Transcriptional regulation of the murine Connexin40 promoter by cardiac factors Nkx2-5, GATA4 and Tbx5

2.1. Cloning and sequencing of the 5′-flanking region of the Cx40 gene

A 2.4 kilobase pair (kb) fragment of the 5′-flanking region of the Cx40 gene was amplified by polymerase chain reaction (PCR) using the Genome Walker Kit (Clontech) and specific primers 12AS and 8AS. The fragment was cloned, sequenced and deposited at GenBank under accession number AF375853. All primers used in this work are described in Supplementary Table 1.

2.2. Cx40 promoter-luciferase constructs

The following plasmids used in this study were previously described elsewhere [12]: −1190/+121Cx40Luc; −889/+121Cx40Luc and −558/+121Cx40Luc. The construct −203/+121Cx40Luc was obtained using Double Strand Nested deletion Kit (Amersham Pharmacia Biotech), while constructs −150/+121Cx40Luc, −50/+121Cx40Luc and −20/+121Cx40Luc were generated by PCR and cloned into the pGL3-Basic (Promega). Site directed mutagenesis of the putative Nkx2-5 site (NKE) was performed using QuickChange Site-Direct Mutagenesis Kit (Stratagene) and confirmed by sequencing.

2.3. Cell culture and transient transfections

The embryonic rat smooth muscle cell line derived from the thoracic aorta, A7r5, COS7 and CH310T1/2 fibroblasts were obtained from American Type Culture Collection (Rockville, MD). Rat neonatal cardiomyocytes were isolated as previously described [15]. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM-GIBCO) supplemented with 10% (vol/vol) fetal bovine serum (GIBCO) and maintained in a humidified 10% CO₂ incubator at 37 °C. 10T1/2 cells were transfected as previously described [12]. A7r5 cells were seeded into 24-well plates to reach 70% confluence by the next day and transiently transfected using Lipofectamine 2000 (Invitrogen) with 0.5 μg of each test plasmid and 0.1 μg of the control pRL-CMV (Promega), carrying the Renilla luciferase gene. Cells were harvested after 48 h. The results were expressed as fold activity after normalization for Renilla or β-Galactosidase activity. Transfections were performed in triplicate in at least three independent experiments.

2.4. Preparation of nuclear extracts and GST fusion proteins

Nuclear extracts were prepared 48 h after transfection; 100 mm² plates were homogenized in 300 μl of buffer I (10 mM NaCl; 20 mM Hepes pH 7.4; 1.5 mM MgCl₂; 0.1% v/v TritonX-100; 20% v/v Glycerol; 1 mM DTT; 0.5 mM PMSF), chilled on ice for 10 min, and precipitated by centrifugation at 2800×g for 1 min at 4 °C. Nuclear fraction was resuspended in 300 μl of buffer C (150 mM NaCl; 50 mM Hepes pH 7.4; 1.5 mM MgCl₂; 1 mM EGTA; 10 mM Na pyrophosphate; 1% v/v Triton X-100; 10% v/v glycerol; 1 mM DTT; 0.5 mM PMSF) plus 41.5 μl of 5 M NaCl and incubated for 1 h at 4 °C. The nuclear extracts were then centrifuged at 14,000×g for 10 min and stored at −70 °C. Recombinant Tbx5 or the Nkx2-5HD proteins were purified on glutathione-Sepharose columns (Amersham Pharmacia Biotech) and eluted by cleavage with 5 U/ml of thrombin (Amersham Pharmacia Biotech) generating GST-free proteins used in the EMSA. Protein concentration was determined by Bradford method (BIORAD).

2.5. Electrophoretic mobility shift assay (EMSA)

Five to ten micrograms of nuclear extracts were mixed with binding buffer (25 mM Hepes pH 7.4; 75 mM NaCl; 1 mM MgCl₂; 0.2 mM EDTA; 0.1% v/v NP-40; 1 mM DTT; 10 ng BSA; 200 ng poly dI/dC) and incubated for 10 min at room temperature. Annealed oligonucleotides (5 pM) were labeled using [γ³²P] ATP and T4 polynucleotide kinase (NEB); 50,000 cpm were added to each sample and incubated at room temperature for 20 min; 10 μl of the mixture was submitted to electrophoresis on a 5% PAGE gel 0.5× TBE or 1× Tris Glycine gels, dried and subjected to autoradiography. Supershift assays were performed as described above except that 0.5–2 μg of antibody against Sp1 (Sigma) or GATA4 C20 (Santa Cruz Biotechnology) were added to the reaction for 10 min before incubation with the ³²P-labeled oligonucleotide.

2.6. RNA extraction and RT-PCR

RNA was extracted from hearts of wild-type or homozygous knockout Nkx2-5 mice littermates at 9 days post coitum (dpc) stage using TRIZOL method (Invitrogen), treated with RQ1 RNase-free Dnase and reverse transcribed using M-MuLV transcriptase with oligo-dT primers. Samples were amplified using specific primers. PCR conditions consisted of 40 cycles at 94 °C for 1 min, 52 °C for 1 min, 72 °C for 1 min using 5 U of Taq polymerase (Merck).

3.1. Transcriptional activity of the Cx40 promoter

To identify cis-acting regulatory elements of the Cx40 gene, a 2.4 kb DNA fragment was amplified from a mouse genomic library and several deletions, ranging from −1190 to −20 relative to the transcription start site were linked to a luciferase reporter gene (Fig. 1A). The resulting series of constructs were transiently transfected into A7r5 cells that express high levels of Cx40 [16] and several other cardiac connexins, including Cx43 and Cx45 (Supplementary Fig. S1). An increase of 55 fold, above control plasmid, was observed for the −1190/+121Cx40Luc construct in A7r5 (Fig. 1A). Similar levels of activity were observed for 203/+121Cx40Luc (Fig. 1B), suggesting that the sequences between −203 and +121 contain important regulatory modules controlling expression of Cx40. Removal of an additional 50 bp from this construct reduced the activity of the −150/+121Cx40Luc plasmid to only 28% of the activity observed for the −1190/+121Cx40Luc construct and further deletions of 100 bp led to the complete silencing of the reporter gene. These data indicate that in A7r5 cells maximal activity for this promoter requires only the first 203 bp of the promoter and further that a basal promoter is found within the first 150 bp upstream of the transcriptional start site of this gene. Importantly, the −150/+121Cx40Luc construct displays equal activity to −1190/+121Cx40Luc when transfected into embryonic primary cardiomyocytes (Fig. 1C).

3.2. Nuclear factor Sp1 binds specifically within the proximal region of the Cx40 promoter

Our transfection data indicates that the elements required for the basal activity of the Cx40 promoter are located between −150 and −50 relative to the transcriptional start site. Within this region, several putative binding sites for transcription factors were predicted by computer analysis. To validate these sites functionally, EMSAs were performed using the identified sequences as probes (Fig. 2A).

Initial experiments investigated the binding site for the Sp1/Sp3 family members since a highly homologous region of the rat Cx40 promoter (Fig. 2A) was recently shown capable of specific interaction with members of the Sp1/Sp3 proteins [14]. Specific bands were identified in EMSAs of cardiomyocyte and A7r5 nuclear extracts (Fig. 2B and data not shown). Furthermore, addition of an antibody to Sp1 resulted in the appearance of a slower migrating band (Fig. 2B, arrow), confirming that these complexes contained Sp1. Thus, functional Sp1 sites are found in the promoters of both the rat and mouse Cx40.

Since Sp1 and Sp3 are ubiquitous transcriptional factors, these results raised an important question regarding Cx40 expression control: what are the unique proteins that regulate the cell-specific expression of Cx40? The role of the ubiquitous Sp1/Sp3 factors in the transcriptional activation of Cx40 appears to be related to stimulation of basal activity, indicating that additional interactions are necessary for correct transcriptional activation of the Cx40 gene in muscle cells.

3.3. Cardiac transcription factors Nkx2-5 and GATA4 confer specific Cx40 promoter transcriptional activation

The proximal promoter of Cx40 has putative DNA-binding sites for two important cardiac specific transcriptional factors, Nkx2-5 and GATA4. Since these factors are both capable of supporting synergistic activation [15,17] and are present in the heart during the onset of Cx40 expression (Fig. 3A), we decided to investigate whether they could influence Cx40 promoter activity. The co-expression of either Nkx2-5 or GATA4 induced an approximate three- to six-fold increase in luciferase activity from −1190/+121Cx40Luc (Fig. 3B). Addition of both factors together led to a 10-fold increase in activation. The same effect was observed when the minimal promoter −150/+121Cx40Luc construct was used (Fig. 3C), indicating that this region contained the sites required for activation by both Nkx2-5 and GATA4 transcription factors.

3.4. Nkx2-5 and GATA4 are both capable of specific interaction within the minimal promoter of Cx40

The minimal promoter contains putative sites for interaction with NK and GATA family members. To test if the cardiac factors Nkx2-5 and GATA4 could interact with these sites, EMSA was performed. The homeodomain of Nkx2-5 was capable of specific interaction with its putative site within the Cx40 proximal promoter (Fig. 4B, lanes 7–10) in a dose dependent manner. Competition experiments with an excess of unlabeled self-oligonucleotide competitor or the high affinity multimerized site for Nkx2-5, 3xHA or control oligonucleotides (NKE mutant or nonspecific oligonucleotide, Fig. 4C, lanes 2–6) demonstrated that the observed mobility shift was due to binding of the Nkx2-5 homeodomain to the corresponding recognition site in the Cx40 promoter. GATA4 was also able to interact specifically with its putative site, as indicated by the competition with self-oligonucleotide or its closely related site in the ANF promoter (Fig. 4C, lanes 3–5), and this complex was supershifted with GATA4 antibodies (Fig. 4C, lane 8).

3.5. Tbx5 has a repressive role on the Cx40 promoter in 10T1/2 cells

T-box transcription factors are important regulators of early cell fate decision in the embryo. One member of the family, Tbx5, is expressed at high levels in the embryonic heart and is an essential component of the myogenic program. Mouse homozygous for a null Tbx5 allele die at mid gestation with severe cardiac defects, and haploinsufficiency in humans results in the autosomal dominant disorder, Holt Oram Syndrome [18].

The presence of a putative site for T-box factors that overlapped the NKE site within the proximal Cx40 promoter led us to investigate if Tbx5 could also be involved in Cx40 regulation. Tbx5 was unable to activate the Cx40 minimal promoter construct in transient transfections of 10T1/2 cells. Interestingly, however, Tbx5 strongly inhibited activation of the proximal promoter mediated by Nkx2-5/GATA4 complex (Fig. 5B). This was specific for the Cx40 promoter since Tbx5 elicited a strong activation of the ANF promoter and displayed synergism with Nkx2-5 and GATA4 or either factor alone (Fig. 5C). Despite these differences, Tbx5 was capable of specific interaction with both Cx40 and ANF proximal sites as detected by EMSA (Fig. 5D).

3.6. Mutation of proximal NKE site impairs Cx40 expression in A7r5

To confirm the role of Nkx2-5 in the regulation of Cx40 promoter, the NKE site present in the minimal promoter −150/+121Cx40Luc was mutated (Fig. 6A) and the ability of this factor to activate transcription assessed in A7r5 cells. When this mutated region was used as competitor oligonucleotide, there was no displacement of Nkx2-5 binding from its site on the proximal promoter (Fig. 4B, lane 5). A significant decrease in promoter activity around 50% was observed when the mutant promoter was assayed in A7r5. Co-transfection of Nkx2-5 in A7r5 cells expressing the minimal promoter region led to a significant increase in promoter activity that was also abolished when the NKE mutated promoter was used (Fig. 6). Together, this data suggests that binding of Nkx2-5 is critical for normal expression of Cx40.

3.7. Cx40 expression is downregulated in vivo in Nkx2-5 knockout mice

Overexpression of a dominant negative isoform of Nkx2-5 in the heart of transgenic mice leads to severe atrioventricular conduction defects that were correlated with decreased Cx40 expression [10]. To further confirm the modulation of Cx40 expression by Nkx2-5, we analyzed the levels of Cx40 mRNA in 9 dpc hearts of Nkx2-5 knockouts by RT-PCR. Indeed, Cx40 expression was markedly decreased in the knockout animals when compared with wild-type littermates (Fig. 7). Interestingly, Cx43 mRNA was also downregulated in the knockout embryos, in agreement with previous results indicating that both connexin genes might be regulated in cardiac tissues by Nkx2-5 [10].

Together these data support a role for the cardiac transcriptional factors Nkx2-5 and GATA4 in the regulation of Cx40 expression.