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

Abstract

The Drosophila wings-up A gene encodes Troponin I. Two regions, located upstream of the transcription initiation site (upstream regulatory element) and in the first intron (intron regulatory element), regulate gene expression in specific developmental and muscle type domains. Based on LacZ reporter expression in transgenic lines, upstream regulatory element and intron regulatory element yield identical expression patterns. Both elements are required for full expression levels in vivo as indicated by quantitative reverse transcription-polymerase chain reaction assays. Three myocyte enhancer factor-2 binding sites have been functionally characterized in each regulatory element. Using exon specific probes, we show that transvection is based on transcriptional changes in the homologous chromosome and that Zeste and Suppressor of Zeste 3 gene products act as repressors for wings-up A. Critical regions for transvection and for Zeste effects are defined near the transcription initiation site. After in silico analysis in insects (Anopheles and Drosophila pseudoobscura) and vertebrates (Ratus and Coturnix), the regulatory organization of Drosophila seems to be conserved. Troponin I (TnI) is expressed before muscle progenitors begin to fuse, and sarcomere morphogenesis is affected by TnI depletion as Z discs fail to form, revealing a novel developmental role for the protein or its transcripts. Also, abnormal stoichiometry among TnI isoforms, rather than their absolute levels, seems to cause the functional muscle defects. INTRODUCTIONContractile protein systems are widely represented in most cell types as a force generator device (Davison et al., 2000). In muscles, troponin I (TnI) is a key element in the protein complex that regulates sliding of thin over thick filaments (Farah and Reinach, 1995;Geeves and Lehrer, 1998;Squire and Morris, 1998;Maytum et al., 2003). Several TnI protein isoforms are generated, either from transcription of independent genes (e.g., vertebrates) or from differential splicing of a single gene primary transcript (e.g., Drosophila). Amino acid substitutions in constitutive or alternatively spliced exons in TnI can lead to pathological conditions such as familial hypertrophic cardiomyopathy (Carrier et al., 1993;Coonar and McKenna, 1997) and distal arthrogryposis (Sung et al., 2003), due to abnormal interactions with other sarcomere components. Also, TnI is a relevant indicator of heart failure (Lewinter and Vanburen, 2002) and a potent angiogenesis inhibitor through its interaction with polycystin-2 (Li et al., 2003). The potential applications that this knowledge could provide, however, are handicapped by the scant information on the regulatory mechanisms of TnI gene expression. This issue is particularly relevant in the context of future gene therapy strategies and justifies this in vivo study of the regulatory mechanism of the Drosophila homologue. In addition, this study takes advantage of the fact that TnI in Drosophila is encoded by a single gene, wings up A (wupA), and that isoform replacem...

Transcriptional regulation of the Drosophila melanogaster muscle myosin heavy-chain gene

“…However the intron element in the troponin T gene is more important for expression in adult muscle, particularly indirect flight muscle. Enhancer redundancy in the troponin genes is proposed to be important for yielding proper levels of gene expression (Marin et al, 2004;Mas et al, 2004), as may be the case for redundant elements in Mhc.…”

“…Each has a large first intron that interrupts the 5′ noncoding region or the first two codons of the gene. This structure is present in both Drosophila (Basi and Storti, 1986;Falkenthal et al, 1984;Geyer and Fyrberg, 1986;Marin et al, 2004;Mas et al, 2004;Parker et al, 1985) and vertebrate muscle genes (Baldwin et al, 1985;Chang et al, 1985;Fornwald et al, 1982;Nabeshima et al, 1984;Strehler et al, 1986). Transcriptional enhancer elements are located in the first intron of a number of such muscle genes (Konieczny and Emerson, 1987;Marin et al, 2004;Mas et al, 2004;Meredith and Storti, 1993;Ng et al, 1989;Yutzey et al, 1989).…”

“…Hanke and Storti (1988) noted that a 26 bp element beginning at -76 of the Drosophila Mhc gene is quite similar to sequences upstream of the Drosophila tropomyosin I and II genes. This sequence could serve as a position-dependent enhancer in all three genes.Other Drosophila muscle genes are interrupted by an intron near their 5′ ends (Basi and Storti, 1986;Falkenthal et al, 1984;Geyer and Fyrberg, 1986;Marin et al, 2004;Mas et al, 2004;Parker et al, 1985), and these introns often contain transcriptional activation sequences. The ß3-tubulin gene has a visceral muscle-specific enhancer element in its first intron (Gasch et al, 1989), as well as a somatic muscle element (Hinz et al, 1992).…”

“…However the intron element in the troponin T gene is more important for expression in adult muscle, particularly indirect flight muscle. Enhancer redundancy in the troponin genes is proposed to be important for yielding proper levels of gene expression (Marin et al, 2004;Mas et al, 2004), as may be the case for redundant elements in Mhc.It is particularly striking that removal of the first intron from Mhc allows transcription in the 28 large cells of the TDT, but not the 4 small cells. This result is intriguing in that we previously documented another difference between the two TDT cell types in Mhc 7 and Mhc 9 mutants (O'Donnell et al, 1989).…”

“…Transcriptional regulatory regions for a number of Drosophila muscle genes have been mapped, resulting in the identification of both unique and shared cis-acting elements (Arredondo et al, 2001;Kelly et al, 2002;Marin et al, 2004;Mas et al, 2004;Meredith and Storti, 1993). The latter includes E-boxes that bind helix-loop-helix transcription factors and MEF2 sites that bind MEF2 protein (Bour et al, 1995;Lilly et al, 1995).…”