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Tissue-specific in vivo transcription start sites of the human and murine cystic fibrosis genes
Introduction
Results
Identification of murine Cftr transcription start sites by 5[prime] RACE
Identification of human CFTR transcription start sites by 5[prime] RACE
Identification of a novel exon of murine Cftr
Exon -1 expression is testis specific
Germ cell-specific expression of Cftr exon -1
Discussion
Materials And Methods
Sources of mouse tissues for total RNA preparation
5[prime] RACE amplification
RNase protection
In situ hybridization
DNA sequence analysis
Acknowledgements
References
Tissue-specific in vivo transcription start sites of the human and murine cystic fibrosis genes
The in vivo transcription start sites of the human cystic fibrosis transmembrane conductance regulator gene (CFTR) and its murine homologue (Cftr) have been mapped in a range of tissues using the technique of 5[prime] rapid amplification of cDNA ends (5[prime] RACE). These are the first in vivo transcription start sites for CFTR or Cftr to be reported. Distinct, tissue-specific patterns of CFTR start site usage were identified in both mouse and human. In particular, striking variation in the position of the murine Cftr transcription start site was seen along the length of the intestinal tract; different start sites being utilized in ileum and in duodenum. In humans, distinct transcription start sites are utilized in adult and foetal lungs. In addition, a novel 5[prime]-untranslated exon of murine Cftr, denoted exon -1, was identified and shown to be expressed exclusively in mouse testis. Expression of exon -1-containing Cftr transcripts was shown by mRNA in situ hybridization to be confined to the germ cells and to be regulated during spermatogenesis.
INTRODUCTION
Cystic fibrosis is a common, autosomal recessive disease characterized by intestinal blockages, exocrine pancreatic insufficiency, male infertility and progressive, degenerative lung disease (1). These diverse symptoms result from mutation of a single gene, the cystic fibrosis transmembrane conductance regulator or CFTR (2,3), the product of which functions as a cAMP-regulated, low conductance chloride channel (4).
CFTR exhibits complex patterns of tissue-specific expression being expressed at low levels in specialized epithelial cells of the intestinal crypts, airway submucosal glands, pancreatic ducts, sweat gland ducts and the male reproductive tract (5-8). In addition, the observed patterns of expression indicate that CFTR expression is regulated by hormones (9) and during development and differentiation (8,10,11). Although the patterns of regulated CFTR gene expression are well known, surprisingly little is known of the genetic elements and transcription factors that regulate the tissue-specific expression of CFTR. Both the mouse and human CFTR genes have promoters which resemble those of `housekeeping' genes (12-14), an observation that is inconsistent with the tissue and cell type specificity of CFTR expression.
Knowledge of the position of the CFTR transcription start site utilized in CFTR-expressing cells in vivo is a prerequisite to understanding the regulation of the CFTR promoter. CFTR transcription start sites previously have only been mapped in a number of immortalized cell lines (12,13,15), and it cannot be concluded that identical sites are used in vivo. In the present study, we used a 5[prime] rapid amplification of cDNA ends (5[prime] RACE) approach to map accurately transcription start sites of human CFTR, and its murine homologue Cftr, in a range of primary tissues. Distinct tissue-specific patterns of transcription start site usage were observed. The implications of these findings with regard to the regulation of CFTR expression are discussed.
RESULTS
Identification of murine Cftr transcription start sites by 5[prime] RACE
In order to identify the major in vivo Cftr transcription start sites utilized in mouse primary tissues, we performed 5[prime] RACE on total RNA isolated from adult mouse lungs, ileum, duodenum, testis, uterus, kidney, and also 16-day-old foetal mouse lungs. First strand cDNA synthesis was performed using a Cftr-specific primer (MC6R; Table 1). cDNAs were then homopolymer tailed and subjected to two successive rounds of hemi-nested PCR amplification with anchor primer complementary to the homopolymer tail and Cftr-specific nested primers (MC5R and MC2.1R; Table 1). Discrete 5[prime] RACE products were amplified from each mouse tissue (Fig. 1 shows representative experiments). These 5[prime] RACE products were excised and either sequenced directly or cloned into the vector pT7 blue (Novagen) and then sequenced. The positions of the observed transcription initiation sites, numbered relative to the A of the initiating methionine codon of the Cftr open reading frame (ORF) at position +1, are shown schematically in Figure 2 and summarized in Table 2. Single, major transcription start sites were observed for mouse adult lung (-82), kidney (-75), uterus (-72) and 16-day-old mouse foetal lung (-72). Variation in Cftr transcription start site usage was seen along the length of the mouse intestinal tract; two start sites were observed in mouse duodenum (-60 and -19), whereas two different major start sites were mapped in mouse ileum (-72 and -75) corresponding to the most abundant 5[prime] RACE products amplified from this tissue (Fig. 1). In addition to the major start sites, a number of minor transcription start sites were observed in the ileum, kidney and uterus. The minor 5[prime] RACE products from the ileum were isolated and found to correspond to transcription start sites at positions +27, -216 and -255.
Figure . The 700 bp of murine Cftr 5[prime]-flanking genomic sequence, spanning exons 1 and -1 and the intervening intron. The nucleotide sequence is numbered relative to the first base of the initiation codon of the Cftr ORF. Exons are shown in the upper case, with the first 17 amino acids of Cftr and the predicted amino acid sequence of a short upstream ORF shown in single letter code. Bent arrows indicate the relative positions of the Cftr transcription start sites utilized in mouse tissues. L, adult lung; FL, foetal lung; T, testis; I, ileum; D, duodenum; U, uterus; K, kidney.
Table 1
As the Cftr transcription start sites were mapped by a PCR-based technique, where more than one start site was observed, no absolute values can be assigned to the percentage of transcript initiating at each of the sites. However, we can conclude that the most abundant products correspond to the major transcription start sites for Cftr and that, collectively, the minor 5[prime] RACE products constitute at most 10% of Cftr transcripts in any tissue. Importantly, the two major start sites mapped in the ileum (-72 and -75) are used with approximately equal frequency while the two start sites mapped in the duodenum (-60 and -19) are used in an ~4:1 ratio, respectively. Finally, the relative intensities of 5[prime] RACE products from foetal and adult lung correspond well with the known down-regulation of Cftr expression in adult mouse lung tissue (unpublished data).
Table 2
Table 3 A significant amount of tissue-specific variation in the pattern of Cftr transcription start site usage was observed in the mouse. In order to investigate whether similar variation in CFTR transcription start site usage also occurs in human tissues, we performed 5[prime] RACE on RNA isolated from human whole small intestine, whole pancreas, whole adult lung and whole foetal lung (pooled from 29 specimens in the age range 18-28 weeks): these tissues represent the major sites of CFTR expression relevant to CF disease pathology. First strand cDNA synthesis was performed using a CFTR-specific primer (HC6R; Table 1). cDNAs were then homopolymer tailed and subjected to two successive rounds of hemi-nested PCR amplification with an anchor primer complementary to the homopolymer tail and CFTR-specific nested primers (HC5R and either HC2R or HC3R; Table 1). Discrete 5[prime] RACE PCR products were amplified from each human tissue (Fig. 3 shows representative experiments). These 5[prime] RACE products were excised from a gel, purified and sequenced directly. The positions of the transcription initiation sites, numbered relative to the A of the initiating methionine codon of the CFTR ORF at position +1, are shown schematically in Figure 4 and summarized in Table 3.
Figure
Figure The 5[prime] RACE products amplified from adult lung, foetal lung and pancreas corresponded to single transcription initiation sites at positions -69, -132 and -68 respectively, whilst the two 5[prime] RACE products amplified from small intestine corresponded to start sites at positions -69 and -114. Because of limited availability of human tissues, it has not been possible to determine whether these two alternative start sites represent variation in start site usage along the length of the human intestinal tract, as is the case with murine Cftr, or whether these alternative start sites are used in different CFTR-expressing cell types all along the length of the intestine [e.g. the epithelial cells lining the intestinal crypt versus the poorly characterized CFTR-expressing cells of the intestinal villus (16,17)]. The results of this in vivo transcription mapping study are broadly consistent with previously published data from immortalized cell lines in that the single transcription start sites utilized in adult lung (-69) and pancreas (-68) and one of the start sites identified in intestine (-69) are close to the major CFTR start site(s) mapped in the colonic epithelial adenocarcinoma cell lines T84, HT-29 and Caco-2 (12,13,15). However, none of the multiple minor start sites observed in T84, HT-29 and Caco-2 cells appear to be used in CFTR-expressing cells in vivo. The observation that distinct CFTR transcription start sites are utilized in adult (-69) and foetal lungs (-132) is intriguing given the previously reported developmental regulation of CFTR expression in the lung (10,11,18). CFTR mRNA levels are substantially higher in foetal lungs than in adult lungs, with CFTR expression declining dramatically after birth, although the precise age at which this down-regulation occurs is not known. It is possible that a switch in transcription initiation from the -132 site to the -69 site correlates with a particular stage in lung development. A correlation between the pattern of CFTR transcription start site usage and the level of CFTR mRNA expression previously has been reported in CFTR-expressing cell lines, suggesting that a mechanism of CFTR regulation operates at the level of start site selection (15). These data suggest that a similar mechanism might operate during human lung development. Figure 1 shows that a major 5[prime] RACE product of 420 bp was amplified from mouse testis. DNA sequencing revealed that this PCR product extended from Cftr exon 2 up to the splice junction with exon 1 and contained 161 bp of Cftr exon 1 and 154 bp of unknown sequence, which was named exon -1. Comparison of the sequence of the major 5[prime] RACE product from testis with the upstream genomic sequence of murine Cftr (19) confirmed the existence of exon -1 as a distinct, untranslated exon and indicated that the major transcription start site for Cftr in mouse testis is at position -549 relative to the initiating methionine codon of the Cftr ORF (Fig. 2). An intron of 285 bp separates exon -1 and exon 1, with the sequences for both splice donor and splice acceptor sites obeying the invariant GT-AG rule (20) at the exon -1/exon 1 splice junction. The position of the exon -1/exon 1 boundary in polyadenylated RNA from mouse testis was confirmed by performing an independent RT-PCR amplification on mouse testis first-strand cDNA primed with oligo(dT) (not shown). The tissue specificity of exon -1 expression was investigated using a dual probe RNase protection assay which simultaneously detected Cftr exon -1 and an internal Cftr sequence (Cftr exons 10-12; base pairs 1580-1865) in samples of mouse tissue total RNA. Cftr exon 10-12 expression was detected in all tissues examined with the exception of pancreas (Fig. 5), in agreement with previous observations that Cftr is expressed at extremely low levels in rodent pancreas (5,21). Significant levels of exon -1 expression were observed only in the testis (Fig. 3). The amounts of each protected fragment were quantitated using a Molecular Dynamics phosphoimaging cassette and Imagequant software, by comparison with the relevant standard curve of known amounts of RNA sense strands (not shown). Approximately 95% of the Cftr transcripts expressed in murine testis contain exon -1, whilst in the other mouse tissues examined <5% of Cftr transcripts contained exon -1.
Figure
Cftr exhibits a cell type-specific pattern of expression in murine testis, with levels of expression varying during the different stages of spermatogenesis (22). Cftr is expressed first in pachytene spermatocytes, with expression being maintained as these cells undergo meiotic division, to produce round spermatids, and then declining as the round spermatids elongate (22). In order to determine whether Cftr exon -1 shows the same cell type-specific pattern of expression, we examined the in vivo expression of Cftr exon -1 in mouse testis by mRNA in situ hybridization. A series of consecutive sections of adult mouse testis were hybridized with probes specific for exon -1 (Fig. 6) and for an internal region of Cftr (data not shown). Identical patterns of cell type-specific expression were observed with both probes, demonstrating that expression of the exon -1-containing Cftr transcript is regulated during stages of spermatogenesis, in agreement with our previous observations of Cftr expression in mouse testis (22).
Figure
CFTR expression in human testis has been reported to be extremely low and not associated with any particular cell type or with any particular stage of spermatogenesis-strikingly different from the rodent expression pattern (23). Nevertheless, the existence of a human equivalent of the testis-specific murine Cftr exon -1 was investigated by performing 5[prime] RACE PCR in order to identify the CFTR transcription start site utilized in adult human testis. The 5[prime] RACE procedure was repeated a total of three times, but no CFTR-specific product bands could be amplified. As a positive control, CFTR expression in human testis was confirmed by RT-PCR using an internal CFTR primer set (not shown). It was concluded, therefore, that the level of CFTR expression in human testis is extremely low but detectable by RT-PCR, but that this low level expression is below the limit of detection by 5[prime] RACE due to the latter method's inherently lower sensitivity. Thus, the position of the CFTR start site utilized in adult human testis remains unknown, and no conclusions can be drawn regarding the expression of a human equivalent of murine Cftr exon -1 in human testis. In this study, we have mapped the in vivo transcription start sites for human CFTR and its murine homologue Cftr. Using a sensitive 5[prime] RACE technique, we were able to map CFTR start sites in a number of different mouse and human tissues. These are the first in vivo CFTR transcription start sites to be mapped for any species. In the case of murine Cftr, the use of distinct, tissue-specific transcription start sites leads to the production of Cftr transcripts which differ in the length of their 5[prime]-untranslated regions (5[prime] UTRs). The observation that distinct transcription start sites are used in different tissues suggests that the tissue-specific regulation of Cftr expression may, in part, operate at the level of transcription start site selection. Analysis of the primary sequence of the 5[prime]-flanking region of murine Cftr provides some clues as to how tissue-specific start site selection might be achieved. No sequence likely to correspond to a TATA box element could be found upstream of any of the in vivo transcription start sites identified for murine Cftr (Fig. 2). However, Table 4 illustrates that sequences surrounding three of the Cftr transcription start sites, the -82 site utilized in lung and the -60 and -19 sites utilized in duodenum, are similar to the consensus sequence for the mammalian initiator (Inr) element, PyPyA(+1)N(T/A)PyPy, that has been suggested to maintain the fidelity of transcription initiation in promoters devoid of TATA boxes (24). It is likely, therefore, that an Inr-dependent mechanism is responsible for directing the formation of a transcription pre-initiation complex in these tissues.
Table 4
Tissue-specific variation in the position of the CFTR start site was also observed in human tissues. Most notably, distinct start sites appear to be used in adult and in foetal lungs. However, the exact position of the CFTR transcription start site relative to the translation initiation codon does not appear to be conserved between equivalent tissues of mice and humans, suggesting that the precise mechanisms responsible for correct start site selection are not conserved cross-species. This is perhaps not surprising given the apparent lack of primary nucleic acid sequence homology between the mouse and human proximal promoter regions (19,25). The identification of a testis-specific, untranslated exon of murine Cftr (exon -1) implies that Cftr expression in this tissue is under the control of a testis-specific alternative promoter. Murine Cftr falls into a group of genes demonstrating cell type-specific expression in the testis, with expression being regulated during spermatogenesis (22). Other genes in this group include many whose protein products are thought to play a particular role in the complex process of spermatogenesis (reviewed in 26), and a potential role for the Cftr chloride channel during spermatogenesis has been suggested (22). Genes such as Cftr that are expressed both post-meiotically in the testis and in other tissues often produce testis-specific transcripts, generated by the use of testis-specific promoters, altered polyadenylation and/or tissue-specific alternative exon splicing (26). We have shown that the testis-specific Cftr transcript is generated through the use of an alternative promoter and alternative splicing in the 5[prime] UTR, to include exon -1 at the extreme 5[prime] end of the transcript. The vast majority (95%) of Cftr transcripts in mouse testis contain this additional untranslated exon, implying an important role for exon -1 with regard to either the function of Cftr in testis or the regulation of Cftr expression in this tissue. The latter alternative would appear to be more plausible as a number of genes expressed post-meiotically in rodent testis exhibit negative translational regulation mediated by the binding of regulatory proteins to specific 5[prime] UTR sequences (reviewed in 27). One of the families of germ cell-specific proteins proposed to mediate translational regulation in the testis are the Y-box proteins (28). Y-box proteins may function both as DNA-binding transcription factors, which bind to specific, cis-acting Y-box promoter elements, and as RNA-binding proteins, which mediate translational regulation via mRNA masking in vertebrate gametes (29). In this context, it is interesting to note that a Y-box consensus sequence, previously suggested to act as a positive regulator of transcription in the murine Cftr promoter (14), is contained within exon -1 and thus will be present exclusively in the 5[prime] UTR of testis-specific Cftr transcripts. Another feature of the murine Cftr 5[prime]-flanking region suggests that translational regulation may be a more general mechanism controlling Cftr expression. An additional AUG codon is present at position -58, upstream of the Cftr ORF (Fig. 2). This upstream AUG precedes a short upstream ORF (uORF) that, if recognized by the translation machinery, would produce a small peptide of five amino acids in length. This uORF will be present in the 5[prime] UTR of Cftr transcripts initiating upstream of position -58, including the major Cftr transcripts expressed in lung, foetal lung, ileum, kidney, uterus and testis. The upstream AUG occurs in a weak Kozak context, with neither the critical purine at position -3 nor the G at position +4 being present (30), whereas the AUG of the Cftr ORF occurs in a much stronger Kozak context (31). The scanning model predicts that translation will usually initiate at the first AUG in a favourable Kozak context (30). Nevertheless, interaction between the translation machinery and the upstream AUG may have the effect of reducing the translation efficiency of the Cftr ORF. The presence of the short uORF would therefore be predicted to reduce the translation efficiency of the major Cftr transcripts expressed in lungs, ileum and testis but not those expressed in duodenum, as the uORF is absent from the 5[prime] UTR of the duodenum transcript initiating at position -19 and is too close to the m7G cap site to exert an effect in the transcript initiating at position -60 (31). Thus, by the use of distinct transcription start sites, the expression of Cftr might also be modulated at the translational level. The identification of the major Cftr transcription start sites utilized in vivo is an essential prerequisite to understanding the function of the Cftr promoter. It will now be possible to define the promoter elements, transcription factors and mechanisms directing the complex patterns of in vivo Cftr expression. All human RNA samples were purchased from Clontech. Mouse tissues were pooled from six male and six female adult mice, all of strain MF1. Foetal lung tissue was pooled from three 16-day-old foetuses removed from a single timed pregnant female. Total RNA was isolated using a guanidinium thiocyanate/acid phenol-chloroform extraction method (32). 5[prime] RACE was performed using an adaptation of the method of Frohman (33). First-strand cDNA synthesis was performed on 2 µg of total RNA using 10 pmol of primer MC6R (Table 1) and 200 U of superscript II reverse transcriptase (BRL). RNA template strands were digested with 2 U of RNase H (BRL), and first-strand cDNAs were purified using QIAquik PCR purification columns (QIAGEN) and homopolymer tailed using 0.2 mM dCTP and 20 U of terminal transferase (Promega). cDNAs were again purified using QIAquik columns and then subjected to two rounds of hemi-nested PCR amplifications using primers MC5R and MC2.1R and a 5[prime] RACE anchor primer specific for the homopolymer tail (purchased from BRL), with 10 pmol of each primer and 2.5 U of AmpliTaq Gold polymerase (Perkin Elmer) per PCR reaction. 5[prime] RACE products were either sequenced directly or ligated into the vector pT7 blue and the recombinant plasmids sequenced. Sequencing reactions were performed using an AmpliTaq Dye Terminator Cycle Sequencing Kit (Applied Biosystems) and analysed on an ABI prism 377 automated sequencer. Dual probe RNase protection assays were performed using the RPA II system from Ambion. Two probes were used to detect different regions of a single Cftr mRNA: the first, CFTR 10-12, corresponded to an internal region of Cftr spanning exons 10-12 [base pairs 1580-1865 of the Cftr cDNA (19)] in the vector Bluescript KS (Stratagene); the second, CFTR -1, contained 150 bp of mouse Cftr exon -1 and 50 bp of RACE anchor primer sequence, amplified from the mouse testis major 5[prime] RACE product by PCR using anchor primer (Gibco-BRL) and the primer MC -1R (5[prime]-ACCGTGGCCAATCTGCGGGACTTAGAACCC), and cloned into Bluescript SK (Stratagene). The Bluescript vectors contain opposing T3 and T7 promoters to allow the synthesis of both 32P-labelled antisense RNA probes and control unlabelled sense strand RNAs by in vitro transcription. Samples (20 µg) of mouse tissue total RNA were hybridized overnight with equal, excess amounts (0.5-1×105 c.p.m.) of 32P-labelled, antisense CFTR -1 and CFTR 10-12 probes. Following RNase digestion and precipitation, protected products were resolved on 6% denaturing polyacrylamide gels and visualized by exposure to Kodak X-omat AR film. In situ hybridization was performed essentially as described previously (17). Probes specific for two regions of mouse Cftr were used for in situ localization of Cftr mRNA: the first, CFTR 10-13, corresponded to a fragment of Cftr exons 10-13 [bases 1580-2108 of the Cftr cDNA (19)] in the vector Bluescript KS; the second, CFTR -1 was the same as that used for RNase protection (above). Both probes were cloned in Bluescript vectors containing opposing T3 and T7 promoters for the generation of [35S]UTP-labelled sense and antisense strand RNA probes by in vitro transcription. Cryostat tissue sections (10 µm) were hybridized overnight with 35S-labelled single-stranded RNA probes in 50% formamide at 55-60°C. Sections were then digested with RNase A to remove excess probe and other single-stranded RNA, and washed at a stringency of 0.1× SSC at 60°C. Slides were dipped in Kodak NTB-2 emulsion, exposed for 2 weeks at 4°C, developed and counterstained with haematoxylin and eosin. Sections were photographed under brightfield and darkfield illumination using a Nikon Optiphot-2 microscope equipped with a Nikon UFX-DX automatic camera. Multiple tissue sections were hybridized with either sense or antisense probes for each of the two regions of Cftr and, in all cases, results were seen to be consistent for each probe. Searches of the GenBank database to look for sequence homology were performed using FastA from the GCG program. N.L.W. was supported by a Wellcome Trust Prize Studentship and A.E.O.T. by a Beit Memorial Research Fellowship. Additional support for this work came from the Cystic Fibrosis Research Trust, ICRF, EU, Henry Smith Foundation and the University of Oxford. C.F.H. is a Howard Hughes International Research Scholar.
Primer
Exon
Sequence
HC6R
6
5[prime]-CCAGCTCTCTGATCTCTGTACTTCATCATC
HC5R
5
5[prime]-TTATCTAGAACACGGCTTGACAGC
HC2R
2
5[prime]-TTTGGTATATGTCTGACAATTCCAGGCGCT
HC3R
3
5[prime]-ACATCGCCGAAGGGCATTAATGAGTTTAGG
MC6R
6
5[prime]-GCAGCTCTTTGATCTCTGTACTTCACCATG
MC5R
5
5[prime]-GTTCAGGTTGTTGGAAAGAAGACTAACAA
MC2.1R
2
5[prime]-ACAAGTGGTCAGCTGAATCAGCAGAAGGGG
Tissue
Major start site(s)
Minor start site(s)
Testis
-549
Lung
-82
Foetal lung
-72
Duodenum
-19, -60
Ileum
-75, -72
+27, -216, -255
Kidney
-75
Uterus
-72
Tissue
Position of start site
Adult lung
-69
Foetal lung
-132
Intestine
-69
Intestine
-114
Pancreas
-68
Identification of human CFTR transcription start sites by 5[prime] RACE
Identification of a novel exon of murine Cftr
Exon -1 expression is testis specific
Germ cell-specific expression of Cftr exon -1
DISCUSSION
Position of start site
Sequence context
Inr consensus
PyPyA(+1)N(T/A)PyPy
-549
AGAAGGA
-82
GCATTGA
-75
CCTGGTC
-72
GGTCCTG
-60
CCAGATG
-19
TCATTGC
MATERIALS AND METHODS
Sources of mouse tissues for total RNA preparation
5[prime] RACE amplification
RNase protection
In situ hybridization
DNA sequence analysis
ACKNOWLEDGEMENTS
REFERENCES
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