©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Two Regulatory Elements within the Promoter Region of the Mouse Connexin 43 Gene (*)

(Received for publication, November 9, 1994)

Zhi-Qing Chen (1)(§) Diana Lefebvre (1)(§)(¶) Xiao-Hui Bai (1) Andrew Reaume (1)(**) Janet Rossant (1) (3)(§§) Stephen J. Lye (1) (2)(¶¶)

From the  (1)Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, and the Departments of (2)Obstetrics and Gynaecology and (3)Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

To define the minimal sequences required for expression of the connexin 43 gene (cx43) in myometrial cells, we generated 5` deletion constructs of a fragment extending 1686 base pairs upstream and 162 base pairs downstream of the transcription start site and determined their ability to drive expression of the chloramphenicol acetyltransferase reporter gene in transfected myometrial cell lines. Our investigation revealed two cis-acting regulatory elements within this fragment. Deletion of a region extending from -102 to -92 led to an increase of the promoter activity by greater than 10-fold, indicating a presence of a repressor element. Deletion of a region extending from -72 to -62 caused a decrease of the promoter activity of a similar extent, implying the existence of a positive element. Electrophoretic mobility shift assays demonstrated that synthetic oligonucleotides derived from these two small regions can each bind with a nuclear protein(s) prepared from myometrial cells, and an introduction of three and two base substitutions into each of these oligomers was sufficient to abolish their protein binding capability. These same mutations, when incorporated in the chloramphenicol acetyltransferase constructs, diminished regulatory functions of the negative and positive elements, and the protein(s) that bind to these functional elements was found in several tissues known to express cx43 gene.


INTRODUCTION

Connexins are the major structural proteins of gap junctions, which permit the exchange of small metabolites and ions between neighboring cells. The connexins are encoded by a multigene family dispersed within the genome. Several cDNAs coding for connexins have now been isolated, each of which has a tissue-specific pattern of distribution. cx43 is one of the most widely expressed members of this gene family being expressed in numerous tissues including uterus, heart, lens epithelium, kidney, brain, mammary gland, ovary, placenta, and intestine(1, 2, 3, 4) . Altered expression of cx43 has been reported in several physiologic and disease states including cancer, preterm labor, ovarian follicular growth, and preimplantation embryo development(3, 5, 6, 7) . Of particular interest to our laboratory is the dramatic increase in gap junctions that occurs in the myometrium with the onset of labor(8) . The appearance of gap junctions is associated with a decrease in input resistance and increase in electrical conductivity in the myometrium (8) and is thought to enable the development of the highly coordinated, intense contractions that result in delivery of the fetus. The close association between the presence of myometrial gap junctions and both term and preterm labor has led to the suggestion that the synthesis of these structures by myometrial smooth muscle cells is essential for labor(8) . We and others have reported that cx43 mRNA is low during pregnancy but increases markedly at term, remains high throughout labor, and declines rapidly following delivery in the rat (9, 10, 11) , sheep(12) , and human myometrium(6) . Moreover, we found a close association between the rate of increase in mRNA and protein during labor, which would be consistent with the level of mRNA being an important regulatory means of myometrial cx43 expression (9) . We have recently shown that cx43 expression in myometrium of both non-pregnant and pregnant rats can be modulated by estradiol and progesterone levels in the plasma(13) . Estrogen causes an increase and progesterone causes a decrease of cx43 expression. Moreover, maintenance of elevated plasma progesterone levels at term blocked both the increase in cx43 expression and the onset of labor, while administration of a progesterone antagonist increased cx43 expression and was associated with preterm labor(9, 13) . These data contrast strikingly with observations in the heart where the expression of cx43 does not change during labor or following steroid treatment (14) and suggest complex cell-specific regulatory mechanisms.

The structure of the 5`-flanking region of the mouse, rat and human cx43 genes has recently been reported although no specific elements that regulate transcription of these genes have been described (15, 16, 17) . The objective of the present study was to locate cis-acting elements involved in the transcriptional control of the mouse cx43 gene within myometrial cells. We demonstrate that the promoter of the mouse cx43 gene, when introduced into myometrial cell cultures, can efficiently drive the expression of the CAT reporter gene. We report the identification of a positive regulatory element and a negative regulatory element located within 100 base pairs (bp) (^1)upstream of the transcription start site by both deletional mapping and electrophoretic mobility shift assay (EMSA) experiments, and we have further confirmed the functional role of these two elements by mutational analysis.


MATERIALS AND METHODS

Cloning and Sequencing of the 5`-Flanking Region of the cx43 Gene

A mouse genomic library was prepared from 129Sv female kidney DNA partially digested with Sau3A, size-selected on a sucrose gradient, and inserted into the BamHI site of Dash (Stratagene, La Jolla, CA). Mouse connexin genomic clones were isolated from this library using a rat cDNA probe (G2A, (1) ), which represents the 5` end of the rat cx43 mRNA. Four clones were thoroughly restriction mapped for BamHI, EcoRI, and HindIII sites (Cx3, Cx6, Cx8, and Cx9; Fig. 1A). Small fragments (200-300 bp) of the 2-kb rat cDNA were used to map the mouse cx43 gene structure relative to this restriction map by probing BamHI, EcoRI, and HindIII digests of the genomic DNA. A 3.5-kb BamHI fragment containing exon 1, derived from Cx8, was fully sequenced on both strands by the dideoxynucleotide termination method(18) .


Figure 1: Restriction map and sequence of the 5`-flanking region of the mouse cx43 gene. A, representative restriction sites of the 20-kb mouse genomic fragment are shown on the top, with the positions of exon 1 and 2 indicated by openboxes. Relative positions of the four overlapping phage clones are indicated as lines underneath the map. B, nucleotide sequence of a SspI/NaeI DNA fragment from the mouse cx43 5` region is aligned with that of the human (16) and rat (17) cx43 genes. Gaps are introduced to allow maximum alignment. Sequences that resemble an AP1 site and a TATA box are indicated. Regions containing positive and negative elements are also underlined. Position of the primer that was used in the primer extension experiment is indicated by a double underline. In the mouse and human sequences, we and the authors of (16) have assigned +1 as the first nucleotide of the transcription start site. In the rat, +1 is assigned to the first nucleotide of the translation start site(17) .



S1 Nuclease Mapping

Mapping of the start site of cx43 transcript was performed as described previously(19) . A 526-bp SspI/NaeI fragment of the cx43 genomic clone (see Fig. 1B) was labeled at its 5` end by T4 polynucleotide kinase and [-P]ATP and purified through a Sephadex column. An aliquot (2 times 10^5 cpm) of labeled DNA fragment was mixed with 50 µg of total RNA at 50 °C for 16 h, then ethanol-precipitated. The sample was digested with S1 nuclease for 1 h at 37 °C. The protected DNA fragments were analyzed on a denaturing acrylamide gel.

Primer Extension

For primer extension analysis, an oligonucleotide complementary to the first exon of the mouse cx43 gene was used (Fig. 1B). Its sequence is as follows: 5`-ACCTGTCTGTTTAAAGTTTCAAAGTCTGCTGCTGTTGGG-3`. It was labeled at its 5` end by T4 polynucleotide kinase and [-P]ATP, and 2 times 10^5 cpm of the probe was incubated with RNA at 42 °C for 1 h in the presence of reverse transcriptase. RNA was obtained from mouse myometrium and heart. As a negative control, mouse liver RNA and yeast tRNA were also included. The extended products were analyzed on a polyacrylamide denaturing gel.

Construction of Plasmids

A 1.8-kb BamHI/NaeI fragment was isolated from the phage clone Cx8, blunt-ended using Klenow, and subcloned into the XhoI site of the vector pSV0ATCAT(20, 21) . The resulting plasmid, -1686, contains 1686 bp upstream and 162 bp downstream of the transcription start site of the cx43. Inserts in plasmids -361, -153, -112, -104, 102, -92, -75, -72, -62, and -54 are the same as that in -1686 except that they deleted various lengths of the upstream portion of the promoter. These inserts were generated either by convenient restriction enzyme digestion or by polymerase chain reaction amplification of the appropriately sized fragments from -1686/+162. Base substitutions in the mutants -104m and -75m were generated by polymerase chain reaction. All the plasmids were sequenced to confirm their identities.

Tissue Culture, Transfection, and CAT Assay

SHM (Syrian hamster smooth muscle) cells were a gift from Dr. K. Riemer (San Francisco, CA) and were originally derived from an androgen/estrogen-induced uterine smooth muscle cell tumor of the Syrian hamster(22) , and Myo14LTR (human myometrial leiomyoma smooth muscle) cells were a gift from Dr. J. McDougall (Seattle, WA; (23) ). Caco2 (24) was kindly provided by Dr. T. C. Suen, Genetics Department, Hospital for Sick Children (Toronto, Ontario, Canada). All the cells were cultured in Dulbecco's modified essential medium (Sigma), supplemented with 10% fetal bovine serum (PDI Biosciences Inc, Canada). Transfections were carried out by the DNA-calcium phosphate coprecipitation method(25, 26) . Cells were plated at approx1.5 times 10^6 cells/10-cm plate 24 h prior to transfection. In each experiment, cells were cotransfected with 2 µg of pRSVbetagal (containing the E. coli lacZ gene under the Rous sarcoma virus promoter for normalization of the transfection efficiency), 7 µg of a CAT construct, and 7 µg of carrier DNA. Cells were harvested approximately 48 h after the transfection, and extracts were prepared by three cycles of freeze-thawing. Approximately 50 µg of protein/sample were used for CAT assay, following normalization by beta-galactosidase activity provided by these extracts. The CAT assay was performed as described(27) ; the reaction products of ^14C-labeled acetylated chloramphenicol were separated by ascending thin layer chromatography (TLC), visualized by autoradiography, excised from the TLC plate, and quantified by scintillation counting.

Nuclear Extract Preparation and EMSA

Nuclear extracts were prepared as described by Dignam et al.(28) with some modifications. Female rats were sacrificed either on day 15 gestation or during labor (day 23). Brain, heart, kidney, and myometrium were excised, minced, and homogenized in 2 ml of homogenization buffer (20 mM HEPES (pH 7.9), 10 mM NaCl, 1.5 mM MgCl(2), 0.2 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin) by Ultra-Turrax T25. Large chunks of tissues and cell debris were removed by filtering through layers of cheesecloth and centrifugation at 500 rpm for 10 min. The resulting cell homogenates were treated the same as tissue culture cells.

The standard EMSA was performed in a final volume of 10 µl of solution containing 200 pg of [-P]ATP-labeled double-stranded oligonucleotide (10,000 cpm), 500 ng of poly(dI-dC), 12% glycerol, 12 mM HEPES, 4 mM Tris (pH 7.9), 1 mM EDTA, 0.6 mM dithiothreitol, 0.3 mg/ml bovine serum albumin, and 3 µg of nuclear protein. After incubation for 30 min at room temperature, the mixture was loaded onto a 6% polyacrylamide gel and electrophoresed for 40 min at 15 V/cm. The gel was dried and autoradiographed. For the competition experiments, 4-, 16-, and 64-fold molar excess of unlabeled competitor oligonucleotides were included in the reactions. Oligomers used in the EMSA were as follows: W1 contains wild-type sequence from -104 to -86 (5`-CTCCTCCCCGCCTTTTCT-3`, 5`-AGAAAAGGCGGGGAGGAG-3`), M1 contains a 3-base substitution of W1 (5`-CTCCTCTTTGCCTTTTCT-3`, 5`-AGAAAAGGCAAAGAGGAG-3`), W2 contains wild-type sequence from -75 to -57 (5`-TTCTCCTAGCCCCTCCTT-3`, 5`-AAGGAGGGGCTAGGAGAA-3`), and M2 is a mutant version of W2, with two 1-base substitutions (5`-TTCTCCTATTCCCTCCTT-3`, 5`-AAGGAGGGAATAGGAGAA-3`).


RESULTS

Genomic Structure

To determine the regulatory mechanisms that influence cx43 gene expression in myometrial cells, we cloned a mouse genomic DNA fragment containing the entire gene encoding for connexin 43 using a rat cx43 probe (Fig. 1). A 3.5-kb BamHI fragment containing the exon 1 was fully sequenced. As shown in Fig. 1, the mouse sequence is about 90% identical to the corresponding region of both human and rat cx43 genes from the start of transcription to position -120(16, 17) . Within this region there is a ``TATA box'' motif and a putative AP1 protein binding site. Upstream from -120, there is evidence for DNA rearrangement including multinucleotide deletion/insertion; upstream of -200, the mouse and human sequences are not comparable, although the rat and mouse maintain considerable sequence identity (Fig. 1). This suggests that there may be conserved regulatory elements downstream of -120. To identify the major transcription start site in mouse myometrium, both S1 nuclease mapping and primer extension experiments were performed. As shown in Fig. 2, the majority of transcription starts from 24 ± 3 bp downstream of the TATA box, which is similar to previous observation made on human, rat, and mouse cx43 genes in other cell types where transcription starts from around 23 bp downstream of the same TATA box(15, 16, 17) .


Figure 2: Determination of transcription start site of the Cx 43 gene. A, primer extension products from yeast tRNA, mouse liver RNA (lanes 1 and 2, negative control), mouse heart RNA (lane3, positive control), and term mouse myometrial RNA (lane 4) were separated on a polyacrylamide gel. Location of the radiolabeled primer is indicated in Fig. 1B. The length of the extension products was determined by comparison to a sequencing reaction product running alongside. B, yeast tRNA (negative control, lane1), mouse heart RNA (positive control, lane2), and term mouse myometrial RNA (lane 3) were hybridized with an end-labeled DNA probe and subsequently digested by S1 nuclease. The probe is the SspI/NaeI DNA fragment from the mouse cx43 5` region, and nucleotide sequence of this probe is shown in Fig. 1B. Protected fragments are visualized by autoradiograph of a sequencing gel.



A Positive and a Negative Regulatory Element Are Contained within Two 10-bp Fragments of the 5` Upstream Region of the cx43 Gene

To identify cis-acting regulatory elements in the cx43 promoter, a DNA fragment containing sequence from -1686 to +162 was linked in cis to a CAT reporter gene. In addition, a series of 5` deletion constructs -368, -153, -112, and -54 were prepared and transient assays were performed in SHM and Myo14LTR cells, both originating from myometrium, and also in Caco2, a colon carcinoma cell line (Fig. 3). The relative transcriptional activity conferred by each of these constructs was measured as the percentage of the transcriptional activity produced by the -1686 construct. The promoter activity of this construct is comparable to that driven under SV40 early promoter in transfected cell lines (data not shown). Fig. 3shows that progressive deletion from -1686 to -54 resulted in a gradual decrease in CAT activity. This implies that, in the region between -1686 and -54, there may exist positive regulatory elements. The fact that the expression pattern of these constructs is similar in each of the three cell lines examined suggests that there may not be elements within the region between -1686 and +162 that confer tissue-specific promoter activity within myometrial cells. We subsequently constructed plasmids -102, -92, -72, and -62 to establish a series of deletion constructs each with a 10-base pair difference in length in order to scan for the putative cis-acting elements in the region between -112 and -54. Whereas plasmids -112, -102, and -62 all produced a similar basal level of CAT activity in SHM cells, the level of transcription driven by both constructs -92 and -72 were high and comparable with that of -1686 (Fig. 4). These results indicate that in the region between -102 and -92 there exists a negative regulatory element, and deletion of this element led to a 10-fold elevation of the promoter activity. In addition, they point out that in the region between -72 to -62 there exists a positive regulatory element required for high level expression of cx43, and deletion of this element reduced the promoter activity to the basal level. We chose to perform further functional analysis on the negative element in the region between -102 and -92, and the positive element in the region between -72 and -62.


Figure 3: 5` deletion analysis of the cx43 promoter region. The recombinant CAT constructs including DNA fragments from the cx43 promoter region are shown on the left. The respective promoter activities are shown in the histogram on the right. The activities are expressed as a percentage of that of construct -1686 within each cell line. Black, gray, and whitebars present results obtained from SHM, Myo14LTR, and Caco2 cells, respectively.




Figure 4: Detailed deletion mapping of the region between -153 and -54 of the cx43 promoter. The recombinant CAT constructs including DNA fragments from the cx43 promoter region are shown on the left. The respective promoter activities (expressed as a percentage of that of construct -1686) in transfected SHM cells are shown in the histogram on the right.



Nuclear Factors in Myometrial Cells Bind Specifically within the Putative cis-Acting Elements

We performed EMSA using nuclear extracts prepared from both hamster and human myometrial cells to determine the ability of the putative cis-acting elements to bind nuclear protein. Oligonucleotide W1 includes the sequence from -102 to -92: 5`-CCTCCCCGCC-3`. The presence of slower migrating DNA-protein complexes indicates that oligonucleotide W1 can interact with myometrial nuclear proteins (Fig. 5, lane 1). Competition experiments showed that 16- and 64-fold molar excess of unlabeled oligonucleotide W1 was able to compete for the protein binding in a concentration-dependent fashion (lanes 2-4). The specificity of the DNA-protein interaction was tested further by mutational analysis. Synthetic oligonucleotide M1 contains a mutant version of the sequence from -102 to -92 such that the trinucleotide CCC in W1 is replaced with TTT in M1. As revealed in Fig. 5(lanes 5-7), the mutation of W1 to M1 resulted in a total loss of the ability of this oligonucleotide to compete for the protein binding. Similar experiments were performed to test the protein binding ability of the positive element (Fig. 6). Oligonucleotide W2 includes the sequence from -72 to -62, 5`-TCCTAGCCCC-3`, and M2, a mutant version, contains the sequence 5`-TCCTATTCCC-3`, in which the dinucleotide GC in W2 is replaced with TT in M2. Oligonucleotide W2 can interact with a myometrial nuclear protein as indicated by the presence of slower moving DNA-protein complexes and also by the ability of excess unlabeled oligonucleotide W2 to compete for the protein binding (lanes 1-4). The mutant oligonucleotide M2 could still compete for the protein binding to the probe W2. However, by comparison with W2, the competing ability of M2 is much less efficient. This suggests that this particular mutant version abolished only part but not all of the protein binding capability of the positive element. Taken together, these data not only demonstrate the specificity of protein binding conferred by W1 and W2, they also define nucleotides that are directly involved in binding of the trans-acting factor. Fig. 7shows that the trans-acting factors are present in extracts of rat brain and myometrium obtained from rats during delivery. We were unable to detect trans-acting factors in nuclear protein extracts prepared from kidney and detected very low levels of protein in heart and in myometrial tissues from pregnant rats, not in labor.


Figure 5: Protein binding to the negative regulatory element. Nuclear extract (3 µg) from Myo14LTR (A) and SHM cells (B) was incubated with a radiolabeled oligonucleotide W1 and protein-DNA binding activity was measured by EMSA. The position of the protein-DNA complexes and that of probe were indicated on the left. Lane1, binding reaction in the absence of competitor DNA. Lanes 2-4 are 4-, 16-, and 64-fold molar excess of the unlabeled W1 as competitor included during binding. Lanes5-7 are 4-, 16-, and 64-fold molar excess of the unlabeled M1, a mutant version of W1, included during binding.




Figure 6: Protein binding to the positive regulatory element. Nuclear extract (3 µg) from Myo14LTR (A) and SHM cells (B) was incubated with a radiolabeled oligonucleotide W2 and protein-DNA binding activity was measured by EMSA. The position of the protein-DNA complexes and that of probe are indicated on the left. Lane1, binding reaction in the absence of competitor DNA. Lanes 2-4 are 4-, 16-, and 64-fold molar excess of the unlabeled W2 as competitor included during binding. Lanes 5-7 are 4-, 16-, and 64-fold molar excess of the unlabeled M2, a mutant version of W2, included during binding.




Figure 7: Tissue-specific distribution of the DNA binding factors associated with probe W1. Nuclear extract from rat tissues was incubated with a radiolabeled oligonucleotide W1 and protein-DNA binding activity was measured by EMSA. These tissues were myometrium of a rat during labor (lane1), and myometrium, kidney, heart, and brain of a rat at day 15 pregnancy (lanes 2-5). The position of the protein-DNA complexes and that of probe were indicated on the left.



Point Mutations Abolished the Regulatory Effects of the cisActing Elements

Function of the regulatory elements in the promoter region of cx43 was further demonstrated by transient CAT assay with mutated promoters. We reasoned that if protein binding to these elements is essential for their regulatory function, then the introduction of point mutations that abolished such protein-DNA interaction should mimic the effect of deletion of these elements. Constructs -104m and -75m are the mutant forms of their wild-type counterparts -104 and -75, respectively, and the mutations introduced into these plasmids are the same base substitutions as those in the mutant oligonucleotides mentioned before. The effect of these mutations on the promoter activity was compared with their wild-type counterparts in transfected SHM cells. Fig. 8showed that, as expected, these point mutations effectively suppressed the regulatory function of these two elements. Construct -104 M drove CAT activity at a similar level to -75, and promoter activity produced by the plasmid -75 M was equivalent to that of -54. Interestingly, mutation of the positive element, -75M, resulted in a complete supression of the function of this element even though EMSA analysis revealed a limited ability of this mutant oligomer to bind nuclear protein in vitro.


Figure 8: Mutational analysis of the two regulatory elements within the cx43 promoter. The recombinant CAT constructs including DNA fragments from the cx43 promoter region are shown on the left. The respective promoter activities in transfected SHM cells are shown in the histogram on the right. Construct -104M contains a 3-base pair substitution within the wild-type -104 construct. Construct -75M contains a 2-base pair substitution within the wild-type -75 construct.




DISCUSSION

This study demonstrates that expression of the cx43 gene in myometrial cell lines is under both positive and negative control at the transcriptional level. Two cis-acting regulatory elements were initially defined by deletion mapping of the promoter and subsequently confirmed by EMSA and mutational analysis. The negative regulatory element lies in a small region between -102 and -92 (Fig. 4). Two lines of evidence suggest that the repressive effect of this negative element on the promoter activity is achieved through its ability to bind with nuclear proteins (Fig. 5). First, a 3-base pair substitution changing the wild-type sequence from 5`-CCTCCCCGCC-3` to 5`-CCTCTTTGCC-3` abolishes its ability to interact with the nuclear protein (as shown in Fig. 5). Second, when introduced into the cx43 promoter, this same mutation suppressed the down-regulatory effect exerted by the element (Fig. 8).

This negative element 5`-CCTCCCCGCC-3` does not share sequence identity with other published repressors and silencers, but does resemble the consensus sequence of the binding site of a positive acting factor, SP1, CCGCCC(29) . We used an SP1 binding site oligonucleotide probe to perform EMSA with HeLa cell nuclear extract, and we found that an excess of unlabeled oligonucleotide W1 was unable to compete the DNA-protein complex shifted by the SP1 probe (data not shown). This suggests that the protein that binds to this negative element may not be related to SP1.

Results from transient transfection assays indicate that the DNA fragment containing 72 bases upstream and 162 bases downstream of the transcription start site of the cx43 gene contains the minimum requirements for high level of transcription of the CAT reporter gene in vitro. The positive element defined in this study constitutes one of the critical regulatory components within this minimal promoter. Mutation of 5`-TCCTAGCCCC-3` to 5`-TCCTATTCCC-3` not only substantially reduces the ability of this positive element to bind with a nuclear protein, it also abolishes its ability to enhance the promoter activity. These data might be interpreted as indicating that the nucleotides not mutated within this oligonucleotide maintain a limited ability to bind the protein in vitro but are not sufficient function as a positive element in vivo (Fig. 8). As with the negative element, this stretch of DNA has no sequence similarity with published regulatory elements.

It is noteworthy that the DNA sequence within -100 bp upstream of the transcription start site is highly similar among human, rat, and mouse cx43 genes (Fig. 1B; (15, 16, 17) ). The two regulatory elements discovered by this study are both localized within this evolutionary conserved region, and each has only one base difference among human, rat, and mouse sequences (Fig. 1B). Moreover, nuclear protein(s) that associate with these elements were found in myometrial cells of both hamster and human origin as well as in tissues of rats. The DNA-binding nuclear proteins were enriched in tissue culture cells of myometrial origin and in nuclear extracts prepared from the brain and in myometrial tissue of delivering rats. In contrast, very little protein was detected in the heart or mid-pregnant myometrium and none in kidney tissue. These results suggest that these DNA-binding proteins are not expressed in all tissues but are not entirely restricted to myometrial cells. Taken together, it is reasonable to speculate that these two elements and their associated proteins may play important regulatory roles in cx43 expression in vivo, especially in myometrium.

The regulation of cx43 gene transcription in cardiocytes has been reported recently. The study by De Leon et al.(16) analyzed transcriptional activity in rat cardiocytes and adult rat heart with a series of deletion constructs of the human cx43 gene promoter. The human deletion constructs and the mouse constructs described here produced a similar pattern of transcriptional activity over a region of greater than 1 kb. Constructs with deletions between -100 to -50 were not analyzed in the cardiocyte study. Consequently it remains to be determined whether the two regulatory elements defined in the present study behave similarly in cardiocytes.

The tissue-restricted expression pattern of cx43, the regulation of its mRNA and protein by steroid hormones (in myometrium; (11) and (14) ) and cyclic AMP (in rat hepatoma; (30) ), and the dramatic up-regulation of expression in myometrium, but not in heart of the animals during delivery(11, 15) , all point to a highly complex machinery that controls cx43 gene expression. The two regulatory elements defined in this study are likely to be components contributing to this complex regulatory machinery.


FOOTNOTES

*
This work was supported by grants from the Medical Research Council of Canada (to S. J. L. and to J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank(TM)/EMBL Data Bank with accession number(s) U17892[GenBank].

§
These authors contributed equally to this work.

Current address: Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia V5Z 1A1, Canada.

**
Current address: Cephalon Inc., West Chester, PA 19380-4245.

§§
Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada and a Howard Hughes International Scholar.

¶¶
Career Scientist of the Ontario Ministry of Health.

(^1)
The abbreviations used are: bp, base pair(s); EMSA, electrophoretic mobility shift assay; kb, kilobase pair(s); CAT, chloramphenicol acetyltransferase.


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