Mouse Connexin40: Gene Structure and Promoter ... - Science Direct

GENOMICS

46, 120–126 (1997) GE975025

ARTICLE NO.

Mouse Connexin40: Gene Structure and Promoter Analysis Kyung Hwan Seul, Peter N. Tadros, and Eric C. Beyer1 Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110 Received June 30, 1997; accepted September 11, 1997

A family of related connexin genes encodes the subunit gap junction proteins that form intercellular channels in different tissues. Connexin40 (Cx40) is one of these proteins, and it exhibits limited expression only in a few cells of the cardiovascular system. To begin to analyze Cx40 expression, we isolated a 3.3-kb rat Cx40 cDNA by hybridization screening of a bacteriophage library prepared from BWEM cells and isolated corresponding mouse genomic clones from a bacterial artificial chromosome library. Restriction mapping, sequencing, and comparison to the rat cDNA showed that the mouse Cx40 gene contained a short first exon, an 11.4-kb intron, and a second exon containing the complete coding region and 3 *-UTR. Exon I contained only 1 base that differed between rat and mouse. Primer extension experiments yielded a single band and confirmed the position of the transcriptional start site. We obtained 1.2 kb of sequence 5* of the transcriptional start site and 400 bp 3 * of exon I. Exon I was closely preceded by a consensus TATA box. The flanking sequences contained a number of potential transcription factor binding sites (including AP-1, AP2, SP1, TRE, and p53). To identify transcriptional regulatory elements in the Cx40 promoter region, a series of DNA deletion fragments flanking exon I was prepared, subcloned adjacent to a luciferase reporter gene, and used for transient transfections of BWEM, SHM, and N2A cells. The resulting luciferase activity determinations suggested that an area of 300 bp 5* of the transcription start site acted as a basal promoter for Cx40 and that there was a strong negative regulatory element in the region from /100 to /297. q 1997 Academic Press

INTRODUCTION

Gap junctions contain channels that allow the intercellular passage of ions and small molecules. In excitSequence data from this article have been deposited with the GenBank Data Library under Accession Nos. AF021806, AF022136, and AF023131. 1 To whom correspondence should be addressed at Division of Pediatric Hematology/Oncology, Washington University School of Medicine, Box 8116, One Children’s Place, St. Louis, MO 63110. Telephone: (314) 454-2492. Fax: (314) 454-2685. E-mail: Beyer@KIDS. WUSTL.edu.

0888-7543/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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able tissues such as myocardium and smooth muscle, electrical coupling through gap junctions facilitates action potential propagation. At least 13 different connexins have been reported, and they show different patterns of tissue distribution and developmental expression, as well as forming channels with differing biophysical characteristics. Unlike some other gap junction proteins, connexin40 (Cx40) has a rather restricted pattern of expression, limited to certain components of the cardiovascular system including atrial myocytes, specialized conducting cells, and some endothelial cells (Bastide et al., 1993; Bruzzone et al., 1993; Davis et al., 1994; Delorme et al., 1995). Cx40 channels exhibit conductance, permeability, and gating properties that are distinct from other connexins found in the heart and elsewhere (Beblo et al., 1995; Veenstra et al., 1995). Due to these unique properties of Cx40, it has been suggested that the abundant Cx40 in Purkinje fibers may contribute to the more rapid propagation of action potentials in these cells compared to working ventricular myocytes (Kanter et al., 1993; Beblo et al., 1995). Of the connexins, there has been only limited analysis of transcriptional regulation. Genomic clones have been isolated for several connexins (Miller et al., 1988; Fishman et al., 1991; Hennemann et al., 1992a,b; Sullivan et al., 1993). All connexin gene structures determined to date appear similar, with two exons, one containing only 5*-untranslated sequence and the other containing the rest of the transcribed sequences including the entire coding region. Reporter gene transfections have identified promoter activity and some possible regulatory elements for Cx32 and Cx43 (De Leon et al., 1994; Yu et al., 1994; Geimonen et al., 1996; Chen et al., 1995). In the course of our cardiac connexin cloning studies, we have isolated portions of the genes for rat, dog, and human Cx40 (Beyer et al., 1992; Kanter et al., 1992, 1994). In view of the unique tissue distribution of Cx40 and its distinctive physiologic characteristics, we were interested in identifying the promoter region of Cx40, which should contain sequences responsible for gene regulation and tissue specificity. We now present data showing the mouse Cx40 gene structure and identification of the promoter region by luciferase reporter gene assays.

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MATERIALS AND METHODS Tissue culture. BWEM cells [which originated from fetal rat myocardial cells (Engelmann et al., 1993)] were cultured in Dulbecco’s minimal essential medium/F-12 (Life Technologies, Inc., Gaithersburg, MD), supplemented with 5% fetal calf serum (JRH Biosciences, Lenexa, KS). Neuro2A cells (N2A, mouse neuroblastoma cells) and SHM cells [Syrian hamster myocytes that were originally derived from an androgen/estrogen-induced uterine smooth muscle tumor of the Syrian hamster (Riemer et al., 1993)] were cultured in minimal essential medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (JRH Biosciences), 11 nonessential amino acids, 2 mM L-glutamine, and 100 U/ml penicillin and 100 mg/ml streptomycin. cDNA preparation and cloning. Total RNA was isolated from BWEM cells. The RNA was reverse transcribed, and double-stranded cDNA with added XhoI and EcoRI linkers was prepared using the cDNA synthesis system from Stratagene (San Diego, CA). This cDNA was used to prepare a library in lambda UNIZAP (Stratagene). Clones were isolated by hybridization screening as described previously (Beyer et al., 1987; Beyer, 1990), using the coding region of rat Cx40 as probe (Beyer et al., 1992). Isolation of genomic clones. A rat genomic library (in lambda DASH) was purchased from Stratagene and was screened by hybridization with the rat Cx40 cDNA as described previously (Beyer et al., 1987; Beyer, 1990). Mouse genomic libraries (prepared from embryonic stem cells) in bacterial artificial chromosome (BAC) and P1 vectors (Genome Systems, St. Louis, MO) were screened using the polymerase chain reaction with primers corresponding to an 393-bp sequence region within the coding region of mouse Cx40 (bases 05 through /388 as numbered from the ATG translational start codon) (Hennemann et al., 1992c). The sequences used were 5*-GCAAGATGGGTGACTGGAGCTTCC-3* (sense primer) and 5*-TTTCGGCTACTGGGTACTCATAGGC-3* (antisense primer). Two positive clones were isolated from each library, and restriction fragments generated with XbaI, NheI, or PstI were subcloned into pBS SK(/) (Stratagene). The full intron was amplified from a BAC clone using the polymerase chain reaction (LA PCR kit; TaKaRa Shuzo Co., Japan) using a sense oligonucleotide derived from rat exon I (5*-GCATAGCGGCCGCAGGTTGAACAGCAGCCAGAGCCTGAAGAAG) with an added NotI site at the 5* end and an antisense primer from exon II (5*-AGGCAGCGGCCGCTTGTCCACACCCTGCCGTGACTTGCCAAA). DNA sequencing. Manual sequencing of plasmid templates was performed using [35S]dATP labeling as described by Beyer (1990) and the Sequenase enzyme (USB, Cleveland, OH) and oligonucleotide primers. Alternatively, automated sequencing was performed with an ABI sequenator on reactions prepared by the dye-termination method using the ABI Prisms Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin–Elmer, Palo Alto, CA). Data were analyzed, and sequences were merged using the PCGene software (Intelligenetics). DNA and RNA blots. DNA amplified from BAC or lambda clones was digested with restriction enzymes, electrophoresed in 1% agarose gels, and transferred to nylon membranes as previously described (Reed et al., 1993). Total cellular RNA was prepared from cultured cells by using total RNA extraction kits (Qiagen GmbH, Hilden, Germany). RNA was separated on formaldehyde/agarose gels and transferred to nylon membranes as previously described (Reed et al., 1993). Hybridization was performed using specific 32P-labeled DNA probes. Primer extension. Experiments designed to identify the Cx40 transcription start site used an oligonucleotide complementary to the beginning of the translated portion of mouse Cx40 (bases 1–33 from the ATG, 5*-AGGAACTCCCCCAGGAAGGCTCCAGTCACCCAT-3*). The oligonucleotide was labeled at the 5* end using T4 polynucleotide kinase and [g-32P]ATP. The primer (2 1 105 cpm) was incubated with RNA at 427C for 1 h in the presence of reverse transcriptase. The extended products were analyzed by electrophoresis on a urea-containing 6% polyacrylamide gel and autoradiography.

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Luciferase-linked Cx40 gene constructs. For Cx40 promoter analysis, a DNA fragment containing exon I and containing 5* and 3* flanking sequence from 01190 to /319 bp (01190//319) was prepared from the mouse genomic Cx40 clone by the polymerase chain reaction and subcloned into pGL3 (Promega, Madison, WI) adjacent to a luciferase reporter gene. A series of 5* deletion constructs that started at 0889 bp (0889//319), 0558 bp (0558//319), and 0293bp (0293//319) was similarly prepared, as was a parallel series of deletions that ended at /121 bp (01190//121, 0889//121, 0558//121, and 0293//121). Transfection and reporter assays. For all transfection/reporter experiments, cells were plated at É 3 1 105 cells/60-mm plate 24 h prior to transfection in triplicate. Transient transfections of BWEM cells were performed using lipofectin (Life Technologies, Inc.) similarly to the stable transfections of these cells that we have performed previously (Davis et al., 1995). Transfections of SHM and N2A cells were performed by DNA–calcium phosphate coprecipitation (Scanta and Adler, 1996). In each experiment, cells were cotransfected with 1 mg of pRSV/b-galactosidase DNA (containing the Escherichia coli lacZ gene driven by the Rous sarcoma virus (RSV) promoter for normalization of transfection efficiency) and 1 mg of the pGL3/Cx40 construct DNA. Cells were harvested 48 h after the transfection, and extracts were prepared by three cycles of freeze–thawing as described (Scanta and Adler, 1996). The luciferase activity was determined from light output measured for 10 s at room temperature in a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). Data were corrected for transfection efficiency according to b-galactosidase activity determined in each extract.

RESULTS

cDNA Cloning of Rat Cx40 and Sequence Analysis To begin to investigate the transcriptional regulation of Cx40, we prepared a cDNA library from BWEM cells [a rat cardiac myocyte-derived cell line that abundantly expresses Cx40 (Laing et al., 1994; Davis et al., 1995)] and isolated two independent Cx40 cDNA clones by hybridization screening with the Cx40 coding region, which we had previously isolated by the polymerase chain reaction (Beyer et al., 1992). The Cx40 cDNA clones contained 3115 bp, including 91 bp of 5*-UTR (Fig. 2), a coding region of 1071 bp, which matched that previously determined by Beyer et al. (1992) and by Haefliger et al. (1992), and a 3*-UTR of 1954 bp (data not shown). To determine the structure of the rat Cx40 gene, we isolated genomic clones for Cx40 by hybridization screening of a genomic bacteriophage library. We isolated a clone that contained portions of the Cx40 coding region starting at an XhoI site corresponding to bp 683 of the rat cDNA. This clone contained uninterrupted sequence that was identical to the remainder of the coding sequence and 3*-UTR determined from the cDNA (data not shown). These observations combined with our previous ability to amplify the coding region from genomic DNA by the polymerase chain reaction (Beyer et al., 1992) confirmed that all of the coding region and the 3*-UTR were contained in a single exon. Unfortunately, we were unable to obtain rat genomic clones corresponding to the beginning of the cDNA. Isolation of Mouse Genomic Clones of Cx40 To obtain mouse genomic clones for Cx40 we screened mouse genomic libraries in BAC and P1 vec-

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FIG. 1. Restriction map and structure of the mouse connexin 40 gene. Restriction fragments and the long PCR product used for sequence determination and restriction mapping are shown above the gene structure. Boxes represent exons; the solid black box represents the translated region. X denotes sites cut by XbaI.

tors using the polymerase chain reaction and primers derived from the Cx40 coding sequence. Four positive clones were isolated. Restriction mapping, DNA blotting, and the polymerase chain reaction were used to determine the structure of the mouse Cx40 gene (Fig. 1), and portions of the sequence were determined (Figs. 2 and 3). The mouse Cx40 gene contained a short first exon containing sequence corresponding to the first 67 bp of the 91 bp of 5*-UTR determined from the rat Cx40 cDNA. There was a single intron of 11.4 kb. There was a second exon containing 34 bp of 5*-UTR, 1077 bp of coding sequence, and a 1.6-kb 3*-UTR. The translated sequences exactly matched the data previously reported by Hennemann et al. (1992c). We sequenced all of exon II, 600 bp 5* of it (into the intron), and 700 bp following the end of the 3* UTR. We sequenced exon I and 1.2 kb 5* and 600 bp 3* of it. The 3*-UTR of rat and mouse Cx40 showed 82.5% identical bases. Comparison of 360 bp determined following the poly(A) ad-

FIG. 2. Exon–intron junction of the mouse Cx40 gene and comparison of 5*-UTR between mouse and rat Cx40 genes.

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dition site showed 82.8% nucleotide identity (data not shown). Exon I contained only 1 base that differed between rat and mouse (Fig. 2). The single intron was bordered by typical splice junctions (Fig. 2). The 3* splice acceptor site was preceded by a polypyrimidine tract. These intronic sequences were similar to the sequences previously determined for dog and human (Kanter et al., 1992, 1994) (comparison not shown). There was no other good consensus splice acceptor within the portions of the intron that we sequenced (data not shown). Sequence analysis of the regions flanking exon I showed that there was a consensus TATA box 38 bp 5* of the transcription site. This 5* flanking region also contained multiple putative transcription factor binding sites, including SP1, AP-1, AP-2, CTF (CCAAT box transcription factor), C/EBP (CCAAT/enhancer binding protein), Y-box, and MRE (metal response element). There were half-palindromic glucocorticoid and thyroid response elements. Near the beginning of the intron, there was a potential C-myc binding site (which differed by a single base from the consensus sequence) and a potential p53 binding site (Fig. 3). Primer extension experiment. To confirm the position of the transcriptional start site for Cx40, we performed primer extension assays using an antisense primer corresponding to the beginning of the mouse Cx40 coding sequence and lung RNA, since it is the richest known source of Cx40 mRNA (Hennemann et al., 1992c). This analysis yielded only a single band (Fig. 4), the electrophoretic migration of which suggested that the transcription start site was 77 bp upstream of the splice donor site of exon I. Reporter gene transfections with Cx40 gene fragments. To analyze transcriptional regulatory elements, we performed luciferase reporter gene transfections. As a negative control, we chose Neuro2A cells, which are a communication-deficient cell line that does not contain any detectable Cx40 mRNA; as positive cell lines, we used BWEM and SHM cells, because they

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FIG. 3. Nucleotide sequence of the mouse connexin40 promoter. The transcription start site is marked by an arrow. Exon I is shaded. Putative regulatory elements (AP-1, AP-2, CTF, C/EBP, SP1, TRE, MRE, Y-box, E2A, p53, and C-myc) are underlined.

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FIG. 4. Primer extension determination of the Cx40 transcriptional start site. A 32P-end-labeled primer complementary to nucleotides 1–33 following the ATG in mouse Cx40 was hybridized with test RNAs and reverse-transcribed. Lane 1, 1.2 kb kanamycin negative control RNA (Promega); lane 2, extended product of Ç142 bp produced from mouse lung RNA, suggesting that transcription starts 77 bp upstream of the splice donor site of exon I; and lane 3, labeled fX174 HinfI DNA standards.

abundantly produce Cx40 mRNA (Beyer et al., 1992, and data not shown). DNA fragments containing exon I, starting 1190 bp 5* of the transcriptional start site and ending either 319 or 121 bp 3* were prepared and subcloned adjacent to a luciferase reporter gene. A series of 5* deletion constructs was also prepared from each of these fragments (Fig. 5A). These constructs were transiently transfected into BWEM cells, and luciferase activity was determined. All constructs produced some luciferase activity. Activity was relatively similar regardless of the amount of 5* sequence included (Fig. 5B). All of the constructs ending at /319 showed rather weak promoter activity which was increased dramatically (as much as sevenfold) by deletion of the region from /121 to /319. A similar pattern of results with the different promoter/deletion constructs was obtained by transfection of a second Cx40-expressing cell line (SHM) (Fig. 5C). In the SHM cells, the most active constructs produced luciferase activities 80–90% as great as those obtained with our positive control, luciferase driven by an RSV promoter. In contrast, transfection of Neuro2A cells (which are communication deficient and do not produce Cx40) gave little luciferase activity (less than 3% of that obtained with the RSV-luciferase) with any of the constructs (data not shown). DISCUSSION

We have presented data demonstrating that the mouse Cx40 gene consists of two exons separated by

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an 11.42-kb intron. Exon II contains coding sequences, whereas exon I contains exclusively 5* untranslated sequences. This structure is similar to the organization determined for other connexins, including Cx32 (Miller et al., 1988), Cx26 (Hennemann et al., 1992b), and Cx43 (Fishman et al., 1991; Sullivan et al., 1993). Unlike Cx32 (Neuhaus et al., 1995), we found evidence for only a single transcriptional start site for Cx40. The sequences of the rat cDNA and the rat and mouse genes may give some insights into Cx40 regulation. The coding sequences that we determined within exon II of mouse or rat Cx40 exactly matched those previously determined (Beyer et al., 1992; Haefliger et al., 1992; Hennemann et al., 1992c). Clearly, portions of the coding sequence contribute to unique protein structure, since Cx40 forms channels with unique regulatory and permeability properties compared to other connexins (Hennemann et al., 1992c; Beblo et al., 1995; Bukauskas et al., 1995). The sequences flanking exon I may have multiple regulatory elements. The Cx40 gene contains a TATA box just upstream of the transcription start site, suggesting that this gene may be transcribed by RNA polymerase II, similarly to many other genes. Cx43 similarly contains a TATA box (Fishman et al., 1991; Sullivan et al., 1993), but this is not a universal feature of all connexin genes, since this site is absent in Cx26 and Cx32 (Miller et al., 1988; Hennemann et al., 1992b). Computer analysis showed that the flanking sequences contained putative binding sites for multiple transcription factors. The significance of most of these is unclear, since there are little available data regarding hormonal, pharmacologic, or developmental regulation of Cx40 expression. Similar to Cx43, the Cx40 gene contained an AP-1 site (at 0848). Such sites bind the transcription factors Fos and Jun. The Cx43 gene also contains putative AP-1 sites within its promoter region (Sullivan et al., 1993; Lefebvre et al., 1995), and these sites have been implicated in the regulation of myometrial Cx43 expression by estrogen (Lefebvre et al., 1995) and in response to activation of protein kinase C (Geimonen et al., 1996). Our transfection experiments using luciferase reporter gene constructs have started to elucidate Cx40 transcriptional regulation. Deletion analysis showed that a small region flanking exon I (0319 to /121) could act as a strong promoter in Cx40-expressing cells; indeed, it was nearly as strong as the RSV promoter in the SHM cells. This suggests that this region includes the basal promoter for Cx40. Furthermore, our data showed that constructs containing the region between /121 and /319 downstream of the transcription start site yielded greatly reduced luciferase activity, suggesting the presence of a strong negative regulatory element within this region. Computer analysis suggested a couple of candidates for elements involved in this regulation, since it contains a sequence that differs by only a single base from the consensus for a C-myc binding site and it contains a single p53 binding site.

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FIG. 5. Luciferase reporter gene analysis of the Cx40 promoter region. (A) A series of fragments of the mouse Cx40 gene flanking exon I and linked to a luciferase reporter gene was prepared. (B) Luciferase activities obtained by transient transfection of BWEM cells. (C) Luciferase activities obtained by transient transfection of SHM cells. Activities are expressed as a percentage of that obtained with pRSVluc (RSV promoter-driven luciferase) and have been normalized for b-galactosidase activity (produced by RSV-b-gal) to correct for transfection efficiency. Bars represent standard deviations.

Regulation by p53 might be surprising, since we identified only a single site while the DNA binding domain of p53 has been shown to bind to two copies of this sequence (Hainaut, 1995) and since typically p53 binding leads to increased gene transcription rather than repression. Finally, characterization of the Cx40 gene and its promoter may provide a useful tool for elucidating regulation of gene expression in an interesting group of cells (since Cx40 expression is largely limited to the atrium, cardiac conducting system, and endothelial cells). The absence of promoter activity in transfections of Neuro2A cells suggests that our identified fragments show some tissue specificity. We hope that with refined analysis, the Cx40 promoter might provide a means to target gene expression to endothelium and the conducting system. ACKNOWLEDGMENTS These studies were supported by NIH Grant HL45466 (to E.C.B.) and an individual NRSA HL-09340001 (to P.N.T.). The authors gratefully acknowledge the work of Karen Reed in the initial rat Cx40 cloning. The authors thank Dr. Jan Albrecht, Ms. Namita AtalParanjothi, and Ms. Mary Ann Mallon for help with performing the promoter/reporter gene assays.

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