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Proc Natl Acad Sci U S A. Feb 1, 2000; 97(3): 1172–1177.
PMCID: PMC15558
Genetics

Comparative genomic sequence analysis of the human and mouse cystic fibrosis transmembrane conductance regulator genes

Abstract

The identification of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in 1989 represents a landmark accomplishment in human genetics. Since that time, there have been numerous advances in elucidating the function of the encoded protein and the physiological basis of cystic fibrosis. However, numerous areas of cystic fibrosis biology require additional investigation, some of which would be facilitated by information about the long-range sequence context of the CFTR gene. For example, the latter might provide clues about the sequence elements responsible for the temporal and spatial regulation of CFTR expression. We thus sought to establish the sequence of the chromosomal segments encompassing the human CFTR and mouse Cftr genes, with the hope of identifying conserved regions of biologic interest by sequence comparison. Bacterial clone-based physical maps of the relevant human and mouse genomic regions were constructed, and minimally overlapping sets of clones were selected and sequenced, eventually yielding ≈1.6 Mb and ≈358 kb of contiguous human and mouse sequence, respectively. These efforts have produced the complete sequence of the ≈189-kb and ≈152-kb segments containing the human CFTR and mouse Cftr genes, respectively, as well as significant amounts of flanking DNA. Analyses of the resulting data provide insights about the organization of the CFTR/Cftr genes and potential sequence elements regulating their expression. Furthermore, the generated sequence reveals the precise architecture of genes residing near CFTR/Cftr, including one known gene (WNT2/Wnt2) and two previously unknown genes that immediately flank CFTR/Cftr.

Cystic fibrosis (CF) is one of the most common inherited disorders in individuals of northern European descent (1). With an autosomal recessive pattern of inheritance, CF has a carrier frequency of ≈1 in 30 Caucasians, with ≈1,000 affected individuals born in the United States each year. The disease is associated with pancreatic insufficiency, repeated pulmonary infections, intestinal blockages, elevated sweat chloride levels, and male infertility. Despite major improvements in therapeutic approaches, CF remains a major and challenging health problem.

One decade ago, investigators elucidated the underlying genetic defect responsible for CF with the identification of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (24). This discovery represents one of the most important triumphs in contemporary human genetics, demonstrating the efficacy of positional cloning as a strategy for identifying genes associated with human disease (5, 6), even in the absence of cytogenetic rearrangements that assist the search. Residing on chromosome 7q31.3 (7) and consisting of 27 exons, the human CFTR gene encodes a 6,129-bp transcript that directs the synthesis of a 1,480-aa protein (2, 3) shown to function as a chloride channel (810).

In the decade since the identification of the CFTR gene, there have been various advances in understanding the function of the encoded protein and the pathogenesis of CF (11). These have included the development of animal models for the disease (1217) as well as new avenues for drug- (18, 19) and gene therapy- (20) based treatment modalities. Less clear are the mechanisms responsible for the characteristic tissue-specific expression of CFTR (e.g., in pancreas, lung, sweat glands, intestine, and liver). A number of studies have pointed to possible CFTR promoter sequences and regulatory elements (2132), including regions with high GC content, Sp1 and AP-1 binding sites, and DNase I hypersensitivity sites. These sequences, some of which constitute the basal CFTR promoter ≈250 bp upstream of the translation start site, resemble regulatory elements more typically found in housekeeping as opposed to tissue-specific genes. Furthermore, studies in other animal species (e.g., mouse) have revealed slightly different expression patterns of the gene (33). In short, the regulatory mechanism(s) responsible for the tissue-specific expression of CFTR remains unclear.

To facilitate studies of CFTR structure and function, we sought to establish the complete sequence of the genomic segments containing the human and mouse genes. Here we report the high-resolution mapping and systematic sequencing of an ≈1.6-Mb segment of human chromosome 7 containing CFTR, an effort performed under the auspices of the ongoing Human Genome Project (34, 35). In addition, we performed parallel analyses of the corresponding Cftr-containing region of mouse chromosome 6, producing ≈358 kb of contiguous mouse sequence. The availability of homologous human and mouse sequence has allowed important long-range, cross-species sequence comparisons to be performed.

Materials and Methods

Construction of a Human Bacterial Clone-Based Physical Map.

Human DNA-containing bacterial clones [bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs)] were isolated, analyzed, and assembled in contigs as described (36, 37). Clones are named with a prefix reflecting their library of origin [RG, Research Genetics human BAC library (Huntsville, AL); GS, Genome Systems human BAC library (St. Louis); DJ, Roswell Park Cancer Institute PAC library (Buffalo, NY)].

Construction of a Mouse BAC-Based Physical Map.

Purified inserts from three cDNA clones containing the mouse Cftr coding region (38, 39) (see http://www.atcc.org, nos. 63165, 63166, and 63167) were radiolabeled and hybridized to membrane filters containing high-density arrays of mouse BAC clones [mouse C57BL/6 library from Genome Systems (GS); mouse ES/129Sv library from Research Genetics (RG)]. Positive clones were colony purified, were reanalyzed by hybridization to confirm the presence of probe sequences, and were subjected to restriction enzyme digest-based fingerprint analysis (40).

BAC/PAC Clone Sequencing.

The high-accuracy sequence of all BAC/PAC clones was established by a shotgun sequencing strategy. Human clones were sequenced at the Washington University Genome Sequencing Center by well established methods (4143) (also see http://genome.wustl.edu/gsc). The sequence of each human clone was submitted to GenBank as a separate record. Mouse clones were sequenced at the NIH Intramural Sequencing Center (see http://www.nisc.nih.gov) by similar methods. Individual sequences were edited and assembled with the phred/phrap/consed suite of programs (4446). The sequence of the three overlapping mouse BACs was assembled into one contiguous block and was submitted to GenBank as a single record (GenBank accession no. AF162137). The approaches used for sequencing both human and mouse DNA should produce error rates of less than 1 in 104 bp, something confirmed by recent quality assessment experiments (47).

Human–Mouse Sequence Comparison.

Repetitive sequences were masked with the repeatmasker program (A. F. A. Smit and P. Green, see http://www.genome.washington.edu/UWGC/analysistools/repeatmask.htm), and the resulting sequence was aligned with a modified version of the sim program (48) by using the default parameters (+1 for a match, −1 for a mismatch, and −6  0.2k for a gap length of k). For another view of the alignment, regions between successive gaps were converted into segments of percent identity relative to positions in the human sequence, with the resulting data then drawn as a percent identity plot (PIP) by using the program laps (49). Only segments with an identity of 50% or greater were plotted, so regions that match poorly appear blank. Gaps within an alignment appear as discontinuities between adjacent horizontal lines. We also analyzed the sequences by using the web site http://globin.cse.psu.edu/pipmaker, which utilizes another alignment program with a distinct substitution matrix and permits matching regions within the sequences to be at different positions and orientations. No significant differences were seen between the results of the two analyses.

Results

Bacterial Clone-Based Physical Mapping of the Human and Mouse CFTR/Cftr Regions.

To assemble a BAC/PAC-based contig map of the human CFTR region, mapped sequence-tagged sites (STSs) residing within an ≈2-Mb region encompassing the gene (50) (see also http://genome.nhgri.nih.gov/chr7) were used to isolate corresponding BACs and PACs. Analysis of the isolated clones and subsequent contig expansion resulted in the assembly of a >1.6-Mb contig consisting of ≈169 clones (the complete contig map is available at http://genome.nhgri.nih.gov/chr7/cftr). A minimal overlapping set of 16 clones was selected and sequenced (Fig. (Fig.11A). These efforts yielded ≈1.6 Mb of contiguous sequence. A more focused effort was applied to the mapping and sequencing of the mouse Cftr region, resulting in the assembly of a BAC contig encompassing the gene (see http://genome.nhgri.nih.gov/chr7/cftr). Three clones (from two mouse strains) were selected and sequenced (Fig. (Fig.11B), yielding ≈358 kb of contiguous sequence.

Figure 1
Sequence maps of the genomic segments encompassing the human CFTR and mouse Cftr genes. High-resolution BAC/PAC-based physical maps of the human and mouse CFTR/Cftr regions were assembled, with the complete contig maps available at http://genome.nhgri.nih.gov/chr7/cftr ...

CFTR/Cftr Gene Structures.

The availability of large blocks of homologous human and mouse sequence allowed detailed analysis and comparisons of the CFTR and Cftr genes. Human CFTR spans ≈189 kb, ≈60 kb less than initially estimated (2). Comparison of the genomic sequence with the original long-range restriction map of the region (2) suggests that this discrepancy most likely reflects the previous overestimation of the sizes of large restriction fragments by pulsed-field gel analysis. Comparison of the CFTR genomic and cDNA sequences confirms the presence of 27 exons (Fig. (Fig.22B). Each intron is flanked by the consensus GT-AG splice-site sequence (51), as previously reported (52). Although exon sizes are consistent with previous reports, intron sizes range from 599 bp (intron 22) to 28.1 kb (intron 10), notably different than earlier estimates (52).

Figure 2
Long-range organization of the greater human and mouse CFTR/Cftr regions. (A) Schematic overview of the location of genes identified in both the human and mouse genomic sequence: WNT2/Wnt2, HSA_C7orf7/MMU_Orf3, CFTR/Cftr ...

Mouse Cftr spans ≈152 kb, considerably less than its human counterpart. The repeatmasker program detected 33,142 bp of interspersed repeats within the Cftr introns, compared with 58,168 bp in CFTR introns. All of the 27 exons are highly similar between human and mouse at a sequence level (see below and http://genome.nhgri.nih.gov/chr7/cftr). In addition, the intron/exon structures of the mouse and human genes are mostly the same, with splice sites occurring at identical positions in both species (Fig. (Fig.22B). As with human CFTR, each mouse Cftr intron is flanked by the consensus GT-AG splice-site sequence. Of note, the polymorphic polyT tract located upstream of exon 9, which is implicated in the variable in-frame skipping of exon 9 during transcription (53), is absent in the mouse. Finally, although most of the introns are larger in the human gene, three mouse Cftr introns are notably larger than their human counterparts; specifically, introns 1, 4, and 12 are 27.6 vs. 24.1 kb, 6.3 vs. 3.2 kb, and 9.4 vs. 1.5 kb, respectively (in mouse vs. human).

Neighboring Genes.

The human and mouse genomic sequences were aligned and subjected to comparative analyses. To obtain a graphical overview of the resulting local alignments, we used a PIP in which each gap-free section of an alignment is represented as a horizontal line that indicates the position in the human sequence and the percent nucleotide identity (a more comprehensive summary of the PIP analysis is presented as an electronic supplement to this paper at http://genome.nhgri. nih.gov/chr7/cftr). For instance, the alignment shown in Fig. Fig.33B begins as follows (with the human sequence aligned on top of the corresponding mouse sequence):

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Figure 3
Percent identity plots (PIPs) for human and mouse genomic sequences. Percent identity plots (60) (see http://globin.cse.psu.edu/pipmaker) for four 14-kb segments are provided: (A) a region containing exons 1–3 of WNT2/ ...

The first gap-free section covers human positions 82,270–82,303 at 79% nucleotide identity whereas the second covers 82,305–82,315 at 82% identity. This gives the first two tiny horizontal lines in Fig. Fig.33B.

The region between the first and last aligned nucleotides spanned 422,311 and 356,976 bp in the human and mouse sequence, respectively, including ≈167 and ≈138 kb upstream of the CFTR and Cftr genes, respectively. Within this region, much of the sequence could be aligned. By using the approach described by Endrizzi et al. (54) to assess the degree of conservation between human and mouse DNA, 49.9% of the unique, noncoding human sequence from the CFTR region aligns with the corresponding mouse sequence. Interestingly, this makes the CFTR/Cftr region more highly conserved than 8 of the 10 regions surveyed by Endrizzi et al. (54), which ranged from 6.4 to 78.1%.

Comparative sequence analysis also revealed important insights about genes neighboring CFTR/Cftr. Complete sequence was generated for human WNT2, a gene first identified in 1987 during the search for the CF gene (55) (GenBank accession no. NM_003391). This gene contains five exons, spans ≈45 kb, and is located on the antisense strand relative to CFTR (Fig. (Fig.22 A and C). The generated mouse sequence extends through the first three exons of the mouse Wnt2 gene (Fig. (Fig.22C). The sequence similarity between the human and mouse genes is high (e.g., see positions 35.5, 41, and 43 kb in Fig. Fig.33A, which corresponds to exons 3, 2, and 1, respectively). In addition, sequences flanking exon 3 at 33.5 and 36.5 kb are highly conserved (Fig. (Fig.33A), perhaps indicating the presence of functional intronic elements (see below).

Several other regions with high human–mouse sequence conservation were identified between WNT2/Wnt2 and CFTR/Cftr (Figs. (Figs.33B and and22A). Within this interval, the genscan program (56) detected a series of putative exons on the antisense strand relative to CFTR/Cftr (Fig. (Fig.22D). There were no high-quality matches between these predicted exons and known genes or expressed sequence tags; however, blast analysis (57) of the predicted protein revealed homology to a Caenorhabditis elegans protein (GenBank accession no. U50071). Preliminary PCR-based studies have revealed that this gene (HSA_C7orf7/MMU_Orf3) is expressed in a number of human epithelial tissues, including prostate, colon, and mammary gland (J. C. Zenklusen and E.D.G., unpublished data). Interestingly, HSA_C7orf7 likely corresponds to the conserved, CpG island-containing segment immediately upstream of CFTR that was evaluated as a candidate during the successful search for the CF gene (2).

Immediately downstream of CFTR, genscan predicts a 23-exon human gene spanning ≈108 kb (Fig. (Fig.22 A and E). Some of the predicted exons have high-quality matches to available expressed sequence tags (e.g., GenBank accession nos. W24687, AA740322, and R51798), including those derived from fetal liver and infant brain. This gene (HSA_C7orf8), present on the antisense strand relative to CFTR, is predicted to encode a 1,692-aa protein. The generated mouse sequence (from clone GS196J17) contains the last nine predicted exons of the gene (MMU_Orf4). PIP analysis (see http://genome.nhgri.nih.gov/chr7/cftr) reveals strong interspecies sequence conservation for all of these exons. However, the amino acid sequence encoded by these conserved exons has no convincing match to any known protein. HSA_C7orf8 was not recognized during the successful search for the CF gene because this region was not explored (2).

Potential CFTR/Cftr Regulatory Elements.

We were particularly interested to see whether comparative sequence analysis would reveal conserved sequences that might play a role in regulating CFTR/Cftr expression. Particular attention was paid to segments within intron 1 (Fig. (Fig.33D) and ≈10 kb upstream of exon 1 (Fig. (Fig.33C). The latter includes the minimal promoter region (21, 22) as well as putative regulatory elements previously described in the human and mouse sequence (23, 24).

The most striking feature about the observed human–mouse sequence conservation in the 10 kb upstream of CFTR/Cftr exon 1 is its extreme uniformity (Fig. (Fig.33C). No segment stands out as markedly more conserved than others; thus, there are relatively few long horizontal lines. Even Fig. Fig.33B, which depicts a region with no known or hypothesized functional elements, shows greater variation (e.g., the well conserved segment at 94 kb). A simple statistic for quantifying this phenomenon is the standard deviation of the lengths of the gap-free segments (calculated in base pairs). For the four intervals featured in Fig. Fig.33 AD, the respective computed values are 52, 33, 23, and 27 bp. Comparison of the last two values indicates that the sequence conservation in intron 1 is slightly more irregular than the region immediately upstream of exon 1.

Taken together, these data indicate that there are regions of high sequence conservation present both upstream and within the CFTR/Cftr gene. Although these deserve more careful follow-up analyses, it is difficult to point to any one(s) as a notably strong candidate for being a regulatory element.

Discussion

The human genomic region containing the CFTR gene has been of long-standing interest for intensive study. Specifically, this chromosomal segment was first selected as a model for developing the paradigm of yeast artificial chromosome-based STS-content mapping, resulting in the assembly of a >2.5-Mb yeast artificial chromosome contig containing the CFTR gene (58, 59). The long-range physical map of this interval was later refined as a part of a global effort to map human chromosome 7 (50). We have now extended these studies and assembled a complete sequence map of the human CFTR region, reflected by the generation of high-accuracy sequence for a contiguous set of 16 BAC/PAC clones that together span ≈1.6 Mb and fully encompass the gene. In addition, we also assembled a similar ≈358-kb sequence map of the corresponding mouse Cftr region.

The sequence data reported here provides information about the precise genomic organization of a number of human genes, including CFTR, WNT2, and the previously unknown genes HSA_C7orf7 and HSA_C7orf8. In the case of CFTR, this data gives important insight about the gene's long-range structure. For example, the size of the genomic interval from the CFTR translational start site to the end of the cDNA is ≈189 kb, which is ≈60 kb smaller than originally reported (2). This discrepancy likely reflects the prior assessment based on measuring the sizes of large restriction fragments. Similarly, the genomic sequence data allows more precise measurement of CFTR intron sizes, thereby refining prior estimates (52). The discovery of two previously unknown genes (HSA_C7orf7 and HSA_C7orf8) flanking CFTR is also interesting. Of note, large, CF-causing genomic deletions have not been encountered to date; perhaps this reflects an important role(s) for one or both of these genes, with a heterozygous deletion resulting in a morbid phenotype.

Our studies also provide the ability to perform detailed sequence comparisons between the corresponding human and mouse regions, leading to a number of valuable insights. First, the genomic regions containing CFTR and Cftr are highly conserved throughout. Although conservation of the coding regions and the ≈10 kb of sequence upstream of exon 1 is not unexpected, the large segments of conserved sequences within the introns is intriguing. For example, PIP analysis reveals several areas of allegedly noncoding DNA with particularly high percent identity values between the human and mouse sequences (e.g., see regions at 94, 340, and 393 kb of the PIP at http://genome.nhgri.nih.gov/chr7/cftr). Similarly, the segments harboring WNT2 and Wnt2 contain several striking areas of conserved sequences within the introns flanking exon 3 (Fig. (Fig.33A).

The presence of discrete regions of highly conserved sequence within noncoding human and mouse DNA may suggest that these segments serve important functional roles (60), such as regulating gene (e.g., CFTR/Cftr) expression. It is notable that, despite major advances in understanding numerous aspects of CF biology, the mechanisms underlying the control of CFTR/Cftr expression remain poorly defined. Thus, the above conserved regions represent candidates for possible regulatory elements and deserve more careful study. Interestingly, DNase I hypersensitivity sites have been detected upstream of CFTR/Cftr (27) as well as within intron 1 (31); there is notably high human–mouse sequence conservation at the expected locations of these sites (see http://genome.nhgri.nih.gov/chr7/cftr), especially for the one in intron 1 (near position 210 kb in Fig. Fig.33D). In addition, preliminary data indicates that the conserved regions in WNT2/Wnt2 affect the expression level of a reporter construct when transfected into mammalian cells (L. Mei and R. C. Hardison, personal communication). Whether these or the other conserved sequences function to regulate WNT2/Wnt2, CFTR/Cftr, or another gene(s) in the area remains to be established.

The human–mouse genomic sequence comparison reported here highlights potential limitations of this approach for finding regulatory elements. For example, recognition sites for transcription factors may be as small as 6–8 nucleotides and, therefore, not detectable by PIP analysis. Another possibility is that, because the rate of fixation of mutations at this particular genomic locus is relatively slow, there may not have been sufficient time to accumulate sequence differences between humans and mice within nonfunctional segments; thus, some highly conserved segments may not be functionally important. Toward that end, it would be valuable to also generate the sequence of the CFTR region in more distantly related animal species (e.g., rat, cow, chicken) for comparative purposes. Alternatively, the mouse may be too divergent from human in terms of Cftr expression, relying on different regulatory sequences. Nonetheless, generating the mouse Cftr sequence will likely enhance studies of CF animal models. A number of mouse strains have been generated with defined Cftr mutations (1215); although these animals show some characteristic CF symptoms, pancreatic insufficiency does not occur, and lung disease is minimal (17). These findings may be explained by differences in Cftr expression in mouse tissues. The distribution of submucosal glands in the lung is different in mouse compared with human, and Cftr expression in the fetal lung is notably lower in the mouse (17). In addition, the mouse pancreas does not express Cftr at high levels, again different from the human (33). Thus, the differences seen between humans and mice harboring CFTR/Cftr mutations may partly reflect differences in their tissue-specific expression of the gene. The sequence data reported here may help to clarify the mechanisms underlying such differences as well as to enhance other studies investigating the regulation of CFTR/Cftr expression, such as those involving the use of CFTR/Cftr-containing yeast artificial chromosome constructs (61, 62) (P. J. Mogayzel, Jr. and M. A. Ashlock, personnel communication).

In summary, we have generated just under 2 Mb of mammalian DNA sequence from a medically important genomic region. Our efforts have provided the definitive genetic blueprint for the extensively studied CFTR gene as well as several flanking genes. The additional infrastructure provided by knowledge of the complete CFTR and Cftr sequences should contribute to myriad studies of CF genetics and pathophysiology, including ongoing efforts to develop pharmacological and gene therapy-based treatments. In addition, our comparative analyses have facilitated the detection and characterization of previously unidentified genes residing on either side of CFTR/Cftr and yielded some tantalizing clues about evolutionarily conserved sequences in the region, including those that they may serve a role in the regulation of CFTR/Cftr expression. Our studies in conjunction with an increasing number of examples in which homologous regions of human and mouse DNA have been sequenced (54, 60, 6367) are beginning to illustrate the power of comparative sequence analysis for understanding genome structure and function. Such efforts come at an exciting time in the Human Genome Project, as large amounts of human sequence are actively being generated (35) and an earlier-than-anticipated program to sequence the mouse genome has been launched (68).

Acknowledgments

We thank Drs. Jim Thomas, Bill Pavan, and Melissa Ashlock for critical review of this manuscript. We thank the numerous members of the Washington University Genome Sequencing Center and the National Institutes of Health Intramural Sequencing Center for their dedicated work in generating the sequence data reported here. These studies were supported in part by funds provided to the Washington University Genome Sequencing Center by the National Institutes of Health for human genome sequencing, Grant LM05110 to W.M. from the National Library of Medicine, and funds to E.D.G. and the National Institutes of Health Intramural Sequencing Center by the Cystic Fibrosis Foundation for mouse genome sequencing.

Abbreviations

CF
cystic fibrosis
BAC
bacterial artificial chromosome
PAC
P1-derived artificial chromosome
PIP
percent identity plot
STS
sequence-tagged site

Footnotes

Data deposition: The sequences reported in the paper have been deposited in the GenBank database (accession nos. AC002542, AC003987, AC006326, AC002465, AC003045, AC000111, AC000061, AC004240, AC002431, AC003084, AC004536, AC006389, AC007874, AC004029, AC006926, AC002529, and AF162137).

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