GATA elements control repression of cardiac troponin I promoter activity in skeletal muscle cells
© Di Lisi et al; licensee BioMed Central Ltd. 2007
Received: 27 February 2007
Accepted: 17 September 2007
Published: 17 September 2007
We reported previously that the cardiac troponin I (cTnI) promoter drives cardiac-specific expression of reporter genes in cardiac muscle cells and in transgenic mice, and that disruption of GATA elements inactivates the cTnI promoter in cultured cardiomyocytes. We have now examined the role of cTnI promoter GATA elements in skeletal muscle cells.
Mutation or deletion of GATA elements induces a strong transcriptional activation of the cTnI promoter in regenerating skeletal muscle and in cultured skeletal muscle cells. Electrophoretic mobility shift assays show that proteins present in nuclear extracts of C2C12 muscle cells bind the GATA motifs present in the cTnI promoter. However, GATA protein complex formation is neither reduced nor supershifted by antibodies specific for GATA-2, -3 and -4, the only GATA transcripts present in muscle cells.
These findings indicate that the cTnI gene promoter is repressed in skeletal muscle cells by GATA-like factors and open the way to further studies aimed at identifying these factors.
Establishment and maintenance of cell specification involves both activating and repressive mechanisms. While positive gene regulation by tissue-specific transcription factors is well established, the role of negatively acting transcription factors has been comparatively less investigated. For example, cardiac and skeletal muscle cells are known to co-express a large number of muscle genes, but the regulatory regions in the gene promoters differ between the two cell types. Both common regulatory factors, such as MEF2, and cardiac- or skeletal muscle-specific transcription factors, such as GATA-4 and MyoD, respectively, have been implicated as tissue-specific activators of muscle gene expression. A few studies have described the repression of muscle gene as a mechanism that control cardiac muscle restricted gene expression. Mutations in the HF-3 element (TAACCTTGAAGGC) of the ventricular myosin light chain 2 promoter leads to marked up-regulation of promoter activity in skeletal muscles of transgenic mice . The transcription factor Nishéd, which binds to a GAAG/CTTC sequence, appears to account for the repression of the chicken cardiac myosin light chain 2 in skeletal muscle .
The cardiac troponin I (cTnI) gene encodes for the cardiac specific inhibitory subunit of the troponin complex, one of the few sarcomeric proteins that is expressed in cardiac but not skeletal muscle . We have reported previously that this specificity is controlled by the proximal 5'-flanking region of the promoter (-230/+16), which drives cardiac-specific expression both in cultured cardiac cells, in terminally differentiated cardiac muscle cell in vivo and in transgenic mice . Different types of transcription factors control the expression of the cTnI promoter in cardiac muscle cells. Among these, three GATA elements, that are targets of GATA-4 transcription factor, are required for promoter activity, as disruption of these elements inactivates the cTnI promoter in cultured cardiomyocytes . Here we report that, surprisingly, the activity of the cTnI promoter is markedly increased in skeletal muscle by mutation or deletion of GATA sites. Nuclear extracts of muscle cells contain proteins distinct from canonical GATA factors that form specific complexes with the GATA elements present in the cTnI promoter. These findings point to a novel mechanism of repression of cardiac gene promoters in skeletal muscle involving GATA sites.
cTnI promoter activity is increased by disruption of GATA elements in skeletal muscle cells
The proximal promoter of the cTnI gene (-230/+16) is sufficient to drive cardiac-specific expression of reporter genes both in cultured cardiomyocytes and in transgenic mice . Promoter analysis showed that three types of regulatory elements are required for optimal gene activation. These include three GA-rich elements binding Sp1 and located betweeen -175 and -133, an A/T-rich motif binding MEF2 and Oct1 and lying at position -36, and three GATA elements, two of which are closely associated between -67 and -57 of the promoter whereas the third is located just upstream of the transcriptional start site. Deletion and mutation analyses showed that mutation of GATA elements inactivated the cTnI promoter in cultured cardiomyocytes .
cTnI GATA binding activity is present in nuclear extracts of skeletal muscle cells
GATA-2, -3 and -4, but not GATA-1, -5 and -6 transcripts are present in skeletal muscle cells
GATA-2, -3 and -4 proteins are not responsible for cTnI GATA binding activity in skeletal muscle cells
The results reported here indicate that the cTnI gene promoter is actively repressed in cultured skeletal muscle cells and in regenerating adult skeletal muscle by factors that binds GATA elements. Both mutation and deletion of GATA elements in the cTnI promoter strongly up-regulate promoter activity in skeletal muscle cells. In addition, EMSA experiments show that 1) nuclear extracts of muscle cells contain proteins that form specific complexes with GATA elements present in the cTnI promoter, and 2) these proteins do not correspond to GATA-2, -3 and -4, the only GATA transcripts expressed in skeletal muscle cells. The nature of the GATA-like factor(s) responsible for the repression of the cTnI promoter in skeletal muscle cells thus remains to be established.
A role of GATA elements in the regulation of skeletal muscle gene promoters has been previously proposed. Mutation of a GATA motif in the myosin light chain 3 promoter was found to attenuate its activity in primary cultures of neonatal rat skeletal muscle, however, the nature of the factor(s) binding to this site was not determined . Deletion of a GATA binding sequence in the dihydropyridine-sensitive receptor (DHPR) α1 subunit promoter leads to reduction in promoter activity and antisense experiments for GATA-2 induced functional changes in L-type calcium channels which are consistent with decreased expression of α1 . However, direct interaction of GATA2 with DHPR promoter was not demonstrated. An interaction between GATA factors and MyoD in the regulation of the FGF4 gene promoter in embryonic myotomes has also been described . However, EMSA assays were performed in HeLa cells and no evidence was presented for the presence of GATA factors in the myotomes. Thus direct evidence for the presence of GATA factors in nuclear extracts of skeletal muscle cells was not reported in these studies.
GATA factors are best known as transcriptional activators and inducers of cell differentiation. However, several studies indicate that they can also act as transcriptional repressors in different cell systems, including heart , adipose tissue  and erythroid cells . Proteins distinct from canonical GATA transcription factors can also bind GATA elements and inhibit gene expression. The ubiquitous nuclear DNA binding protein Ku70 was shown to have a repressor activity in the transcriptional regulation of the GBP and KEL promoters by binding WGATAG, but not WGATAA elements [21, 22]. However, it is unlikely that Ku70 is responsible for the repression of the cTnI promoter in skeletal muscle since all of the GATA elements in the cTnI promoter have a GATAA consensus sequence. GATA-like protein-1 (GPL-1), which is expressed at high levels in the somatic cells of the developing gonads, but not in skeletal muscle, acts as a transcriptional repressor of GATA factor function .
In conclusion, our results suggest that the transcription of cTnI gene is actively repressed in skeletal muscle cells by proteins binding to GATA motifs in the proximal promoter. The finding that the transcription of the cTnI gene promoter is actively repressed in skeletal muscle cells raises the possibility that under certain conditions this inhibitory mechanisms may be relieved and cTnI transcription may take place also in skeletal muscle. Indeed, cTnI mRNA has been detected in skeletal muscle biopsies of patients with Duchenne muscular dystrophy at the mRNA level  though not at the protein level .
The present study shows that cTnI promoter activity is markedly increased in skeletal muscle by mutation or deletion of GATA sites and that these sites form specific complexes with proteins present in nuclear extracts of muscle cells but different from canonical GATA factors. Although the GATA-like factors involved in the repression of the cTnI promoter have not yet been identified, our results suggest a novel mechanism of repression of cardiac promoters in skeletal muscle.
The -230/+16 cTnI promoter linked to chloramphenicol acetyl transferase (CAT) and the mutated constructs have been described . Deletion of the two GATA elements (-67/-57) was obtained by double PCR as described . Briefly, the cTnI promoter was amplified in two separate reactions: from -230 to -67 and from -56 to +16 with the oligos -67 and the -56 designed with a partial overlap. Each PCR product was gel purified and combined in an equimolar ratio. Three cycles without primers were performed, and subsequently 20 cycles of PCR were performed with addition of the 5'-upstream and 3'-downstream primers. The final amplified product was digested with HindIII and SalI and cloned into the plasmid pCAT enhancer (Promega). The construct was confirmed by sequencing.
Muscle regeneration and in vivo transfection
Male Wistar rats (200–250 g) were anesthetized with Isoflurane (FORANE, Abbott SpA) at 1–1.5% with oxygen. The rat soleus muscle was exposed and muscle degeneration/regeneration was induced by injecting 0.5% bupivacaine (Marcaine) in saline solution. Intramuscular injection of plasmid DNA (50 μg) was performed at day 3 after bupivacaine treatment as described previously . Muscles were removed at 7 days after injury and frozen in isopentane cooled in liquid nitrogen. Animals were kept in conventional facilities with free access to food and water. Adequate care for their health and well-being was provided in accordance with the Italian Animal Act (Law 116/92). These studies were conducted under the supervision of the Institutional Ethics Committee.
Muscle cell cultures and transfections
Primary cultures of cardiac and skeletal muscle cells were prepared from neonatal rat heart and hind limb muscles as described previously . Skeletal muscle cells were grown for 3 days in DMEM supplemented with 20% fetal calf serum, 2% glutamine and 12.5 U/ml penicillin-streptomycin, then transfected with 10 μg of the cTnI-CAT constructs and 1 μg of CMV-lacZ, as an internal control. After 24 h, the cells were induced to differentiate by changing the medium to DMEM supplemented with 4% horse serum, 2% glutamine, 12.5 U/ml of penicillin-streptomycin. The cultures were harvested after 2 days in the differentiation medium. A similar procedure was used to obtain differentiated cultured from C2C12 cells.
Muscles were homogenized in 200 μl ice-cold lysis solution containing 100 mM potassium phosphate pH 7.8, 0.1% triton and 1 mM dithiothreitol (added just before use). Tissues were homogenized with an Ultra Turax T25. The homogenates were centrifuged at 10.000 rpm for 30 min at 4°C; the supernatants were frozen in liquid nitrogen and stored at -80°C. CAT activity was assayed by TLC scintillation counts as described . Values (means ± S.E.) were expressed as fold increase in CAT activity over the promoterless construct. Each construct was tested in 4 to 8 independent experiments. For muscle cell cultures β-Galactosidase and chloramphenicol acetyl transferase (CAT) assays were performed with 25 μl of muscle cell lysate. Values (means ± S.E.) were expressed as fold increase in normalized CAT activity over the promoterless construct. Each construct was tested in 3 to 5 independent experiments.
For real-time PCR assays, total RNA was prepared from C2C12 myotubes with SV Total RNA Isolation reagent (Promega). 400 ng of RNA was converted to cDNA using random hexamers and Superscript II (Invitrogen). PCR amplification was performed with an IQ5 real time PCR system (BioRad) using SYBR green. The primers were designed to span an intronic sequence and were validated by PCR and gel analysis. Primer sequences were as follows (forward & reverse):
F: 5' GCTCAGCAGCCTATTCTTCC 3'
R: 5' CGTTGCTCCACAGTTCACAC 3'
F: 5' ACGCCTGTGGCCTCTACTAC 3'
R: 5' GGATTTGCTGGACATCTTCC 3'
F: 5' ACCACGTCCCGTCCTACTAC 3'
R: 5' AGAGATCCGTGCAGCAGAG 3'
F: 5' CTGTCATCTCACTATGGGCAC 3'
R: 5' CCAAGTCCGAGCAGGAATTTG 3'
F: 5' TTTGAAGGCAGAGTCCAGTC 3'
R: 5' AGGCAAAGTCTTCAGGTTCG 3'
F: 5' TTCTACACAAGCGACCACCTC 3'
R: 5' GCCGTCTTGACCTGAATACTT 3'
The data were normalized using oligonucleotide primers for cyclophilin A as an internal standard.
Nuclear extracts (20 μg) from C2C12 myotubes or other cell types were separated by SDS-PAGE using 8% gels and blots were incubated with monoclonal antibodies specific for GATA-2, -3, -4 and a polyclonal antibody specific for MEF2 (Santa Cruz Biotechnology Inc.). Antibody binding was revealed by use of SuperSignal West Pico chemiluminescent substrate (Pierce).
Gel Mobility Shift Assay
Oligonucleotides used in gel shift assays *
5' CGCCTGTTATCGCTTATCCTGGG 3'
GCGGACAATAG CGAATAG GACCC
cTnI mut GATA → CACA
5' CGCCTGTTG TG GCTTG TG CTGGG 3'
GCGGACAA C A C CGAA C A C GACCC
cTnI mut GATA → GCCA
5' CGCCTGTTGG CGCTTGG CCTGGG 3'
GCGGACAA CC G CGAA CC G GACCC
5'GATCCCAGGAATGTGTCTGATA AATCAGAGATA ACCCA 3'
Data are expressed as means ± SE (represented as error bars). Comparisons were made using the Student's t test, considering P < 0.05 statistically significant.
This work was supported by grants from the European Union (Network of Excellence MYORES, contract LSHG-CT-2004-511978, and Integrated Project HeartRepair, contract LSHM-CT-2005-018630) to SS, and from the University of Padova (Progetto di Ateneo 2005 CPDA054298) to SA. We thank Elisa Calabria and Paola Del Piccolo for help in construct preparation and Western blotting.
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