between 6.8mut^oligo and Caco2 nuclear extract are marked by *.

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.

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 DHS6.8^oligo (represented by a rectangle) and pNI-6.8mut (6.8mut) contains the mutant version DHS6.8mut^oligo (represented by a rectangle with a cross in it). pNI-6.8NdeI (6.8NdeI) contains the wild-type DHS6.8^oligo sequence cloned upstream of the HS2 enhancer (as opposed to between HS2 and the γ-neo reporter). The number of Neo^R 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.

Figure 5.

3C analysis of the CFTR locus. (A) Scale figure of the CFTR gene with exons marked by vertical bars and the translation start site represented by a bent arrow. Small vertical lines above the gene denote HindIII sites and half arrow heads show the locations of 3C primers. Due to space constraints, primers are not drawn to scale, and the fixed CFTR promoter HindIII fragment is expanded, showing the location of Taqman probe (joined ball and star) and the reverse primer. The –20.9 kb DHS and +6.8 kb DHS locations are also indicated. (B) 3C data from primary human skin fibroblast cells. (C) 3C data from primary human epididymis cells. The x-axis for each chart is drawn to scale, with units representing base pairs relative to the CFTR translation start point (0). Both charts are aligned with the CFTR gene figure above. Vertical dashed lines represent HindIII sites. The interaction frequency between a fixed HindIII fragment at the CFTR promoter (shaded grey) and HindIII fragments at various regions across the CFTR gene was measured by Taqman quantitative PCR. The interaction frequency at each point is expressed relative to the interaction between two HindIII fragments within the ubiquitously expressed ERCC3 gene. Data shown represent the average of two independent experiments. Each real-time PCR reaction was performed three times and averaged. Error bars denote SEM.

Figure 6.

Looping model for CFTR gene. In CFTR-expressing cell types, such as primary epididymis cells, elements in the CFTR 3' flanking region are in close proximity with the CFTR promoter. This 3' flanking region includes the tissue-specific +6.8-kb DHS, shown here to bind CTCF, as well as other previously described DHS (13,6). Protein factors bound at each of these sites interact with the promoter-bound transcription machinery, thus forming an active chromatin hub (ACH) and helping regulate expression of the CFTR gene. In addition to DHS from the 3' flanking region, intronic DHS such as the intestine-specific intron 1 element (31,42,43) and others (represented by the elements marked ‘?’), may contribute to the CFTR ACH in a tissue-specific manner.