The human Na+/H+ exchanger NHE2 gene: genomic organization and promoter characterization

Jaleh Malakooti, Refka Y. Dahdal, Pradeep K. Dudeja, Thomas J. Layden, and Krishnamurthy Ramaswamy

Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois at Chicago and Westside Veterans Affairs Medical Center, Chicago, Illinois 60612


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Na+/H+ exchanger (NHE) 2 belongs to a family of plasma membrane transporters involved in intracellular pH and cell volume regulation. We recently reported cloning of human NHE2 (hNHE2) from a colonic cDNA library. Northern blot analysis has identified NHE2 mRNA only in small intestine, prostate, kidney, colon, and skeletal muscle. In this study, we describe the structure and 5'-regulatory region of the hNHE2 gene. The hNHE2 gene spans >90 kb and is organized in 12 exons intervened by 11 introns. All introns contain the conserved GT and AG dinucleotides at the donor and acceptor sites, respectively. The hNHE2 gene was mapped to chromosome 2q11.2. Primer extension analysis revealed a single transcription initiation site in human colonic adenocarcinoma cell lines. Analysis of the DNA nucleotide sequences of a 1.4-kb fragment of the 5'-flanking region shows no canonical TATA or CAAT boxes. However, the promoter region contains several potential cis-regulatory elements such as Sp1, early growth response-1, activator protein-2, MyoD, p300, nuclear factor-kappa B, myeloid zinc finger protein-1, caudal-related homeobox (Cdx) gene A, and Cdx protein-2 binding sites. In transient transfection studies, a reporter construct containing the 1.4-kb promoter region exhibited low luciferase activity levels. However, after deletion upstream of -664, its activity increased approximately threefold. Thus our data suggest that an inhibitory element may exist in the NHE2 promoter 5'-upstream region.

exon-intron junction; chromosome 2q11.2; transcription factor; C2BBe1; transfection


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

STUDIES IN THE HUMAN ILEUM and colon have established the involvement of an electroneutral Na+/H+ exchanger (NHE) in the process of Na+ absorption. Recent studies (2, 7, 25, 37, 38, 41, 50, 53) have identified six molecular isoforms of the NHE gene family. NHE1 to NHE5 are plasma membrane proteins, whereas NHE6 is found in the mitochondrial membrane. Studies (14) of the NHE1 isoform have demonstrated its ubiquitous nature as well as its localization in the basolateral membrane of the polarized epithelial cells. The NHE4 isoform has also been implicated in the basolateral Na+/H+ exchange activity of the rat kidney cells (11). The NHE2 and NHE3 isoforms, on the other hand, are localized to the apical membrane and have been implicated in apical Na+ absorption (21, 50, 51). In contrast to the NHE1 isoform, the expression of the other family members is tissue and cell specific (2, 7, 12, 25, 37, 38, 50). All six isoforms show similar structural features, including a membrane-spanning domain known to be involved in ion exchange (17) and a cytoplasmic domain that is highly divergent among all isoforms and contains several putative regulatory consensus sequences (52).

Chromosomal mapping has shown a wide distribution of members of this gene family on the genome of different species. In humans, NHE1 (28, 32), NHE3 (6), and NHE5 (25) have been reported to map to chromosomes 1p35, 5p15.3, and 16q22.1, respectively. To date, only the genomic organization of the human NHE1 (hNHE1) (33) and NHE5 has been reported (2). Also the 5'-regulatory region of the NHE1 has been characterized to some extent (26, 33). However, no information is available on the other isoforms. To characterize the hNHE2 isoform and its contribution to apical Na+/H+ absorption in the intestine, we (29) have cloned and sequenced this isoform and shown that its gene product is capable of Na+/H+ exchange activity in a heterologous system. In the present study, we have determined the genomic organization of the hNHE2 gene, identified its exon-intron boundaries, and mapped the gene to the chromosome 2q11.2. We have also cloned a 3.3-kb fragment of the NHE2 promoter region. Sequence analysis of the promoter and the 5'-flanking region revealed the presence of numerous potential transcription factor response elements as well as intestinal-specific cis-acting regulatory elements that might be involved in tissue-specific expression and regulation of the NHE2 gene.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Materials. All chemicals were purchased from Sigma Chemical (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). Restriction endonuclease and other modifying enzymes were from either New England Biolabs (Beverly, MA), GIBCO BRL (Gaithersburg, MD), or Promega (Madison, WI). The T-A cloning kit, JM109 competent cells, and luciferase assay system were from Promega.

Isolation of hNHE2 genomic clones. Two recombinant phage clones containing hNHE2 genomic DNA sequences were obtained by screening a placental genomic library (Stratagene, La Jolla, CA) with a 32P-labeled NHE2 cDNA fragment. These clones were characterized by restriction enzyme analysis and Southern blot hybridization (data not shown) using standard methods (1). The clones, lambda NHE2A and lambda NHE2B, contained 13.7- and 19-kb genomic DNA inserts, respectively, and overlapped by 4 kb. Together, they spanned ~28 kb of genomic DNA harboring the 3' end of the hNHE2 cDNA. To reveal the gene structure, we subcloned the genomic DNA insert of the phage clones into the cloning vector pGEM-13 using Not I restriction enzyme sites that flanked the 5' and 3' ends of the inserts. The new constructs containing 13.7- (pRD137) and 19.0-kb (pRD190) genomic DNA fragments were used for partial sequence analysis of the genomic DNA, using primers designed based on the hNHE2 cDNA (29). Comparison of the nucleotide sequences of the genomic clones and the cDNA revealed that the 13.7-kb genomic insert contained sequences corresponding to exons 8-12 and that the 19-kb DNA fragment contained sequences from exons 6 to 9. Our attempts to isolate the remaining genomic DNA region corresponding to the 5' end of the gene by using the same DNA library and screening method were unsuccessful. Therefore we used a long-distance extension PCR method to clone the remaining introns.

Identification of introns by PCR. The introns were identified and cloned by PCR amplification using the Expand long template PCR kit (Boehringer) in 50 mM Tris · HCl, pH 9.2, 16 mM (NH4)2SO4, 1.75 mM MgCl2, 0.35 mM of each dNTPs, 10 µM of each primer, 2.5 U rTth polymerase, and 200 µg human genomic DNA (Clontech). PCR amplification conditions were as follows: one cycle of preamplification denaturation at 95°C for 2 min followed by 35 cycles of two-step cycling at 94°C for 30 s and 5 min of annealing and extension at 68°C followed by a final extension of 10 min at 68°C. If needed, the annealing temperature and/or extension time was changed based on primer specification and size of the amplification products, where a three-step PCR cycling procedure was employed. The PCR primers (Table 1) were chosen from the vicinity of the NHE2 cDNA sequences that resembled the known splice junction sequences at the exon level (35).

                              
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Table 1.   Primers used for cloning of the introns

The sizes of PCR reaction products obtained were compared with those expected from cDNA amplification using the same primers. When the sizes of PCR reaction products were different, nested PCR was performed using inner primers to amplify and clone the introns. Using this scheme, we cloned all the introns except for intron 1, which we were unable to amplify due to its large size (see below). Therefore, to determine the exon-intron junctions of intron 1, we cloned the 5' and 3' ends of this intron using the Genome Walker kit (Clontech, Palo Alto, CA). Five different restriction enzyme-generated genomic DNA pools were linked to anchor sequences and screened by the 5' and 3' rapid amplification of cDNA ends method. Anchor primers in conjunction with the 5' or 3' gene-specific primers from exon 1 and exon 2, respectively, were utilized to clone a 1.6-kb fragment at the 5' end of intron 1 and a 10-kb fragment at the 3' end of intron 1. All PCR products were analyzed on a 1% agarose gel, DNA bands were excised, and DNA was extracted by the Sephglas BandPrep kit from Amersham Pharmacia Biotech (Piscataway, NJ). The purified DNA fragments were cloned in pGEM-T, T-A cloning vector (Promega) and sequenced from each end by the dideoxy chain termination method using the Sequenase version 2.0 kit (US Biochemical). The exon-intron junction sequences were identified by comparison with the hNHE2 cDNA sequence and the presence of exon-intron consensus sequences.

Chromosomal localization. Fluorescence in situ hybridization (FISH) was performed by Genome Systems (St. Louis, MO) using a hNHE2 genomic DNA clone (pRD190) as the hybridization probe. The labeled probe was combined with sheared human DNA and hybridized to normal metaphase chromosomes from peripheral blood lymphocytes. The initial experiment resulted in specific labeling of the long arm of a chromosome believed to be chromosome 2 on the basis of size, morphology, and banding pattern. A second experiment was conducted in which a genomic probe from the N-myc locus, which has previously (43) been mapped to 2p23, was cohybridized with the NHE2 probe. This experiment resulted in the specific labeling of the short and long arms of chromosome 2. Eighty metaphase cells were analyzed, with sixty-six exhibiting specific labeling.

To confirm the chromosomal localization, genomic DNA from a rodent-human somatic cell hybrid cell line containing only the human chromosome 2, NA11712 (Coriel Cell Repository) was subjected to PCR amplification using hNHE2-specific oligonucleotides. The primers used were sense primer from exon 2, 5'-CCATCTGTATCACAAGTTCG-3', and antisense primer from intron 2, 5'-GTCATGCAGTGTGAATGTG-3'. The thermocycling parameters were an initial denaturation for 2 min at 95°C, followed by 35 cycles at 94°C (30 s) and 54°C (30 s) and 2 min at 68°C. As control genomic DNA from parental lines, a normal human genomic DNA (NAIMR91) and mouse A9 DNA (NA00346B) also were subjected to the PCR amplifications using the same primers and PCR conditions.

RNA isolation. Total RNA was isolated from the postconfluent T84 or C2BBe1 cells using RNA STAT-60 (Tel-Test, Friendswood, TX) according to the manufacturer's suggested protocol.

Primer extension analysis. The transcription initiation site of hNHE2 was determined by primer extension using SuperScriptII RT (GIBCO BRL). An antisense oligonucleotide complementary to nucleotides 172-195 upstream from the translation initiation codon was synthesized and end labeled with [gamma -32P]ATP and T4 polynucleotide kinase. Free [gamma -32P]ATP was removed by using mini Quick Spin oligo columns (Boehringer Mannheim). For primer extension reaction, 105 counts/min of the end-labeled oligonucleotides and 15 µg of total RNA from T84 or C2BBe1 cells were coprecipitated and dissolved in diethyl pyrocarbonate-treated water and heated at 75°C for 5 min and then brought to 42°C. The reaction was complemented with 200 µM dNTPs and reaction buffer to a final concentration of 100 mM Tris · HCl (pH 8.3), 150 mM KCl, 6 mM MgCl2, 10 mM dithiothreitol, and 200 U of SuperScript II in a reaction volume of 20 µl. The reaction was carried out for 50 min at 42°C. The extension products were phenol-chloroform extracted and ethanol precipitated, and pelleted nucleic acids were dissolved in stop solution (US Biochemical). The samples were heated at 90°C for 3 min and analyzed on a 6% polyacrylamide-7 M urea denaturing gel. The gel was dried and exposed to X-Omat AR film. A sequencing ladder was used as a size marker to determine the size of the extended primer.

Cloning of 5'-flanking region. The 5'-regulatory region of the NHE2 gene was cloned using the Genome Walker kit (Clontech). PCR amplifications were performed with genomic DNA fragment pools as template, an anchor primer that hybridizes to the 5' end of the genomic fragments, and a gene-specific primer from hNHE2 cDNA. This resulted in amplification of a 1.4-kb DNA fragment. After gel purification, the 1.4-kb DNA fragment was cloned in pGEM-T cloning vector (Promega) and designated pJM1.4N2. Subsequent to similar nested PCR amplifications, a larger fragment (3.3 kb) of the 5'-flanking region of the NHE2 gene was cloned in pGEM-T and named pJM3.3 N2. DNA nucleotide sequences at the 3' end of the 3.3-kb fragment were determined and compared with the 5'-untranslated region (UTR) of the hNHE2 cDNA. This confirmed that the 3.3-kb fragment was the 5'-flanking region of the NHE2 gene.

Reporter plasmid construction. Plasmids used for transfection were generated using pGL2-Basic (Promega), which contains a promoterless luciferase reporter gene. To clone the 1.4-kb DNA fragment in pGL2-Basic, pJM1.4N2 was digested with Nco I restriction enzyme, which cuts the NHE2 sequence at the translation initiation site, and the ends were filled in with Klenow (1) and digested again with Sac I to release the 1.4-kb DNA fragment. After gel purification, this DNA fragment was cloned upstream from the luciferase structural gene in pGL2-Basic that was cut at the Sac I site and blunt ended at the Hind III site. This clone was named pJM1.4-N2P. pJM664-N2P was constructed by digesting the 1.4-kb fragment of the 5'-flanking region with Sma I, which generates a 664-bp fragment (bases -415 to +249), and subcloning into pGL2-Basic.

Cell culture and transfections. C2BBe1 cells, a subclone of the Caco-2 cell line, were obtained from Dr. M. Rao of the Department of Physiology, University of Illinois at Chicago. The C2BBe1 cells were maintained in collagen-coated culture dishes in DMEM supplemented with 10% FCS, 50 U/ml penicillin, 50 µg/ml streptomycin, 10 µg/ml transferrin, and 2 mM glutamine. Transfection experiments were performed in six-well plates on preconfluent cells using Lipofectamine Plus reagent (GIBCO BRL) according to the manufacturer's instructions. The cells were cotransfected with one of the NHE2 constructs and pRSV-beta gal. The latter plasmid served as an internal control for transfection efficiency. After 48 h, cells were lysed using a kit from Promega and total cell extracts were harvested. Luciferase activity was assayed using a Packard liquid scintillation analyzer (1900 TR) and normalized to beta -galactosidase activity.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Genomic organization of hNHE2 gene. The nucleotide sequences at the exon-intron junctions and the sizes of introns were determined as described in MATERIALS AND METHODS. On the basis of comparison of the nucleotide sequences at the exon-intron junctions to the hNHE2 cDNA sequence (29), the genomic structure of the hNHE2 gene was established. Figure 1 depicts the genomic organization of the hNHE2 gene. This gene consisted of 12 exons, which were interrupted by 11 introns. The first exon contained the 5'-untranslated region (UTR), as well as the transcription and translation initiation sites. Exon 12 contained the last 122 amino acids, the stop codon, and the 3'-UTR. Exons 1-5 and a portion of exon 6 code for the NH2-terminal domain of the polypeptide containing the transmembrane segments, and exons 6-12 encode the COOH-terminal regulatory domain (Fig. 2). The sizes of the introns were determined by sequencing or PCR amplification of the human genomic DNA and are presented in Table 2. All introns were located within the coding region, and all splice junctions conform to the GT/AG splice donor/acceptor rule (8, 44) (Table 2). Introns 2, 5, 6, 9, and 10 are in phase 0; introns 1, 4, and 11 are in phase 1; and introns 3, 7, and 8 are in phase 2 (Table 2). Therefore only exons 6, 8, and 10 are symmetrical, whereas all other exons are asymmetrical (39).


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Fig. 1.   Structure of the human Na+/H+ exchanger (hNHE) 2 gene. The two overlapping clones, pRD13.7 and pRD19.0, were cloned by genomic DNA library screening. Together, these clones contain the introns 6-11. The introns 2, 3, 4, and 5, indicated by thick lines and Roman numerals, were cloned by PCR amplification of the genomic DNA using primers from the adjacent exon sequences. The dashed line in intron 1 donates the DNA region, which was not cloned. The size of intron 1 was deduced based on the comparison with GenBank clone no. AC007239. The 5' and 3' ends of the intron 1 and the 5'-flanking region (5'-FR) were cloned with the Genome Walker kit (Clontech). An arrow shows the transcription start site. Arabic numbers indicate exons.



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Fig. 2.   Comparison of the amino acid sequences and intron insertion sites of hNHE2 and hNHE1. Arrows show the site of the splice junctions. The membrane spanning segments are overlined. * Amino acid sequence identity.


                              
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Table 2.   Organization of human NHE2 gene

The hNHE1 isoform is also composed of 12 exons and 11 introns (33). A comparison of the exon-intron boundaries of hNHE2 to those of hNHE1 revealed that the exon-intron splice junctions occur at the same position for the first nine introns with respect to the amino acid sequences of the two polypeptides (Fig. 2).

Subsequently, in the process of characterizing the NHE2 promoter region (see below), we found out that the nucleotide sequence of a portion of the human chromosome 2 was deposited into the GenBank (accession no. AC007139). This clone contains the entire NHE2 gene. On the basis of nucleotide sequence comparison between this sequence and the hNHE2 cDNA, we calculated the size of intron 1 to be 37.4 kb.

Chromosomal localization. The chromosomal localization of the NHE2 gene was determined by FISH using the hNHE2 genomic DNA clone, pRD190, as a probe. These studies resulted in the specific labeling of a position immediately adjacent to the centromere on the long arm of chromosome 2 (Fig. 3A). The identity of the chromosomes exhibiting a specific signal was confirmed by cohybridization with N-myc (43) genomic DNA, which is a chromosome 2-specific probe (Fig. 3B). The specifically hybridized chromosomes demonstrated that NHE2 was located in an area that corresponds to band 2q11.2 (Fig. 3C). To further confirm the results of the FISH assignment, we analyzed genomic DNA from the somatic cell hybrid cell line NA11712 by PCR using a forward primer from exon 2 and a reverse primer from the 5' end of intron 2 (Fig. 3D, lane 1). Control genomic DNA from parental cells, a human genomic DNA (NAIMR91; Fig. 3D, lane 3), and genomic DNA from mouse A9 (NA00346B; Fig. 3D, lane 4) were also subjected to PCR amplification using the same parameters. The expected PCR reaction product of 485 bp was obtained from the hybrid cell line NA11712, which retains only human chromosome 2, and from parental human genomic DNA, whereas no specific amplification product was produced using mouse A9 genomic DNA as a template. In agreement with our chromosomal mapping studies, Szpirer et al. (48) had assigned the NHE2 gene to the human chromosome 2, using a rat NHE2 cDNA as probe.


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Fig. 3.   Chromosomal assignment of the hNHE2 gene. A: a 19-kb hNHE2 genomic DNA was used as probe to identify the location of the gene on the human chromosome. NHE2-specific fluorescence in situ hybridization signals are shown on both chromatids of chromosome 2. B: cohybridization of the NHE2 probe and a genomic probe specific to the short arm of the human chromosome 2 (N-myc). Arrows indicate the hybridization signals. C: based on R-banding pattern generated by bromodeoxyuridine after cell synchronization, the NHE2 signal was assigned to 2q11.2. Arrowhead indicates the location of the gene. D: genomic DNA from a hybrid human-rodent cell line, NA11712 (lane 1), which retains only human chromosome 2. NA11712 was used for PCR verification of NHE2 locus on chromosome 2 using hNHE2-specific primers. As controls, human genomic DNA from Clontech (lane 2), human parental line NAIMR91 (lane 3), and mouse A9, NA00346B (lane 4) were also subjected to PCR amplification using the same parameters.

Determination of transcription initiation site. The transcription initiation site of the hNHE2 gene was determined using total RNA from intestinal epithelial cell lines T84, Caco-2, and C2BBe1. However, before primer extension experiments, the expression of the NHE isoforms NHE1, NHE2, and NHE3 in these cell lines was investigated by RT-PCR as shown in Fig. 4. The results of these experiments indicated that both NHE2 and NHE3 genes were expressed in C2BBe1 (Fig. 4, lanes 3 and 4) and Caco-2 cells (Fig. 4, lanes 11 and 12) but not in the mouse fibroblast NIH/3T3 cell line (Fig. 4, lanes 7 and 8). The NHE1 isoform was expressed in all three cell lines (Fig. 4, lanes 2, 6, and 10). The T84 cell line was shown to express the NHE2 isoform by Northern blot analysis (data not shown). Subsequently, primer extension analysis was performed using an antisense oligonucleotide corresponding to cDNA sequences at 172-195 bases upstream from the translation start codon and total RNA from the T84, Caco-2, and C2BBe1 cells. A 143-nucleotide primer extension product was identified in all three cell lines, whereas no signal was found in control yeast transfer RNA (Fig. 5). The primer extension product overlaps with a deoxyadenosine residue 315 nucleotides upstream from the translation initiation site. The nucleotide pair at the transcription start site, an A residue preceded by a C, is the most common dinucleotide pair at the eukaryotic transcription initiation sites (10, 23). Based on these data, we have designated this adenosine residue as +1 for the transcription initiation site of the hNHE2 gene (Fig. 6).


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Fig. 4.   Expression of the NHE isoforms in intestinal epithelial cell lines and NIH/3T3 was studied by RT-PCR. RT reaction was performed with 5 µg of total RNA from the respective cells, oligo(dT) as primer, and SuperScript II. Isoform-specific primers were utilized to amplify 2 µl of the RT reaction from each cell line. Lane 1, DNA size marker. Lanes 2-5, 6-9, and 10-13, RT-PCR products of C2BBe1, NIH/3T3, and Caco-2 cells, respectively. Lanes 2, 6, and 10, PCR products generated by NHE1-specific primer. Lanes 3, 7, and 11, PCR products generated by NHE3-specific primer. Lanes 4, 8, and 12, PCR products generated by NHE2-specific primers. In lanes 5, 9, and 13, the RT reactions contained no RT and were amplified with NHE2-specific primers. Lane 14, positive control using an NHE2 cDNA clone as template and the same gene-specific primers in lanes 4, 8, and 12. The faint band in lane 8 was generated by nonspecific PCR amplification as determined by sequence analysis.



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Fig. 5.   Mapping of NHE2 transcription initiation site by primer extension analysis. An oligonucleotide complementary to the 5'-untranslated region of the hNHE2 cDNA was annealed to 15 µg total RNA from T84 (lane 1), Caco-2 (lane 2), and C2BBe1 (lane 8) cell lines or yeast tRNA (lane 7) and extended with SuperScriptII RT. A sequence ladder of the NHE2 5'-flanking DNA region primed with the same primer as in primer extension reactions is shown in lanes 3-6 (representing A, C, G, and T ladders, respectively). * The transcription initiation site on the sense strand.



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Fig. 6.   5'-Upstream sequence of the hNHE2 gene. The nucleotide sequence of the 5'-flanking region and part of the first exon of the NHE2 gene is shown. Numbers at right are based on the transcription initiation site (+1) as determined by primer extension analysis. Consensus sequences for binding to the regulatory factors are shown. The NHE2 translation start site is underlined. A thick line marks the complementary strand of the oligonucleotide used for primer extension (PE) experiment. The nucleotide sequences are submitted to GenBank under accession no. AF273748. TRE, thyroid hormone receptor; PEA3, polyoma viral enhancer 3; GRE, glucocorticoid response element; Oct-1, octamer binding protein-1; CdxA, caudal-related homeobox (Cdx) gene A; Cdx-2, Cdx protein-2; CRE-BP1, cAMP responsive element-binding protein-1; NF-kappa B, nuclear factor-kappa B; MZF-1, myeloid zinc finger protein-1; Egr-1, early growth response-1; AP-2, activator protein-2.

Cloning and sequence analysis of 5'-flanking region. Using the genome walking technique, we cloned a 3.3-kb DNA fragment upstream of the NHE2 translation initiation site. Nucleotide sequence analysis of the 3' end of this clone confirmed that the cloned DNA fragment overlaps with the 5'-UTR of the hNHE2 cDNA reported previously (29). Subsequent to sequence determination of 1.4 kb of the 5'-flanking region of the hNHE2 gene, a databank homology search revealed that this DNA sequence overlapped with nucleotide sequences of a 139-kb clone of the human chromosome 2 deposited into GenBank (accession no. AC007239). The nucleotide sequence of our clone was in complete agreement with that of AC007239 except for a single discrepancy at position -689 (Fig. 6), where a G replaces a T in the chromosome 2 sequence. This G residue was present in two independent clones that we sequenced.

The DNA nucleotide sequence 5' of the NHE2 gene and a portion of the first exon are shown in Fig. 6. The hNHE2 gene lacks the typical TATA and CAAT boxes, but it has numerous potential DNA motifs for various transcription factors that might be responsible for regulation of the NHE2 gene expression. These include guanine-cytosine (GC)-rich sequences that constitute the potential binding sites for Sp1 (3, 9) at -20 and -335; activator protein-2 (AP-2) (18) at -21; two early growth response-1 sites (20) at -339 and -20, both overlapping Sp1 sites; sequences similar to the p300 binding site (16) in reverse orientation at -581 that overlap with a nuclear factor-kappa B (NF-kappa B) motif (47) at -587; two octamer binding protein-1 (Oct-1) sites (45) at -787 and -849; two myeloid zinc finger protein-1 binding sites (34) at -105 and -374, both in reverse orientation; MyoD (4) at -91, also in reverse orientation; a CACCC site at -206; and several polyoma viral enhancer 3 sites (54, 55) at -527, -926, -505, and -856, of which the last two are in reverse direction. Moreover, binding sites for two caudal-related homeobox (Cdx) family members, Cdx gene A (CdxA) (30) and Cdx protein-2 (Cdx-2) (46), were found at -785 and -506, respectively. In addition, a palindromic site for glucocorticoid response element (GRE) at -854 to -840 and a number of half sites for both GRE and thyroid hormone receptor are also present, suggesting that the regulation of this gene involves a complex array of regulatory factors.

Recently (36), the rat NHE2 promoter has been cloned and characterized. As in the hNHE2 promoter, the rat promoter lacks canonical TATA and CAAT boxes and is highly GC rich. A comparison of the human and rat NHE2 promoter sequence is shown in Fig. 7. The optimal alignment of the two sequences exhibited an overall 59% sequence identity. Subsequent analysis of the highly conserved regions demonstrated that some transcription binding sites, such as Sp1, AP-2, CACCC, NF-kappa B, and Oct-1 were conserved (Fig. 7). However, there are other cis elements that are not conserved, suggesting that different mechanisms are involved in the expression of NHE2 in these species.


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Fig. 7.   Comparison of the hNHE2 and rat NHE2 5'-flanking region. The nucleotide sequences surrounding the transcription initiation site and the 5'-flanking region were compared. The conserved nucleotide sequences are boxed. The putative binding sites for transcription factors, which are conserved in both species, are specified. The arrow and star indicate transcription initiation sites of the hNHE2 and rat NHE2 genes, respectively.

Functional promoter analysis of 5'-flanking region. To investigate whether the putative promoter region is functional, we cloned the 1.4-kb DNA fragment 5' of the luciferase reporter gene in pGL2-Basic (pJM1.4-N2P). This fragment contains 1 kb of the 5' promoter region and 315 bp of the 5'-UTR. In addition, a Sma I fragment harboring nucleotides -415 to +249 was also subcloned into pGL2-Basic (pJM664-N2P). The resultant fusion plasmids and the control vectors, pGL2-Basic, pGL2-Promoter, and PGL2-Control, were used for transient transfection of the C2BBe1 cells. As shown in Fig. 8, pJM1.4-N2P containing sequences from -1050 to +318 exhibited a moderate promoter activity, which was threefold of that obtained with the promoterless vector. However, deletion of sequences upstream from position -415 resulted in higher luciferase activity in C2BBe1 cells transfected with pJM664-N2P. The luciferase activity of the NHE2 promoter construct containing bases -415 to +249 was 13% of that found with pGL2-Promoter vector, which uses the SV40 promoter and was significantly lower, over 2-log range, than the pGL2-Control containing the SV40 promoter and enhancer sequences (data not shown). The moderate increase in expression of luciferase activity in the construct containing the bases -415 to +249 of the NHE2 promoter may be attributed to the loss of an inhibitory element contained within the deleted DNA region. Several putative response elements for transcription factors are located within this region (Fig. 6), and the question of whether any of these regulatory factors is involved in transcriptional repression of the NHE2 promoter will require further study.


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Fig. 8.   Measurement of NHE2 promoter activity. DNA fragments containing different lengths of the 5'-regulatory region were cloned upstream from the luciferase gene, and promoter activity of the cloned DNA was established by transiently expressing promoter-reporter constructs in C2BBe1 cells. Corrections for transfection efficiency were made by cotransfection with pRSV-beta gal vector. Promoter activity was calculated by fold increase over the luciferase activity of the control pGL2-Basic. The inserts in the promoter constructs are indicated based on the numbering scheme of Fig. 6. Values are means ± SE; n = 4.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we established the complete structure of the hNHE2 isoform, including the transcription initiation site. Using a combination of genomic DNA library screening and PCR amplification techniques, we have determined the position and sizes of all the NHE2 introns. These studies revealed that the hNHE2 gene encompasses a region >81 kb of the genome and consists of 12 exons and 11 introns. The first exon contains the 5'-UTR, as well as the transcription and translation initiation sites. The last exon contains the last 122 amino acids of the polypeptide, the stop codon, and the 3'-UTR.

Similar to hNHE2, the NHE1 isoform is also composed of 12 exons and 11 introns (33), and exon-intron junctions are well conserved between the two genes (Fig. 2). Moreover, with respect to intron splice phasing, the two genes are totally identical (Table 2). In all higher eukaryotes, the intron splice phasing type 0, in which introns are located between two codons, is found more frequently than types 1 and 2, in which introns interrupt the reading frame (19). In both NHE2 and NHE1, by insertion of introns 1, 4, and 11, the coding triplets are interrupted between the first and second nucleotides (type 1) and between the second and third nucleotides by introns 3, 7, and 8 (type 2). On the other hand, there are no interruptions in the coding triplets by insertion of the introns 2, 5, 6, 9, and 10 (type 0). These data indicate that, despite differences in the amino acid sequences and the spatial localization of the NHE2 and NHE1 proteins, the genomic organization of their respective genes is quite similar. Unlike hNHE2 and hNHE1, the rat NHE3 and hNHE5 are organized in 17 exons and 16 introns (24) and 16 exons and 15 introns (2), respectively. These studies, along with further elucidation of the gene structure of the other NHE isoforms, may be useful for an understanding of the path of molecular evolution of the members of the NHE gene family.

A single transcription initiation site was mapped to an adenosine residue 315 bp upstream from the ATG translation start codon. The 5'-flanking region of the hNHE2 gene lacks a canonical TATA box (8) or CAAT sequence (13) in the expected proximity of the transcription initiation site. However, the region spanning from a Sma I restriction enzyme site at -415 to ATG translation start codon at +316 is composed of 77% G + C nucleotides. The presence of such a GC-rich sequence within the 5'-flanking region is a feature of TATA-less promoters that usually contain Sp1 binding sites (40). In fact, two Sp1 binding sites were identified in close proximity to the transcription start site (Fig. 6), both of which interact with Sp1 transcription factor family members (Malakooti and Ramaswamy, unpublished data). Thus the NHE2 gene promoter belongs to the subclass of TATA-less RNA polymerase II promoters.

We have also identified several other potential cis-acting regulatory elements in the promoter region. Of particular interest was the identification of the CdxA and Cdx-2 homeodomain protein binding sites. CdxA, a chicken homeobox gene, belongs to the caudal family of vertebrate homeobox genes. The expression of these genes is restricted to the endoderm-derived epithelia during embryognesis (15). Cdx-2 is expressed in the intestinal epithelial cells and has been reported (31) to be involved in differentiation of these cells. The binding site for Cdx-2 was identified in the 5'-regulatory region of several genes that are specifically expressed in enterocytes, such as sucrase isomaltase (46) and lactase-phlorizin hydrolase (49) genes. Because NHE2 mRNA is predominantly found in epithelial cells of the gastrointestinal tract, the Cdx homeodomain family members may be involved in the regulation of the NHE2 gene expression during enterocyte differentiation.

A comparion of hNHE2 and rat NHE2 promoter sequences demonstrated that several transcription factor binding sites were conserved between the two species. A knowledge of the identified conserved elements as well as of those not conserved in the hNHE2 and rat NHE2 promoters may provide the basis for detailed studies leading to the identification of the mechanisms regulating the expression of the NHE2 gene.

Transient transfection studies revealed significant activity of the NHE2 promoter in C2BBe1 cells. The expression of the reporter constructs containing different lengths of the NHE2 promoter fragment suggested that an inhibitory element is present between -1050 to -415 bp from the transcription start site. The identification of a repressor element in the upstream DNA region parallels the finding for the rat NHE2 gene (36), in which a similar decrease in gene expression was noted on deletion of the upstream promoter sequences.

The mapping of NHE2 to 2q11.2 did not indicate linkage to any known diseases. A defect in NHE function has been implicated in human diseases such as essential hypertension (22) and congenital diarrhea (5). Studies by Lifton et al. (27) on families exhibiting essential hypertension have ruled out the involvement of hNHE1 in this disease. Other NHE gene products that may be involved in these disorders are NHE2 and NHE3, both of which have been suggested to play major roles in transepithelial Na+ absorption. Recently, Schultheis et al. (42) have demonstrated in NHE2 knockout mice that the NHE2 isoform may be required for the parietal cell long-term viability and not for its acid secretion. However, as yet no human disease has been mapped definitely to any of the NHE isoforms.

In summary, the genomic structure and the promoter region of the hNHE2 gene were characterized and mapped to the chromosome 2q11.2. Knowledge of the genomic organization, chromosomal loci, and the promoter region of all the NHE isoforms will facilitate the search for mutations in these genes and identification of their role in human diseases, including essential hypertension, congenital diarrhea, and other diseases of the gastrointestinal tract.


    ACKNOWLEDGEMENTS

We thank V. C. Memark for technical help.


    FOOTNOTES

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-33349 and by the Department of Veterans Affairs.

Address for reprint requests and other correspondence: J. Malakooti, Section of Digestive and Liver Diseases, Dept. of Medicine, Univ. of Illinois, 840 S. Wood St., Chicago, IL 60612 (E-mail: malakoot{at}uic.edu).

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.

Received 17 July 2000; accepted in final form 24 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 280(4):G763-G773