Cloning of the Multipartite Promoter of the Sodium-Calcium Exchanger Gene NCX1 and Characterization of Its Activity in Vascular Smooth Muscle Cells*

Timo SchellerDagger , Alexander KraevDagger , Sven Skinner, and Ernesto Carafoli§

From the Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), 8092 Zürich, Switzerland and § Department of Biochemistry, University of Padova, 35121 Padova, Italy

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The sodium-calcium exchange activity is mediated by proteins encoded in a small gene family, of which the gene NCX1 is ubiquitously expressed in mammalian tissues. In this study, the multipartite promoter of this gene was analyzed in the human and rat genomes by means of DNA cloning, reverse transcriptase-polymerase chain reaction, and transient transfection of fusion constructs with the firefly luciferase gene into cultured rat aortic smooth muscle cells. The gene-proximal promoter, located 30 kilobase pairs (kb) away from the first coding exon 2, has features of a GC-rich housekeeping promoter and is apparently always active; in specific tissues, however, it is augmented by one or two additional promoters, located either within 1.5 kb upstream of it, or 35 kb upstream. The gene proximal promoter shows the highest activity in aortic smooth muscle cells. In mammalian species transcripts from all three promoters undergo splicing via an intermediate, containing two noncoding exons, of which the downstream one is normally not present in the terminal splicing product.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Sodium/calcium exchange was first discovered in invertebrates (1), but received much attention in the last decade because of its implied role in maintaining calcium homeostasis in mammalian tissues, particularly heart, brain, and kidney. Two generic activities have been described in animal tissues, one exchanges only sodium and calcium ions, the other also co-transports a potassium ion along with a calcium ion (for a review, see Nicoll et al. (2)). Following cDNA cloning, coding for the protein of the first type, a "cardiac exchanger" (3), structurally related cDNAs were discovered in numerous animal species, from nematodes to man (see Kraev et al. (4) and references therein). It was shown that in mammals this protein is encoded by a small multigene family (5), with one gene, NCX1, being nearly ubiquitously expressed and featuring complex tissue-specific alternative splicing, the other two genes having relatively restricted tissue distribution of expression and limited splicing options (6). Although it was readily obvious from data accumulated in sequence data bases, a study in rat (7) first conclusively demonstrated that NCX1 transcripts in different tissues have at least three different 5'-untranslated leaders, which are spliced to a common core containing the protein coding sequence. A recent publication linked these leaders to three "heart," "kidney," and "brain" alternative promoters (8) in the cat NCX1 gene. While most eukaryotic genes are controlled by a complex array of cis-regulatory elements modulating transcriptional activity at a single site, a substantial number of genes have recently been found to utilize multiple promoters (9-14). In some cases, such an organization was proposed to underlie downstream translational regulation mechanisms (15). Here, we present data on the cloning and functional characterization of the colinear multipartite promoters of the rat and human NCX1 genes. The three transcription start sites of the gene, spread over 35 kb,1 apparently act together in a specific quantitative pattern, in which one ubiquitous promoter drives a low level of unspecific expression, augmented in specific tissues by two additional, spatially distinct promoters. Transcript initiated from any of the three promoters undergoes splicing via a transient intermediate, containing two noncoding exons, of which the downstream one is removed from the terminal splicing product. An alignment of rat and human three-exon cluster that includes two promoter regions has also revealed unusually conserved areas downstream of the alternative first exons, a feature previously observed in the the calcium pump PMCA1 gene (16).

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Genomic Library Screening, DNA Isolation, and Sequence Analysis-- Bacterial strains and rat genomic library in lambda  FIX II were purchased from Stratagene AG (Basel, Switzerland). Methods for nucleic acid isolation and genomic library screening have been described previously (4). DNA sequencing was performed on an automated sequencer Li-Cor 4000L (Li-Cor Inc., Lincoln, NE), using a modified thermostable polymerase dCode (a generous gift of Dr. D. Clark, DNamp Ltd., Farnborough Hunts, UK). The sequencing data were processed by a sequence assembly program Sequencer (Gene Codes Corporation, Ann Arbor, MI) and further analyzed by a GCG package version 8.0 (17). BLAST (18) searches of DNA and protein sequence data bases were run on a WWW server of the Swiss Cancer Institute (ISREC, Epalinges s/Lausanne).

Isolation and Analysis of YAC and BAC Clones-- Human YAC clone 809_b_6, containing the entire NCX1 gene, was described previously (4). Human BAC clones, overlapping STS DS2329 (accession no. X92368) were received from Center d'Etude du Polymorphisme Humain (Paris, France). BAC DNA was isolated on a Qiagen tip-100 column, following a protocol provided by the manufacturer for low copy number plasmids, except that the elution buffer was prewarmed to 65 °C. BAC DNA was mapped by essentially standard procedures (19), except that digests were performed with 100-ng DNA portions, and gels were stained with SYBR Green I (Molecular Probes BV, The Netherlands). Gels were blotted to positively charged nylon membranes and hybridized to PCR fragments, labeled with digoxigenin-11-dUTP (Boehringer Mannheim AG, Rotkreuz, Switzerland). Blots were visualized by a digoxigenin-chemiluminescent detection kit (Boehringer) and exposed to Fuji RX films. Fragments of interest were subcloned into plasmid vectors, using standard procedures (19).

RT-PCR Analysis-- Whole cells RNA was prepared from frozen rat tissues or from fresh pellets of cultured rat cells using Trizol reagent (Life Technologies, Inc.). Poly(A)+ RNA was prepared with an Oligotex kit (Qiagen AG, Basel, Switzerland). Human brain and heart poly(A)+ RNA and human kidney poly(A)+ RNA were purchased from CLONTECH Laboratories (Palo Alto, CA). In the "standard protocol" first strand cDNA was prepared either from 10-20 µg of total RNA or from 0.5-1 µg poly(A)+ RNA using a cDNA synthesis kit (Pharmacia Biotech, Duebendorf, Switzerland) and amplified with AmpliTaq Gold (Perkin-Elmer Int., Rotkreuz Branch, Rotkreuz, Switzerland). In an "enhanced protocol" cDNA was synthesized with Expand reverse transcriptase (Boehringer), and the cDNA was immediately amplified (without purification or freezing) either as above or with Expand thermostable polymerase mixture. Typically, 10% of a cDNA synthesis reaction was amplified in a 50-µl PCR reaction for 40 cycles, using 0.3 µM exon 2-specific primer and one of several primers, specific for alternative first exons. The primer sequences are available on request. PCR products were resolved on 2% agarose gels and stained with SYBR Green I or blotted to a positively charged nylon membrane, as described above. Gel images were recorded with a Minolta RD-175 digital camera and processed with Adobe Photoshop 3.0 software. Blot images were recorded on a PhosphorImager model 425 and processed with ImageQuant 1.1 software (Molecular Dynamics, Sunnyvale, CA).

RACE Analysis-- cDNA, synthesized from 1 µg of poly(A)+ RNA, was treated with 0.2 unit of RNase H (Pharmacia Biotech) for 15 min at 37 °C and purified by ethanol precipitation in the presence of 20 µg of glycogen. The single-stranded cDNA was treated with terminal transferase (Boehringer) for 15 min at 37 °C, essentially as suggested by the manufacturer, except that dATP was used at 50 µM. The reaction was again purified by ethanol precipitation, and the entire pellet was taken into a 50-µl PCR reaction, made up with a 1 µM exon 2-specific primer, 0.01 µM Not-dT20 primer (included in the Pharmacia cDNA synthesis kit), and 1 µM anchor primer, specific for the 5'-terminal part of the Not-dT20 primer. The reaction was subjected to the following cycling program: five times (94 °C for 30 s, 40 °C for 1 min, 72 °C for 2 min), then 35 times (94 °C for 30 s, 63 °C for 30 s, 72 °C for 30 s), using Expand thermostable polymerase mixture (Boehringer). The resultant product was cloned into a pUCBM20 vector (Boehringer), and clones were screened with an exon 2-specific probe. Positive clones were sequenced, using an exon 2-specific dye primer.

Cell Culture-- Vascular smooth muscle cells (VSMC) were isolated from rat aorta (female, Wistar-Kyoto strain, 6-8 weeks old) and recultured several times essentially as described previously (20) on Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 units/ml penicillin/streptomycin, and 1% nonessential amino acids. The cells were passaged weekly and used between passages 5 and 15.

Production of Promoter Constructs and Transfection Analysis-- Relevant rat DNA fragments covering the three 5'-flanking regions of NCX1 leader-encoding exons were amplified from lambda  clones or plasmid subclones and inserted into a reporter vector pGL3 (Promega, Madison, WI) upstream of the firefly luc gene. DNA was prepared from these constructs, using Qiagen tip-100 columns and quantitated by fluorimetry (minifluorometer TKO100, Hoefer Instruments, San Francisco, CA). A second normalizing plasmid pRL-TK (Promega), containing the Renilla luc gene under control of the thymidine kinase promoter was prepared in a similar way. A mixture of a promoter construct plasmid and a normalizer plasmid (weight ratio of 20:1) was introduced into cultured aortic cells, prepared as above, using a lipotransfection reagent Tfx (Promega) according to the manufacturer's instructions. Transfections were made as triplicates in a 24-well plate. The SV40 promoter-luciferase plasmid pGL3 served as a positive control, and the same plasmid with the SV40 promoter deleted served as a negative control. Following transfection the cells were incubated in serum-containing medium for about 48 h. Cell extracts were prepared using a lysis buffer supplied with the Dual-Luciferase kit (Promega). Luciferase activities in extracts were assayed according to the manufacturer's instructions and recorded on a Berthold luminometer model LB 9507. Values of the firefly luciferase activities were normalized against the Renilla luciferase activity and averaged to produce a value that was taken as a measure of a construct's activity. The dexamethasone (Sigma Chemie, Buchs, Switzerland) treatment was done essentially as described elsewhere (21).

Mapping of the Transcript Start Point in Transfected Cells by RNase Protection-- A riboprobe was designed that contained a complete exon 1c and 124 bases upstream of it (see Fig. 2). To synthesize the probe, a T7 promoter sequence was incorporated into the respective fragment by PCR, and the probe was subsequently synthesized, using a Maxi-Script kit (Ambion Inc., Austin, TX) and [alpha -32P]CTP (Amersham). The probe was gel-purified, and dilutions of it were used to analyze samples of transfected and nontransfected VSMC with a Direct Protect Lysate RPA kit (Ambion). The protected products were resolved by electrophoresis on 6% Sequagel (National Diagnostics, Atlanta, GA), and the gel was dried and exposed to a phosphorimaging screen for 2 days.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Cloning of the NCX1 Putative Promoter Region from Rat Genomic DNA-- Previous studies in rat (7) have characterized four alternative 5'-untranslated regions from various tissues, of which three (Br1, Br2, and Kc1) apparently corresponded to distinct sites in the genome. Initially, these sequences were used to design primers to retrieve short (100-300 bp) fragments from rat genomic DNA using PCR. The fragments were cloned into an M13 vector, and clones were subsequently used to prepare high specific activity probes by limited second strand synthesis in the presence of radiolabeled dNTPs (22). These probes were used to screen a rat genomic library in lambda  FIXII. Isolated clones were checked for cross-hybridization, and it was found that those hybridizing to the Kc1 5'-leader (accession no. U04934) also hybridized to the Br1 5'-leader (U04935). The subcloning of relevant areas into plasmid vectors was followed by sequence analysis. About 4 kb of DNA covering the areas, hybridizing to the Kc1 and Br1 5'-leaders, were sequenced, as well as about 2 kb covering the Br2 5'-leader (accession no. U04936). All three 5'-leader sequences were found to represent distinct exons, and the 3'-exon/intron junction sequences complied with the standard consensus sequence. Since lambda  clones containing Kc1/Br1 and Br2 exons did not overlap, the distance between them was assumed to be the same as that determined by long range mapping on the human YAC clone (see below).

Cloning of the NCX1 Putative Promoter Region from Human Genomic DNA-- A human exon similar to the rat Br1 exon had previously been mapped about 30 kb upstream of the first "coding" exon 2, in proximity of the STS D2S2328 (4). This allowed the isolation of corresponding BAC clones from a CEPH human genomic library in BeloBACII. Two independent clones with 140-kb inserts were studied in detail, using standard mapping methods, and a 6-kb EcoRI fragment, hybridizing to the human exon 1 (accession no. X92368X92368), was subsequently cloned into a plasmid vector and completely sequenced. BamHI sites were mapped within the 70-kb XhoI fragment, covering this exon and exon 2 (Fig. 1), using a probe hybridizing to the area proximal to the conserved XhoI site in exon 2. A portion of the alternative exon coding for the 5'-leader, similar to the rat Br2 exon, was isolated by RACE (see below) from human heart mRNA and mapped on the previously characterized YAC clone 809_b_6, essentially as described previously (4). The data obtained in the cloning and mapping experiments are summarized in Fig. 1.


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Fig. 1.   Physical map of the human genomic DNA, covering the multipartite promoter of the NCX1 gene. The scale in kilobases is shown above the map. Alternative first exons are shown as thick vertical lines, numbered 1a through 1e, and exon 2 is shown as a closed rectangle. The position of the human STS D2S2328, as well as the two other GT/AC dinucleotide repeat sequences are shown by closed squares. The extent of the genomic sequence cloned in the BAC and the YAC clone is shown by thick horizontal lines below the map. The nonredundant rat clones, isolated from a genomic library in lambda  vector, are aligned with the human genomic DNA map. Filled circles indicate repetitive sequences, one of which was identified as a LINE element. B, BamHI; C, ClaI; and X, XhoI.

Identification of Alternative First Exons in Rat and Human Genomic Sequences-- Genomic sequences around the Br1 exon in the rat gene and around the human "exon 1" were determined from the subcloned fragments of a lambda  clone and a BAC clone, respectively. These sequences were analyzed for similarity to each other and to other NCX1 5'-leaders found in nucleotide sequence data bases. Since the similarity of the Br1 exon to human exon 1 was 65-70%, only regions having this degree of similarity among mammalian species were considered meaningful.

Several regions of similarity to other mammalian genomic and cDNA sequences were found in the human genomic sequence (Fig. 2): a region of about 240 bp, previously defined as "exon 1," similar to the rat Br1 5'-leader; a region of about 120 bp similar to a 5'-leader (accession no. L35846) isolated from feline heart; and a region of about 200 bp similar to the Kc1 5'-leader sequence, isolated from rat kidney (accession no. U04934U04934), and at the same time similar to clone p17, isolated from bovine heart (accession no. L06438). Close examination of genomic DNA sequences within a few bases around the left border of similar regions revealed the presence of a plausible exon-intron consensus sequence. It was hypothesized that the regions of similarity, some of which have been identified as alternative 5'-untranslated regions in different mammalian tissues, represented alternative exons spliced to the common core encoded in exon 2, located 30 kb downstream (Fig. 1). However, although the position of the clustered three putative first exons is very similar in rat and human genomic DNA (Figs. 1 and 2), the pattern of their expression in different tissues (see below and also under "Discussion") does not seem to agree with the simple model recently presented in Barnes et al. (8). Since the previously used terms "brain-," "heart-," and "kidney"-specific exons appear confusing, they were replaced by a neutral designation "exon 1a, 1b, 1c," and so forth. Specifically, human exon 1/rat Br1 was designated as "exon 1a," the exon similar to the bovine heart/rat kidney 5'-leader as "exon 1c," and the feline kidney exon (also found in rat kidney in this work, see below) as "exon 1b" (Fig. 2). The region of similarity to rat Br2 5'-leader (accession no. U04936U04936) was not found in the vicinity of exons 1a-1c. However, a human heart RACE product having the structure shown in Fig. 2 was isolated in this work; it apparently contained an incomplete exon 1c intervening between exon 2 and a new sequence, similar to the rat exon Br2. The new sequence, designated exon 1d (although it was not actually sequenced on human genomic DNA), was mapped ~35 kb upstream of exons 1a-1c on the human YAC clone 809_b_6 (Fig. 1).


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Fig. 2.   Schematic representation of the splicing options of the exons coding for the NCX1 5'-untranslated leaders in mammalian species. The genome structure in rat and man is shown at the top. Exons are shown as boxes, introns as thin connecting lines. Primers used to detect exon expression are shown by arrows above the exons; exon and intron length is shown below. In addition to the data obtained in this work, the following EMBL/GenBankTM nucleotide data base entries were used to compile the figure: U04936, U04934, U04935, X68812, X68813 (rat), L06438 (calf), L35846, and U67072-U67075 (cat). Suggested terminal splicing products are shaded.

Expression of Alternative 5'-Leaders in Tissues and Cell Lines-- It was initially assumed that regions of similarity between rat and human DNA around exon 1 represented potential alternative exons and primers were therefore designed to test if this region was indeed found spliced to the common core of exon 2 in various human and rat tissues (Table I). The approach was adopted based on experience that RACE analysis of rare transcripts, such as NCX1 transcript, is conducive to preferential isolation of 5'-end RNA sequences that are more easily amplified by PCR instead of providing a faithful representation of alternative 5'-ends from the original RNA pool. It was also prompted by the apparent inconsistency of the finding that, while there was no sequence similarity between cat and rat "kidney-specific" exons, regions of similarity to both could still be mapped upstream of the obviously analogous exon 1a in rat and human DNA. Finally, it was necessary to confirm that the human alternative first exons, initially identified by sequence similarity to the corresponding rat exons, were indeed found in the NCX1 transcripts in human tissues. In the first round of experiments a primer complementary to exon 2 was positioned relatively close to the splicing junction (Fig. 2, primer 2-1), so that a relatively small (<1 kb) product could be amplified. To counteract possible differences in the amplification efficiency of relatively short fragments, sharing the same end, an alternative primer (Fig. 2, 2-2) was used in later experiments, which produced ~2-kb fragments, containing the entire exon 2, so that addition of alternative first exons resulted in small size differences between the PCR products.

                              
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Table I
Composition of alternative 5'-leaders and respective RT-PCR product sizes

The results of a rat tissue screening are presented in Fig. 3. Clearly, exon 1a was ubiquitously expressed in all tissues derived from live rat (albeit to a different extent), as expected from published data (7). In contrast, most immortalized or tumor-derived cell lines from brain failed to show expression of any of the 5'-leaders and thus failed to express the NCX1 gene (not shown). Finally, exon 1d was expressed in heart and brain, as predicted by the published data. A more complex situation was observed in the expression of exons 1c and 1b. The use of the promoter region upstream of the tandem exon 1c-1b was implied from the results of the transfection studies (see "Discussion"), as well as from the results of the RT-PCR experiments on human tissues. However, when total RNA was used in RT-PCR experiments according to the "standard protocol" (see "Materials and Methods"), expression of this exon was detected in rat kidney but not in heart or smooth muscle. In contrast, with the "enhanced protocol" a prominent product was detected (Fig. 3) in rat heart and smooth muscle with some of the primers specific for this exon. With the same protocol, both exons 1c and 1b were found in rat kidney mRNA, using exon 1c- or exon 1b-specific primers (Fig. 3B), although the product containing exon 1c was clearly dominant. The conflict between the data on cat and rat kidney-specific exons, evident from the published data, was thus resolved, since both were found spliced to exon 2 in rat kidney (see also "Discussion").


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Fig. 3.   RT-PCR screening of rat tissues for the expression of the alternative first exons. Experiments were done with a splicing site proximal exon 2-specific primer (A) and with a splicing site distal exon 2-specific primer (B). Sense primers, specific for alternative first exons, are indicated above each track of the gel. Southern analysis of RT-PCR products was done with an exon 2-specific oligonucleotide probe. The positions of the length marker bands are indicated at the left side of each panel.


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Fig. 4.   Erratic splicing of NCX1 noncoding exons in rat and human tissues. Negative images of agarose gels, stained with ethidium bromide. A, splicing products of the 1a-driven transcript in human heart. B, splicing products, isolated from rat heart and brain 2 h post mortem. Sense primers, specific for alternative first exons, are indicated above each track.

Erratic Splicing of Alternative 5'-Leaders in Mammalian Tissues-- Unexpected results were obtained in the RT-PCR experiment on human heart cDNA when an exon 2-specific primer, positioned about 1.5 kb away from the splicing junction, was used with an exon 1a-specific primer. Two closely sized products were observed (Fig. 4A). The larger was found to contain an additional sequence inserted between exon 1a and exon 2, this additional sequence representing yet another exon (designated 1e in Fig. 2) bound by standard consensus sequences and located 7 kb downstream of exon 1a. The shorter product was a fusion of exon 1a and exon 2 without intervening DNA stretches. Similarly, a product containing exon 1d and incomplete exon 1c together was isolated from human heart and kidney. Since such splicing products were never detected in rat tissues, it was suggested that a two-exon-containing leader represents a transient splicing intermediate, which accumulates post mortem. To check this possibility, RNA was isolated from a rat that was left at room temperature for 2 h after decapitation. Indeed, a specific pattern of leader expression, observed in a quickly frozen tissue, was largely lost, so that every possible exon combination now could be found in brain and heart (Figs. 3 and 4B).

Analysis of Promoter Activity by Reporter Gene Expression-- Sequences upstream of the rat exons 1a, 1c, and 1d were cloned into reporter vectors, using PCR and/or oligonucleotide adaptors. Constructs containing various fragments upstream of the firefly luciferase gene were contransfected into rat VSMC together with a normalizing plasmid, containing the Renilla luciferase gene under the control of the thymidine kinase promoter. The results of these experiments (Fig. 5) supported the existence of three separate promoters upstream of exons 1a, 1c, and 1d. Since all three constructs were routinely assayed in the same series of transfections, it could be concluded that the 1.6-kb fragment upstream of 1a had the highest absolute activity in rat VSMC, followed by the 1c upstream fragment, which was 2-fold less active. The lowest, albeit significant, activity was that of the 1d upstream fragment. The transfection of deletion constructs showed that a minimal level of activity was retained by a 312-bp 1a upstream fragment. These results are partially at variance with the RT-PCR data, which showed only a small amount of endogenous 1c-driven transcript in these cells (Fig. 3A). Mapping of the start point of this transcript showed two low intensity bands, which could not be adequately interpreted. However, upon transfection of VSMC with p1c DNA RNase protection experiments repeatedly revealed a strong band, which did not disappear after DNase I treatment (not shown). Apparently, transcription in this construct was initiated at a site located >110 bases upstream of exon 1c, a finding that implies the existence of a "cryptic" promoter, obviously not used in VSMC in vivo.


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Fig. 5.   Promoter activity of the NCX1 gene regions upstream of exons 1a, 1c, and 1d in rat VSMC. p1c contained 1.5 kb upstream of the exon 1c; p1d contained 460 bp upstream of the exon 1d; pPst, pBgl, pApo, and pSpe contained various fragments upstream of the exon 1a, as indicated by the negative numbers. Positive numbers indicate the portions of the exons included in the constructs. pGL3 is a positive control plasmid, containing the SV40 promoter; pLuc0 was made from pGL3 by deleting the SV40 promoter. Values are ± S.E. of three independent transfections relative to the value for pGL3.

The dexamethasone treatment required the use of a serum-free medium, and the transfection experiment had to be modified to include the media-accommodation step. At the concentration previously used for immortalized cardiac myocytes (21) the addition of dexamethasone to VSMC failed to yield any conclusive data.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Physical Map and Tissue-specific Expression of the NCX1 Exons Coding for the 5'-Untranslated Leaders-- Our previous work had found the "ubiquitous" 5'-untranslated leader of the NCX1 mRNA encoded in exon 1 located about 30 kb away from the remainder of the gene (4). This leader, termed "Br1" by others, is consistently found in rodent, feline, and human brain cDNA clones (see Fig. 2). The analysis of the data on available mammalian NCX1 5'-leaders, together with the results of genomic mapping experiments, has allowed to unambiguously assign them to five distinct exons. The physical map of the human genomic DNA, constructed in this work from the BAC and YAC clone analysis, shows that alternative first exons are spread over a large distance, although three of them are clustered in an 1.5-kb stretch (Figs. 1 and 2). The map, together with the RT-PCR studies, integrates all earlier observations made in rat and cat (7, 23) with the few changes outlined below. These changes affect both the definition of tissue specificity of alternative promoters, as well as the composition of alternative 5'-leaders.

An earlier study implied that different 5'-leaders were spliced to the common core in a tissue-specific way, so that a single leader dominated in NCX1 transcripts found in specific tissues. Consequently, individual 5'-leaders and the presumed exons encoding them were called heart-, brain-, and kidney-specific (8). The main difference between the model of the multipartite promoter presented here and those implied from earlier reports is that no single transcription start can be called "tissue-specific," although expression of exon 1c clearly dominates in kidney and exon 1d in heart (Fig. 3). The alternatively spliced NCX1 transcript in heart is also known to use a unique exon 7 (or E), and only one of the mutually exclusive exons 3/4, in the protein-coding portion of the mRNA (24). The promoter found in front of the "ubiquitous" exon 1a is apparently always active, while in specific tissues one or two auxilliary promoters augment it; however, the relative activity of these auxilliary promoters varies in specific tissues (Figs. 3 and 4). Hence, the extrapolation of the situation prevailing in heart or kidney to other tissues would be, in the best of cases, a simplification. In the case of smooth muscle and brain it would be an outright error, since in these tissues structurally distinct 5'-leaders are generated from at least two transcription start sites in the genome (see below). Existence of additional promoters (e.g. upstream of 1d and between 1d and 1c) also cannot be excluded, since RACE may not be able to reveal all alternative exons.

A second point is the existence of composite leaders, i.e. a family of leaders generated from every one of the three transcription start points. Composite leaders, e.g. encoded in more than one exon, have long been available in public data bases, and have been isolated from both cDNA libraries and RACE experiments. For example, the two-exon 5'-leader "1d-1c" (Fig. 2) was consistently isolated from the heart of several mammalian species. The two-exon leaders were not observed (or observed in only a small amount) in cases when time intervals from only seconds to 2 min elapsed between the decapitation of the rat and the freezing of its tissues. In contrast, deliberate "aging" of a dead tissue destroyed the specific pattern of leader expression, observed in freshly frozen tissues, so that exons 1c and 1b could be observed in rat heart and brain (Fig. 4B). In view of these data, detection of two-exon leaders is a likely result of using a tissue that was stored at room temperature before RNA isolation. Consequently, exon 1b (first isolated in the cat by RACE) is likely to be used in kidney only as a portion of a transient intermediate, because amplification of this species is weak (Fig. 3B) in a fresh tissue, but quite obvious in the tissue stored at room temperature. Available data also suggest that exons 1c and 1b are observed in heart and brain only as portions of two-exon splicing intermediates. From the same point of view expression of exon 1c in aorta is seen only because it forms a portion of a similar splicing intermediate, possibly 1d-1c.

Erratic splicing products have been previously documented for mutually exclusive exons 3 and 4 of NCX1 (25), as well as for positionally similar exons of the human NCX2 genes (4). Hence, the data obtained on commercially available human RNA samples should be viewed with caution, unless they can be compared with data from other mammalian species. It is conceivable, however, that such observations may accidentally uncover the pathways into which the normal splicing process is routed in disease states. Splicing aberrations of tyrosine hydroxylase transcripts (i.e. exon skipping and imprecise splicing site selection) were also documented in post-mortem samples from the adrenal medulla of patients with progressive supranuclear palsy (26). Monitoring expression of exons such as exon 1e may thus be of special interest in certain human disorders.

Finally, the region of about 200 bp, having 80% cross-species similarity between rat and man located downstream of exons 1c and 1a, is of special interest. A similar situation was previously documented for exons 1 and 1* of the human calcium pump gene PMCA1 (16). In analogy with the published work on PMCA1, no expression of this region was detected in this work, at least in the tissues examined (heart and brain). Thus, the significance of these regions remains to be determined. The one downstream of exon 1c formally represents an upstream region of the promoter located 1 kb away and may thus be involved in binding transcription factors. Both regions may also be involved in exon recognition during the alternative splicing process.

Distribution of Transcription Factor Consensus Sequences in the Multipartite NCX1 Promoter-- Of the three transcription start sites characterized genetically in this work, only that upstream of exon 1c contained a typical TATA box. Using a computer screening against the transcription factor data base, consensus sequences for SP1, AP1, and glucocorticoid response elements could be found within a reasonable distance upstream of the three exons (not shown). However, the regulatory elements of this promoter are possibly spread, given its topology, over a ~60-kb distance, of which only about 15% has been actually sequenced in this work. In this case there is no reason to believe that only exon-proximal upstream elements contribute to the complex interaction of transcription factors. The physical map and partial sequence data will help in exploring the issue.

Tripartite Promoter Activity in Cultured Aortic Cells-- Na/Ca exchanger activity was demonstrated previously in the immortalized aortic smooth muscle cell line A7r5 (27), and the protein was localized by immunofluorescence microscopy (28). The results of the work described here on the relative activity of the regions upstream of the three putative transcription starts measured by the transfection of luciferase fusion constructs into cultured rat VSMC were partially at variance with the RT-PCR data, since they showed simultaneous activity of the three promoters, while RT-PCR detected only small amounts of 1c-driven endogenous transcript in these cells. However, the quantitative ratio need not reflect the situation in vivo, because certain long range interactions may have been lost in the reporter constructs. The mouse actin gene has been shown to be regulated by cardiac- and smooth muscle-specific enhancer elements located 7-10 kb upstream of the transcription start (29). Moreover, the proximity of the 1a and 1c promoters may lead to cross-interaction of the regulatory factors, while in the constructs used in this work the two regions were separated. Finally, since exon 1c is also used as an intermediate in the splicing of the 1d-driven transcript, long range interactions of the two promoters may be involved in the concomitant transcription and splicing of the transcripts. Experiments with much larger upstream regions are clearly required to further explore this issue.

Despite the large size and complex pattern of its expression, the NCX1 promoter does not represent a unique example. Since a similarly complex gene, the Antennapedia of Drosophila (30-32), has been described, such genes were considered exceptions. However, an extremely similar organization was recently described for the multipartite promoters of the human furin (9), the interleukin-1 receptor (14), the NPY-Y1 receptor (10), and the chloride/carbonate exchanger AE2 (13) genes. However, in the case of the NCX1 gene, the existence of such a complex promoter was not predicted by prior biochemical observations on the modes of its regulation.

RT-PCR screening of the 5'-leader expression has shown that exon 1a is always expressed in live tissues. However, when the same set of primers was used on stable cell lines, i.e. PC12 and p19, no expression of this leader was seen. Apparently, the NCX1 transcription from this promoter may be silenced by the immortalization. This is not necessarily always so, since the transcription is active in certain immortalized lines derived from smooth muscle (27); however, the usefulness of immortalized lines for studies of the NCX1 promoter regulation is evidently questionable. Transgenic cell lines and transgenic organisms, containing a fusion of the entire promoter region to a reporter gene, have become instrumental in the analysis of complex promoters (33-36). The large size of the NCX1 promoter and the apparently multistep 5'-leader alternative splicing complicate the study of this problem; however, the adaptation of BAC vectors (37) for eukaryotic cell transfection may eventually overcome the present technical limitations. Alternatively, the nematode Caenorhabditis elegans, which contains NCX-like genes (four candidates have already been identified in the genome project data base) and which is readily amenable to genetic manipulation, could become a paradigm in future studies of the NCX gene regulation.

    ACKNOWLEDGEMENTS

We are grateful to our colleagues D. Guerini for samples of cell lines and for useful discussions, N. Kraev for assistance in genomic mapping, P. Gazzotti for help in dissections of rats, and R. Moser for assistance in preparing the figures.

    FOOTNOTES

* This work was supported by the Swiss National Foundation Grant 31-30858.91.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y13032-Y13037, Y12878, Y12885.

Dagger The first two authors contributed equally to this work.

To whom correspondence should be addressed: Laboratory of Biochemistry III, Swiss Federal Institute of Technology (ETH), Universitätsstrasse 16, CH-8092 Zürich, Switzerland. Tel.: 41 1 632 30 11/12; Fax: 41 1 632 12 13; E-mail: carafoli{at}bc.biol.ethz.ch.

1 The abbreviations used are: kb, kilobase pair(s); bp, base pair(s); PCR, polymerase chain reaction; RT, reverse transcriptase; RACE, rapid amplification of cDNA ends; YAC, yeast artificial chromosome; BAC, bacterial artificial chromosome; STS, sequence tagged site; VSMC, vascular smooth muscle cells.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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