(Received for publication, November 29, 1995; and in revised form, February 9, 1996)
From the
The cystic fibrosis transmembrane conductance regulator (CFTR) gene exhibits a tightly regulated pattern of expression in human epithelial cells. The mechanism of this regulation is complex and is likely to involve a number of genetic elements that effect temporal and spatial expression. To date none of the elements that have been identified in the CFTR promoter regulate tissue-specific expression. We have identified a putative regulatory element within the first intron of the CFTR gene at 181+10kb. The region containing this element was first identified as a DNase I hypersensitive site that was present in cells that express the CFTR gene but absent from cells not transcribing CFTR. In vitro analysis of binding of proteins to this region of DNA sequence by gel mobility shift assays and DNase I footprinting revealed that some proteins that are only present in CFTR-expressing cells bound to specific elements, and other proteins that bound to adjacent elements were present in all epithelial cells irrespective of their CFTR expression status. When assayed in transient expression systems in a cell line expressing CFTR endogenously, this DNA sequence augmented reporter gene expression through activation of the CFTR promoter but had no effect in nonexpressing cells.
The cystic fibrosis transmembrane conductance regulator (CFTR) ()gene (Rommens et al., 1989; Riordan et
al., 1989) shows a tightly regulated pattern of temporal and
spatial expression (Crawford et al., 1991; Harris et
al., 1991; Denning et al., 1992; Trezise et al.,
1993). Very little is known about the genetic elements and
transcription factors that regulate CFTR expression. The basal promoter
of the CFTR gene has been analyzed in some detail (Chou et
al., 1991; Yoshimura et al., 1991a, 1991b; Koh et
al., 1993), although the data are somewhat inconsistent. The
minimal promoter sequence found between -226 and +98 bp with
respect to the transcription start site defined by Chou et
al.(1991) is sufficient to drive low levels of expression of a
reporter gene (Chou et al., 1991; Koh et al., 1993).
However, Chou et al.(1991) also identified an element
immediately upstream of -277 that repressed reporter gene
transcription in several cell lines.
Analysis of the DNA sequence of the basal CFTR promoter reveals a number of elements that may be involved in regulation of transcription. There are several potential binding sites for the AP-1 transcription factor (GGAGTCAG) and three putative binding sites for the transcription factor Sp1 (GGGCGG). There is evidence for in vitro regulation of CFTR gene expression by phorbol esters (Trapnell et al., 1991). A cAMP-response element (TGACATCA) has been defined within the CFTR promoter at -48 to -41 with respect to the transcription start site defined by Yoshimura et al. (1991a) (McDonald et al., 1995). Two purine/pyrimidine repeat elements have been identified in the 5`-flanking region of the CFTR gene, one of which has been shown to be S1 nuclease-sensitive in supercoiled plasmids, suggesting a non-B DNA structure (Hollingsworth et al., 1994; McDonald et al., 1994).
There is little data on the control of cell specificity of CFTR expression, although there is ample evidence for such regulation. The human CFTR gene is expressed at significant levels mainly in the epithelia lining the pancreas, intestine, bile ducts, male genital ducts, and certain regions of the airway epithelium including the inferior turbinate of the nose, the trachea, and the serous portion of submucosal glands (Crawford et al., 1991; Denning et al., 1992; Engelhardt et al., 1993). There is evidence that expression of the CFTR gene may be hormonally regulated in epithelia within the reproductive system (Trezise et al., 1992; Rochwerger et al., 1994). Some degree of cell type-specific control has been inferred for uncharacterized elements within the immediate 5`-untranslated region. A number of DNase I hypersensitive sites that show some degree of correlation with CFTR expression have been observed between -3,000 bp relative to the transcription start site and +100 bp into intron 1 (Yoshimura et al., 1991b; Koh et al., 1993). However, these sites have only been examined in a few long term cell lines that either do or do not express CFTR mRNA and protein and hence may not adequately reflect cell-specific regulation of expression of the CFTR gene in vivo. Transgenic mouse experiments in which 19 kb of genomic DNA 5` to the CFTR gene were placed 5` of a reporter gene failed to achieve expression (Griesenbach et al., 1994).
Because the expression control elements of the CFTR gene had not been well defined, we screened a larger region of genomic DNA than had been analyzed previously in an attempt to identify these elements. The chromatin structure of 120 kb of genomic DNA 5` to the CFTR gene was analyzed in a number of CFTR expressing and nonexpressing cell types, including primary genital duct epithelial cells in addition to long term cell lines. We identified DNase I hypersensitive sites within this region by screening with probes isolated from cosmid and phage clones (Rommens et al., 1989). Novel DNase I hypersensitive sites were observed at -79.5 and -20.5 kb 5` to the ATG translation start codon of the CFTR coding sequence (Smith et al., 1995). Neither of these sites showed strong correlation with CFTR expression in the cell types investigated. Although they may play an important role in the complex series of events involved in the regulation of CFTR transcription, these data do not support the existence of cell-specific control elements at these sites.
Another DNase I hypersensitive site was observed within intron 1 of the CFTR gene. Detection of this site correlated well, quantitatively and qualitatively, with the levels of expression of the CFTR gene in both long term cell lines and primary genital duct epithelial cells. Nuclear extracts from cells that transcribe the CFTR gene contain specific proteins that bind to DNA in the region of this hypersensitive site. Further, analysis of the putative regulatory element through transient assays of reporter gene constructs showed a positive effect on the activity of the CFTR promoter in cells that express the CFTR gene endogenously.
Figure 1: A, long range map of 70 kb of genomic DNA flanking exon 1 of the CFTR gene. The restriction map for relevant sites for the enzymes BamHI(B), EcoRI(R), HindIII(H), and XhoI(X) is shown on the solid line. The scale is in kilobases, where zero denotes the position of the ATG start codon of the CFTR coding sequence. I denotes the location of exon 1. The cosmid cW44 used as a source of probes is indicated by the horizontal arrow. The solid boxes represent the cW44 XB5.0, H4.0, and EB1.7 probes used to detect the novel DNase 1 hypersensitive site at 181+10kb shown in Fig. 2. The vertical arrow marks the location of this site. B, the restriction map of 800 bp flanking the 181+10kb DNase I hypersensitive site. The sites of the primers used to amplify segments 3/4, 5/6, and 7/8 and the BS0.7 fragment are shown by arrows. The HindIII (H) and EcoRI (R) restriction sites shown close to the hypersensitive site in A are shown as well as the Scrf1 (Sc), Sau3a (Sa), and Alu (A) restriction sites in the 7/8 fragment. C, oligonucleotides used in competition experiments are shown as follows. ASTM1F/R and ASTM2F/R are in italics; ASTM3F/R are in bold italics; ASTM4F/R and ASTM6F/R are underlined; and ASTM5F/R are overlined. The PUT2 and inf.1 transcription factor binding motifs located within the 7/8 element are shaded.
Figure 2: Detection of a DNase I hypersensitive site at 181+10kb. Figure shows autoradiographs of Southern blots of genomic DNA extracted from nuclei treated with DNase I, digested with BamHI, and probed with the cW44 H4.0 fragment shown in Fig. 1. For each cell type (Caco2, MCF7, RVP, primary fetal vas deferens cells, Capan1, HT29, primary fetal epididymis cells, and 37566), lane 1 shows DNA prepared from nuclei that had not been treated with DNase I. Lanes 2-5 show DNA prepared from nuclei treated with increasing amounts of DNase I: lane 2, 15 units; lane 3, 30 units; lane 4, 60 units; lane 5, 120 units DNase I. The cW44 H4.0 probe hybridizes to an approximately 22-kb BamHI restriction fragment. A subfragment of approximately 8 kb can be seen in lanes 2-5 in the cells lines that express the CFTR gene endogenously. This indicates the presence of a DNase I hypersensitive site in this region, located at approximately 181+10kb of the CFTR gene, lying within intron 1 (shown by the arrow in Fig. 1).
In all transfection experiments the pGL2B 245 constructs were co-transfected with the amount of DNA of pdolCMVcat (Ma et al., 1992) as a transfection control. Luciferase and CAT assays were carried out by standard procedures. Each transfection experiment was carried out 6 times (5 for MCF7) with individual constructs being assayed in quintuplicate in each experiment. The results are expressed as relative luciferase activity, with the pGL2B 245 CFTR promoter construct equal to 1, corrected for transfection efficiency as measured by CAT activity. Statistical analyses of results were performed using Minitab Statistical Software, Release 7 (1989, Minitab Inc. 3081 Enterprise Drive, State College, PA 16802).
Figure 3: Gel mobility shift profiles of the 205-bp 7/8 fragment with nuclear extracts from CFTR+ and CFTR- cell lines and competition with the 5/6 and 7/8 DNA fragments. Lanes 1, no nuclear extract; lanes 2, MCF7 (CFTR-); lanes 3, HPAF (CFTR-); lanes 4, HT29 (CFTR+); lanes 5, Caco2 (CFTR+). Complexes a1, c1, and c2 are marked.
Figure 4: Gel mobility shift profiles of the 205-bp 7/8 fragment with nuclear extracts from CFTR+ and CFTR- cell lines and competition with the subfragments of the 7/8 DNA fragment as shown. A and B, lanes 1, no nuclear extract; lanes 2, MCF7 (CFTR-); lanes 3, 37566 (CFTR-); lanes 4, primary epididymis (CFTR+); lanes 5, Caco2 (CFTR+). Complexes a1, a2, b1, b2, c1, and c2 are marked. C, restriction map of the 7/8 fragment.
Further mapping of the location of the DNA-protein complexes detected by gel mobility shift analysis was achieved by competition with subfragments of the 7/8 element (see Fig. 4C). In each case the labeled probe was the entire 7/8 fragment, and all complexes were abolished by the presence of excess unlabeled 7/8 fragment (Fig. 4A). The 38-bp AluI fragment did not compete with any of the protein-DNA complexes (not shown). All protein-DNA complexes were eliminated by an excess of 112-bp ScrfI (Fig. 4B) and 74-bp AluI/ScrfI fragments (not shown), suggesting that all proteins causing gel shifts were binding between the ScrfI and AluI sites. In some competition experiments (not shown) the 93-bp ScrfI fragment showed weak competition with the a1, a2, b1, and b2 complexes, suggesting the relevant protein complex might also involve sites close to the 3` end of this fragment The addition of excess unlabeled 130-bp Sau3a fragment blocked the formation of complexes seen at bands a1, a2, b1, and b2 (Fig. 4A). Adding an excess of the 75-bp Sau3a fragment eliminated the complexes seen at bands c1 and c2 (Fig. 4B). Hence, the complexes a1, a2, b1, and b2 detected in CFTR-expressing cells probably include proteins that bind to DNA sequences lying 5` to the Sau3a site (Fig. 4C), and the complexes seen in all cell lines, c1 and c2, involve DNA-protein interactions primarily 3` to the Sau3a site.
Figure 5: DNase I footprint of the 7/8 element. Protected sequences are shown on the right. Lanes 1 and 14, AG ladder; lane 2, no DNase I; lanes 3 and 8, no nuclear extract; lane 4, 20 µg of nuclear extract from MCF7 (CFTR-); lane 5, 40 µg of nuclear extract from MCF7 (CFTR-); lane 6, 20 µg of nuclear extract from HPAF (CFTR-); lane 7, 40 µg of nuclear extract from HPAF (CFTR-), lane 9, 20 µg of nuclear extract from Caco2 (CFTR+); lane 10, 40 µg of nuclear extract from Caco2 (CFTR+); lane 11, 20 µg of nuclear extract from primary epididymis cell culture i (CFTR+); lane 12, 40 µg of nuclear extract from primary epididymis cell culture i (CFTR+); lane 13, 40 µg of nuclear extract from primary epididymis cell culture ii (CFTR+).
The DNase I footprint data suggest a complex pattern of DNA-protein interactions within this region of the 7/8 fragment. The results obtained with nuclear extracts from the primary cells suggest that the complex of protein(s) may be altering chromatin structure as revealed by the presence of DNase I hypersensitive sites. It is probable, given the complexity of the DNase I footprint and gel mobility shift data, that a number of proteins are interacting with this region of genomic DNA.
Competition with a 100-fold molar excess of oligonucleotides ASTM2F/R resulted in a reduction in the amounts of band a1 on gel mobility shift reactions in Caco2 (Fig. 6A). This suggested that the 20 base pairs of ASTM2F/R encompassed at least one site of DNA-protein interaction for the a1 complex. However, when oligonucleotide ASTM2F/R was labeled and used in gel mobility shift assays (not shown), it was inefficient (in comparison with the whole 7/8 fragment) at generating a DNA-protein complex with nuclear extracts from Caco2 cells, suggesting that nucleotides lying outside this 20-bp sequence (presumably 5` to it, on the basis of the 7/8 restriction fragment competition experiments above) may be important for the formation of complex a1. Further evidence for this was provided by gel mobility shift experiments with the ASTM5F/R oligonucleotide, which generated the a1 complex more efficiently than ASTM2F/R (not shown). ASTM2F/R was also seen to inhibit formation of the a2/b1/b2 complexes seen with primary epididymis cell nuclear extracts (Fig. 6C). Here again, the competition was incomplete, suggesting that other elements outside this sequence (presumably 5` to it) were important in the generation of these complexes. The ASTM5F/R oligonucleotide was also effective in competition of the a1/a2/b1/b2 complexes (competition of a1 is shown in Fig. 6B) with little effect on the noncell-specific c1 and c2 complexes.
Figure 6:
Gel mobility shift profiles of the 205-bp
7/8 fragment with nuclear extracts from Caco2 and primary epididymis
cells and competition with oligonucleotides ASTM1F/R, ASTM2F/R,
ASTM3F/R, ASTM4F/R, and ASTM5F/R as shown. A, Caco2 nuclear
extract and competition with 100 (lanes 1), 200
(lanes 2), and 500
(lanes 3) excess of
oligonucleotide ASTM2F/R and ASTM3F/R as marked. P denotes 7/8
probe only, and 0 denotes no competition. B, Caco2
nuclear extract and competition with 100
ASTM2F/R (lane
2), ASTM4F/R (lane 4), and ASTM5F/R (lane 5) as
marked. 0 denotes no competition. C, epididymis
nuclear extract and competition with 200
ASTM1F/R (lane
1), ASTM2F/R (lane 2), and ASTM3F/R (lane 3) as
marked. 0 denotes no competition. D, diagram to show
locations of oligonucleotides (1, 2, 3, 4, 5) and binding
sites of Caco2 and epididymis-specific proteins and non-cell
type-specific proteins to the 7/8 fragment.
Competition with oligonucleotides ASTM4F/R showed no inhibition of the a1 complex (Fig. 6B), hence it is likely that the a1/a2/b1/b2 complexes are interacting with DNA 5` to the end of oligo 4. This was confirmed by gel mobility shift experiments (not shown) using the MseI fragment lying between 720 and 748 (see Fig. 6D) as a probe, which only generated the c1 and c2 complexes. Competition with oligonucleotides ASTM4F/R showed inhibition (though incomplete) of formation of the c1 and c2 complexes seen in all cell types analyzed (Fig. 6B). Further, the ASTM4F/R oligonucleotide alone was effective at forming the c1 and c2 complexes when used as a probe in gel mobility shift assays (not shown). However, oligonucleotide ASTM3F/R, which overlaps the 3` 19 bp of ASTM4F/R, was only able to cause slight inhibition of the c1 and c2 complex in Caco2 at 500-fold excess (Fig. 6A), suggesting that the important DNA-protein interactions required for generating the c1 and c2 complexes are close to the Sau3a site.
Neither oligonucleotides ASTM1F/R nor ASTM6F/R showed competition with any of the DNA-protein complexes detected in the primary epididymis cell nuclear extracts or those from any of the other cell lines (not shown). Hence the precise nature of the DNase I footprint observed in the region of ASTM1F/R (GTACTTTGGAATC) with the epididymis cell nuclear extracts (Fig. 5) remains obscure.
In summary (Fig. 6D) the DNA-protein interactions that generate the gel mobility shifts c1 and c2 seen with nuclear extracts from all the cell types that we have analyzed occur between the MseI site at 720 and the MseI site at 748 with the key sites in the complex lying closer to the 5` half of this fragment. The DNA-protein interactions that generate the gel mobility shifts a1, seen in Caco2 and HT29 and a2, b1, and b2 seen in primary epididymis nuclear extracts (none of these complexes being seen in the cell lines Panc-1, HPAF, and MCF7 that do not transcribe CFTR) are 5` to the MseI site at 720 but 3` to the end of oligonucleotide ASTM6F/R at 699. The efficiency of the ASTM5F/R oligonucleotide in generating the a1 complex in Caco2 nuclear extracts confirms this localization. The ability of the ASTM2F/R oligonucleotide to form the a1 complex, even if inefficiently, suggests that the base pairs involved in this interaction do not extend greatly to the 5` end of this oligonucleotide at 711.
Figure 7: Transient transfection experiments. The bar chart shows the luciferase activities for each construct relative to the 245 CFTR promoter only construct (=1) in the MCF7, 16HBE14o-, and Caco2 cell lines.
The results
of the luciferase assays showed that none of the cloned fragments from
intron 1 had an effect on the CFTR promoter when transfected into MCF 7
cells. Similar results were obtained in the 16HBEO14o- cell line
that while transcribing CFTR at a high level as a result of SV40
Ori- transformation, does not show the DNase I hypersensitive
site at 181+10kb that is seen in the other cell types that express
CFTR endogenously. However the 7/8 fragment had a positive effect on
CFTR promoter activity in the Caco2 cell line. Although the 5/6
fragment had essentially no effect, 1.1 (S.E.= 0.176)
promoter alone, the 7/8 fragment augmented luciferase activity by a
mean of 2.2-fold (S.E.= 0.311) with respect to the CFTR promoter
alone. The BS0.7 fragment that encompasses the 750-bp fragment of the
putative regulatory element (see Fig. 1B, 1AIR-TSR8)
caused a mean amplification of luciferase activity of 3.4-fold
(S.E.= 0.350). An analysis of variance was performed on log
transformed data (to correct for non-normality) with experiment number
added to the model as a block to control for temporal variation. After
correcting the significance levels for multiple comparisons, the 7/8
and the BS0.7 elements were each seen to have significantly greater
activity than the 245 CFTR promoter element alone (p < 0.01
in both cases). However, the 5/6 element did not differ in activity
from the promoter alone (p > 0.3).
The identification and isolation of element(s) that control expression of the CFTR gene are of particular importance in the context of potential targeted gene therapy for CF. Previous analyses of chromatin structure (and methylation status) of the CFTR gene promoter region have identified a number of DNase 1 hypersensitive sites in a small number of cell lines that show some correlation with CFTR expression in those lines (Koh et al., 1993; Yoshimura et al., 1991a); however, to date the picture is incomplete. We have identified DNase 1 hypersensitive sites at -20.5kb and -79.5kb to the translational start codon of the CFTR gene (Smith et al., 1995); these sites are seen in all cell types we have analyzed. Hence, the search for elements and factors that mediate tissue-specific expression continues. It is likely that regulation of expression of the CFTR gene is complex and involves the interaction of a number of different regulatory factors and elements. We describe here one element that appears to play a role in controlling expression of the CFTR gene.
Through a combination of DNase I footprint analysis and gel mobility shift assays using subfragments of the region (7/8) containing this element and oligonucleotides, we have determined that the regulatory element is located within a sequence of about 40 bp. The 40-bp sequence contains two distinct sites of DNA-protein interactions as illustrated in Fig. 6D. The 5` side of the MseI site at 720 bp contains the sequence AATCCTAACTCTGTCACTTAT. A minimum of 9 bases (in bold) at the end of this sequence are crucial for the binding of the proteins found specifically in the nuclear extracts of the CFTR-expressing Caco2 and primary epididymal cells. It is probable that additional base pairs may be involved in the interaction. On the 3` side of the same MseI site is the sequence TAACAATGTGATCTTAGGCAATTTACTT. A minimum of 13 base pairs (in bold) are likely to be involved in DNA binding of the proteins that generate the c1 and c2 complexes detected in CFTR expressing and nonexpressing cells.
Analysis of the DNA sequence shown in Fig. 1C reveals the presence of consensus binding motifs for several known transcription factors within the 7/8 region. These include PUT2 ATGTACTT (Siddiqui and Brandriss, 1988) and inf.1 AAGTGA (Fujita et al., 1987). The PUT2 protein functions in concert with the proline utilization pathway of Saccharomyces cerevisiae. The regulatory element lies upstream of the TATA box of this gene and is essential in proline induction of the PUT2 gene. The PUT2 homology within the 7/8 region lies within the sequence 671-683 that shows protection on DNase I footprints solely with the primary epididymal cell nuclear extracts (see Fig. 5). The relevance of this putative element to regulation of CFTR gene expression is not inherently obvious.
A region of homology with
inf.1 lies at the 5` end of the ASTM2R oligonucleotide on the reverse
strand and is coincident with the base pairs that appear to be
responsible for binding of the a1 complex observed in Caco2 nuclear
extracts. The inf.1 element is a 6-bp unit of a repeated sequence that
mediates virus-induced transcription of interferon , although any
relevance to CFTR expression is obscure.
The pattern of expression of these proteins in different cell types is of interest. All cell types we have analyzed contain proteins that generate the c1 and c2 complexes that bind to oligonucleotide ASTM4F/R. On the basis of gel mobility shifts, these proteins would appear to be the same in all cells. However, the proteins that bind to the region of the ASTM2F/R and ASTM5F/R oligonucleotides in different cell types are likely not to be all the same. Nuclear extracts from intestinal carcinoma cell lines Caco2 and HT29 and the pancreatic adenocarcinoma cell line Capan1 all produce the same prominent gel mobility shift designated a1. The abundance of this complex is greater in the intestinal carcinomas, and this may reflect the CFTR expression levels in these cells (see Table 1). Nuclear extracts from primary epididymis cells show at least three gel mobility shift bands (a2, b1, and b2) with mobilities differing from the single predominant complex seen in Caco2 nuclear extracts.
Oligonucleotide competition experiments have shown that the proteins that cause the a1/a2/b1/b2 gel shifts are all interacting with the sequence of ASTM2F/R and ASTM5F/R. However, the DNase1 footprinting data show a greater region of protection of the DNA backbone by nuclear extracts from the primary cells. We have not determined whether the other proteins are in fact binding directly to the DNA or to other proteins in the DNA-protein complex. We are further characterizing the nature of these proteins.
The data from the gel mobility shift assays (taken together with the data showing that the DNase I hypersensitive site at 181+10kb is only seen in cell types that express the CFTR gene) lead us to propose the following model for DNA-protein interactions in this region of the gene. Some protein factors that bind to this region are present in nuclear extracts from most cell types, regardless of their status with respect to CFTR expression, and their presence alone does not create a DNase I hypersensitive site. Additional proteins that bind to the DNA in this region and cause conformational changes in the chromatin structure to expose a DNase I hypersensitive site are expressed in many cell types that endogenously transcribe the CFTR gene. There is evidence for such a mechanism existing in regulatory elements associated with other genes (Jenuwein et al., 1993). At this stage the nature of any interactions between the individual proteins themselves and between each of them and the DNA backbone remains to be elucidated.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U47863[GenBank].