(Received for publication, November 9, 1994)
From the
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.
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) ()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.
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) .
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`).
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.
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.
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.
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.
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17892[GenBank].