Regulation of the rat cardiac troponin I gene by the transcription factor GATA-4

Abstract

Troponin I is a thin-filament contractile protein expressed in striated muscle. There are three known troponin I genes which are expressed in a muscle-fibre-type-specific manner in mature animals. Although the slow skeletal troponin I isoform is expressed in fetal and neonatal heart, the cardiac isoform is restricted in its expression to the myocardium at all developmental stages. To study the regulation of this cardiac-specific and developmentally regulated gene in vitro, the rat cardiac troponin I gene was cloned. Transient transfection assays were performed with troponin I–luciferase fusion plasmids to characterize the regulatory regions of the gene. Proximal regions of the upstream sequence were sufficient to support high levels of expression of the reporter gene in cardiocytes and relatively low levels in other cell types. The highest luciferase activity in the cardiocytes was noted with a plasmid that included the region spanning -896 to +45 of the troponin I genomic sequence. Co-transfection of GATA-4, a recently identified cardiac transcription factor, with troponin I–luciferase constructs permitted high levels of luciferase expression in non-cardiac cells. Electrophoretic mobility-shift assays demonstrated specific binding of GATA-4 to oligonucleotides representative of multiple sites of the troponin I sequence. Mutation of a proximal GATA-4 DNA-binding site decreased transcriptional activation in transfected cardiocytes. These results indicate that the proximal cardiac troponin I sequence is sufficient to support high levels of cardiac-specific gene expression and that the GATA-4 transcription factor regulates troponin I–luciferase expression in vitro.

Transcription ofDrosophilaTroponin I Gene Is Regulated by Two Conserved, Functionally Identical, Synergistic Elements

“…When comparing the genomic regions upstream and downstream to the promoter of insect TnI genes, there is a striking conservation of the type and array of putative transcription factor binding sites ( Figure 6). For the rat slow, quail fast, and rat cardiac TnI genes, there are expression studies similar to those reported here, albeit in transfected cells (Yutzey et al, 1989;Banerjee-Basu and Buonanno, 1993;Nakayama et al, 1996;Murphy et al, 1997). With the exception of the rat cardiac gene, two regulatory regions upstream and downstream of the transcription initiation site have been documented.…”

Amphibian cardiac troponin I gene's organization, developmental expression, and regulatory properties are different from its mammalian homologue

“…Of interest, sitedirected mutagenesis of either element eliminated the expression of this promoter. Since a mutation of the GATA element in the rat (Murphy et al, 1997) and human (Bhavsar et al, 2000) TnIc promoters was shown to reduce promoter activity by 94% and 80%, respectively, the observation that GATA transcription factors are involved in initiating/regulating TnIc expression in this amphibian were not unexpected. However, that mutation of a CArG-box motif (the binding site for a serum response factor) in the amphibian TnIc promoter also eliminates its expression was novel.…”

“…Because the only vertebrate TnIc gene sequences reported to date are from mammals (Ausoni et al, 1994;Bhavsar et al, 1996;Murphy et al, 1997), we compared the size and organization of this amphibian gene with its mammalian homologues and found that it is 6 -10 times larger than any of its mammalian counterparts. The R. catesbeiana TnIc gene contains nine introns, whereas only seven introns have been identified in the mammalian TnIc genes.…”

“…On the basis of sequence similarity, we noted several putative regulatory elements (highlighted in Fig. 6) that have been implicated in the cardiac-specific expression of the mouse (Di Lisi et al, 1998), rat (Murphy et al, 1997), and human (Bhavsar et al, 1996(Bhavsar et al, , 2000 TnIc genes. To identify the cis-acting elements involved in regulating the cardiac-specific and developmental expression of this Rana gene, we used the 2,394-bp 5Ј-flanking region of this gene, as well as fragments of it, to drive the expression of a reporter gene (GFP) in the Xenopus transgenic system.…”

“…The gene that encodes TnIc in R. catesbeiana, RcTnIc, is Ͼ35,000 bp in size making it at least 9.5ϫ larger than the rat TnIc gene (Murphy et al, 1997) and at least 6.5ϫ larger than the human TnIc gene . Analysis of the intron/ exon boundaries within the Rana gene reveals that it contains nine introns ( Fig.…”