BACKGROUND
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 mef <dig>  and cardiac- or skeletal muscle-specific transcription factors, such as gata- <dig> 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- <dig> element  of the ventricular myosin light chain  <dig> promoter leads to marked up-regulation of promoter activity in skeletal muscles of transgenic mice  <cit> .
the transcription factor nishéd, which binds to a gaag/cttc sequence, appears to account for the repression of the chicken cardiac myosin light chain  <dig> in skeletal muscle  <cit> .
the cardiac troponin i  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  <cit> .
we have reported previously that this specificity is controlled by the proximal 5'-flanking region of the promoter , which drives cardiac-specific expression both in cultured cardiac cells, in terminally differentiated cardiac muscle cell in vivo and in transgenic mice  <cit> .
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- <dig> transcription factor, are required for promoter activity, as disruption of these elements inactivates the ctni promoter in cultured cardiomyocytes  <cit> .
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.
RESULTS
ctni promoter activity is increased by disruption of gata elements in skeletal muscle cells
the proximal promoter of the ctni gene  is sufficient to drive cardiac-specific expression of reporter genes both in cultured cardiomyocytes and in transgenic mice  <cit> .
promoter analysis showed that three types of regulatory elements are required for optimal gene activation.
these include three ga-rich elements binding sp <dig> and located betweeen - <dig> and - <dig>  an a/t-rich motif binding mef <dig> and oct <dig> and lying at position - <dig>  and three gata elements, two of which are closely associated between - <dig> and - <dig> 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  <cit> .
we have now performed ctni promoter analyses in skeletal muscle, a tissue that does not express ctni  <cit> .
for these experiments we used the regenerating rat soleus muscle, a model that allows efficient gene transfer  <cit>  and can be used for promoter analyses in vivo  <cit> .
as shown in figure  <dig>  in regenerating muscle the - <dig> ctni/cat promoter/reporter construct is expressed at very low levels, similar to the levels seen in non muscle cells  <cit> .
the activity of the promoter is neither affected by deletion of the three sp <dig> binding elements located between - <dig> and - <dig>  <cit>  nor by mutation of the a/t-rich motif.
in contrast, mutation of the proximal gata element  surprisingly leads to 5-fold increase in promoter activity and an even greater effect is observed by mutating the more distal tandem gata elements or all three gata motifs .
to rule out the possibility that this effect is due to generation of new binding sites, we produced two additional mutants: in the first we deleted the two upstream gata elements in the context of the - <dig> promoter, in the other we made a different mutation in all three gata sites .
as shown in fig.
<dig>  promoter activity is also markedly increased in the deleted mutant and in the construct with the gata → gcca mutation.
the activation of the promoter cannot therefore be due to generation of new binding sites for activating transcription factors, but must result from the lack of a repressive effect of factors binding to these elements.
next we asked whether the effect of gata mutations in the ctni promoter is also observed in cultured skeletal muscle cells.
primary cultures from newborn rat heart and skeletal muscle were transfected with wild-type ctni promoter and mutated constructs.
as shown in fig.
<dig>  promoter activity was decreased in cardiac but was markedly increased in skeletal muscle cells by mutations of gata elements.
similar effects were observed with c2c <dig> cells .
ctni gata binding activity is present in nuclear extracts of skeletal muscle cells
these results suggest that factors binding to gata elements are present in skeletal muscle cells and are required for repressing the ctni promoter in these cells.
to support this interpretation, we performed electrophoretic mobility shift assays  with c2c <dig> muscle cell nuclear extracts .
using a dna probe spanning the two distal gata sites of the ctni promoter and nuclear extracts from differentiated c2c <dig> cells, we identified a major retarded complex .
the formation of this complex was prevented by self-competition  and by gata sequences from the b-type natriuretic peptide  promoter .
in contrast, competition with 50-fold or 100-fold molar excess of double stranded oligonucleotides containing the same mutations in the gata sites used for promoter analyses  did not remove the complex.
therefore, a specific gata binding activity is present in nuclear extracts of c2c <dig> muscle cells.
gata- <dig>  - <dig> and - <dig>  but not gata- <dig>  - <dig> and - <dig> transcripts are present in skeletal muscle cells
gata transcription factors have been grouped in two subfamilies based on their expression pattern: gata- <dig>  - <dig> and - <dig> genes are mainly expressed in hematopoietic cells, while gata- <dig>  - <dig> and - <dig> are expressed in various mesoderm- and endoderm-derived tissues, including heart and gut  <cit> .
there is no report on the expression of gata factors in skeletal muscle, except for gata- <dig> and - <dig>  gata- <dig> transcripts are expressed in the somites at e <dig>  and gata- <dig> promoter can also drive lacz expression in somites at e <dig>   <cit> .
gata- <dig> transcript and protein are induced by igf- <dig> in skeletal muscle fibers undergoing hypertrophy both in vitro and in vivo  <cit> .
we investigated the expression of gata factors by rt-pcr in c2c <dig> muscle cells and detected the presence of gata- <dig>  - <dig> and - <dig> but not gata- <dig>  - <dig> and - <dig> transcripts .
quantitative real-time pcr showed that gata- <dig> mrna is relatively more abundant than gata- <dig> and gata- <dig> in muscle cells .
gata- <dig>  - <dig> and - <dig> proteins are not responsible for ctni gata binding activity in skeletal muscle cells
western blotting analysis was performed to determine whether gata- <dig>  - <dig> and - <dig>  which are detected in skeletal muscle cells at the transcript level, are also present at the protein level.
however, with the exception of a very faint band observed for gata- <dig>  these gata factors were not detected in blots from c2c <dig> myotubes incubated with specific antibodies .
gel shift assays were also used to determine whether gata- <dig>  - <dig> and - <dig> proteins are in fact present in skeletal muscle cell nuclear extracts and bind gata elements of the ctni promoter.
as shown in fig.
<dig>  the gata- <dig> antibody, that partially inhibits the formation of the specific complex in control jurkat nuclear extract , does not significantly change the pattern of gel retardation with c2c <dig> extract .
in the presence of the gata- <dig> antibody, a clear supershift is observed with jurkat nuclear extracts , whereas the pattern of gel retardation is unchanged with c2c <dig> extract .
finally, a gata- <dig> antibody produced a strong supershift with cardiac muscle cell nuclear extract  but not with c2c <dig> cell extract .
discussion
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- <dig>  - <dig> and - <dig>  the only gata transcripts expressed in skeletal muscle cells.
the nature of the gata-like factor 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  <dig> promoter was found to attenuate its activity in primary cultures of neonatal rat skeletal muscle, however, the nature of the factor binding to this site was not determined  <cit> .
deletion of a gata binding sequence in the dihydropyridine-sensitive receptor  α <dig> subunit promoter leads to reduction in promoter activity and antisense experiments for gata- <dig> induced functional changes in l-type calcium channels which are consistent with decreased expression of α <dig>  <cit> .
however, direct interaction of gata <dig> with dhpr promoter was not demonstrated.
an interaction between gata factors and myod in the regulation of the fgf <dig> gene promoter in embryonic myotomes has also been described  <cit> .
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  <cit> , adipose tissue  <cit>  and erythroid cells  <cit> .
proteins distinct from canonical gata transcription factors can also bind gata elements and inhibit gene expression.
the ubiquitous nuclear dna binding protein ku <dig> was shown to have a repressor activity in the transcriptional regulation of the gbp and kel promoters by binding wgatag, but not wgataa elements  <cit> .
however, it is unlikely that ku <dig> 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- <dig> , 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  <cit> .
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  <cit>  though not at the protein level  <cit> .
CONCLUSIONS
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.

 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 mef and cardiac- or skeletal muscle-specific transcription factors, such as gata- 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- element of the ventricular myosin light chain 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 in skeletal muscle. the cardiac troponin i 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, 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- 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 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 sp and located betweeen - and - an a/t-rich motif binding mef and oct and lying at position - and three gata elements, two of which are closely associated between - and - 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. we have now performed ctni promoter analyses in skeletal muscle, a tissue that does not express ctni. for these experiments we used the regenerating rat soleus muscle, a model that allows efficient gene transfer and can be used for promoter analyses in vivo. as shown in figure in regenerating muscle the - ctni/cat promoter/reporter construct is expressed at very low levels, similar to the levels seen in non muscle cells. the activity of the promoter is neither affected by deletion of the three sp binding elements located between - and - nor by mutation of the a/t-rich motif. in contrast, mutation of the proximal gata element surprisingly leads to 5-fold increase in promoter activity and an even greater effect is observed by mutating the more distal tandem gata elements or all three gata motifs. to rule out the possibility that this effect is due to generation of new binding sites, we produced two additional mutants: in the first we deleted the two upstream gata elements in the context of the - promoter, in the other we made a different mutation in all three gata sites. as shown in fig. promoter activity is also markedly increased in the deleted mutant and in the construct with the gata → gcca mutation. the activation of the promoter cannot therefore be due to generation of new binding sites for activating transcription factors, but must result from the lack of a repressive effect of factors binding to these elements. next we asked whether the effect of gata mutations in the ctni promoter is also observed in cultured skeletal muscle cells. primary cultures from newborn rat heart and skeletal muscle were transfected with wild-type ctni promoter and mutated constructs. as shown in fig. promoter activity was decreased in cardiac but was markedly increased in skeletal muscle cells by mutations of gata elements. similar effects were observed with c2c cells. ctni gata binding activity is present in nuclear extracts of skeletal muscle cells these results suggest that factors binding to gata elements are present in skeletal muscle cells and are required for repressing the ctni promoter in these cells. to support this interpretation, we performed electrophoretic mobility shift assays with c2c muscle cell nuclear extracts. using a dna probe spanning the two distal gata sites of the ctni promoter and nuclear extracts from differentiated c2c cells, we identified a major retarded complex. the formation of this complex was prevented by self-competition and by gata sequences from the b-type natriuretic peptide promoter. in contrast, competition with 50-fold or 100-fold molar excess of double stranded oligonucleotides containing the same mutations in the gata sites used for promoter analyses did not remove the complex. therefore, a specific gata binding activity is present in nuclear extracts of c2c muscle cells. gata- - and - but not gata- - and - transcripts are present in skeletal muscle cells gata transcription factors have been grouped in two subfamilies based on their expression pattern: gata- - and - genes are mainly expressed in hematopoietic cells, while gata- - and - are expressed in various mesoderm- and endoderm-derived tissues, including heart and gut. there is no report on the expression of gata factors in skeletal muscle, except for gata- and - gata- transcripts are expressed in the somites at e and gata- promoter can also drive lacz expression in somites at e. gata- transcript and protein are induced by igf- in skeletal muscle fibers undergoing hypertrophy both in vitro and in vivo. we investigated the expression of gata factors by rt-pcr in c2c muscle cells and detected the presence of gata- - and - but not gata- - and - transcripts. quantitative real-time pcr showed that gata- mrna is relatively more abundant than gata- and gata- in muscle cells. gata- - and - proteins are not responsible for ctni gata binding activity in skeletal muscle cells western blotting analysis was performed to determine whether gata- - and - which are detected in skeletal muscle cells at the transcript level, are also present at the protein level. however, with the exception of a very faint band observed for gata- these gata factors were not detected in blots from c2c myotubes incubated with specific antibodies. gel shift assays were also used to determine whether gata- - and - proteins are in fact present in skeletal muscle cell nuclear extracts and bind gata elements of the ctni promoter. as shown in fig. the gata- antibody, that partially inhibits the formation of the specific complex in control jurkat nuclear extract, does not significantly change the pattern of gel retardation with c2c extract. in the presence of the gata- antibody, a clear supershift is observed with jurkat nuclear extracts, whereas the pattern of gel retardation is unchanged with c2c extract. finally, a gata- antibody produced a strong supershift with cardiac muscle cell nuclear extract but not with c2c cell extract. discussion 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- - and - the only gata transcripts expressed in skeletal muscle cells. the nature of the gata-like factor 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 promoter was found to attenuate its activity in primary cultures of neonatal rat skeletal muscle, however, the nature of the factor binding to this site was not determined. deletion of a gata binding sequence in the dihydropyridine-sensitive receptor α subunit promoter leads to reduction in promoter activity and antisense experiments for gata- induced functional changes in l-type calcium channels which are consistent with decreased expression of α. however, direct interaction of gata with dhpr promoter was not demonstrated. an interaction between gata factors and myod in the regulation of the fgf 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 ku was shown to have a repressor activity in the transcriptional regulation of the gbp and kel promoters by binding wgatag, but not wgataa elements. however, it is unlikely that ku 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-, 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. 
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. 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 c2c 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- - and - the only gata transcripts present in muscle cells. we reported previously that the cardiac troponin i 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 reported previously that the cardiac troponin i 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 c2c 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- - and - 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. 