Fiber-Type-Specific Transcription of the Troponin I Slow Gene Is Regulated by Multiple Elements

Abstract

The regulatory elements that restrict transcription of genes encoding contractile proteins specifically to either slow-or fast-twitch skeletal muscles are unknown. As an initial step towards understanding the mechanisms that generate muscle diversity during development, we have identified a 128-bp troponin I slow upstream element (SURE) and a 144-bp troponin I fast intronic element (FIRE) that confer fiber type specificity in transgenic mice (M. Nakayama et al., Mol. Cell. Biol. 16:2408-2417, 1996). SURE and FIRE have maintained the spatial organization of four conserved motifs (3 to 5): an E box, an AT-rich site (A/T2) that binds MEF-2, a CACC site, and a novel CAGG motif. Troponin I slow (TnIs) constructs harboring mutations in these motifs were analyzed in transiently and stably transfected Sol8 myocytes and in transgenic mice to assess their function. Mutations of the E-box, A/T2, and CAGG motifs completely abolish transcription from the TnI SURE. In contrast, mutation of the CACC motif had no significant effect in transfected myocytes or on the slow-specific transcription of the TnI SURE in transgenic mice. To assess the role of E boxes in fiber type specificity, a chimeric enhancer was constructed in which the E box of SURE was replaced with the E box from FIRE. This TnI E box chimera, which lacks the SURE NFAT site, confers essentially the same levels of transcription in transgenic mice as those conferred by wild-type SURE and is specifically expressed in slow-twitch muscles, indicating that the E box on its own cannot determine the fiber-type-specific expression of the TnI promoter. The importance of the 5 half of SURE, which bears little homology to the TnI FIRE, in muscle-specific expression was analyzed by deletion and linker scanning analyses. Removal of the 5 half of SURE (؊846 to ؊811) results in the loss of expression in stably transfected but not in transiently expressing myocytes. Linker scanning mutations identified sequences in this region that are necessary for the function of SURE when integrated into chromatin. One of these sites (GTTAATCCG), which is highly homologous to a bicoid consensus site, binds to nuclear proteins from several mesodermal cells. These results show that multiple elements are involved in the muscle-specific activity of the TnIs promoter and that interactions between upstream and downstream regions of SURE are important for transcription in the context of native chromatin.Skeletal muscle commitment, differentiation, and maturation are largely controlled by the transcriptional regulation of genes encoding channels, receptors, metabolic enzymes, and muscle-specific contractile proteins (8, 52). Whereas our understanding of the transcription factors regulating the commitment and differentiation of myocytes has advanced significantly in the past years, little is known about the factors that regulate fiber type diversity during muscle maturation. During postnatal development, following the innervation of muscles by motoneurons, distinct isoforms of myosin heavy chains and ot...

Activity-dependent repression of muscle genes by NFAT

“…Molkentin and coworkers (42) reported that, while CaNA␣ and CaNA␤ mutant mice exhibit a reduction of the slow/oxidative program, the effects were mediated through an NFAT-independent mechanism. Swoap et al (37) concluded from in vitro and in vivo studies that CaN and NFAT are not sufficient to dictate the slow muscle program, consistent with our previous analysis of the TnI SURE (15,27). On the other hand, McCullagh et al (36) showed that transcription from an artificially trimerized IL-2 NFAT site is selectively enhanced by slow-patterned activity, a finding we have reproduced here (Fig.…”

“…S1]. Like other genes encoding contractile proteins, TnI transcription during embryonic/ early fetal development is regulated by the MRF (i.e., MyoD) and MEF-2 families of factors that bind cis elements harbored in the SURE and FIRE enhancers (21,26,27). The differential expression of TnIs and TnIf in diverse fibers is already evident during late fetal and perinatal development (15,20), but their expression becomes more confined to slow-and fast-twitch myofibers after P7 when motoneurons depolarize muscles with distinct activity patterns (Fig.…”

“…Reporter levels were measured from transgenic muscle tissue as described (27), by pulverizing and homogenizing tissues. Tissues and C2C12 cells were resuspended in passive lysis buffer (Promega) supplemented with protease inhibitors (Roche), cleared by centrifugation, and assayed by using the dual luciferase reporter assay.…”

“…A systematic deletion analysis in transgenic mice led to the identification of a 128-bp slow-specific upstream regulatory element (SURE) and a 144-bp fast-specific intronic regulatory element (FIRE) that restrict TnI transcription to either slow or fast muscles (20,26). Interestingly, these enhancers share four homologous cis-acting regulatory motifs: an E-box and MEF-2, CACC and CAGG sites (26,27). The TnIs SURE is bipartite, with the half that contains the first three elements conferring general muscle specificity, whereas the other half harboring the CAGG site and a bicoid-like motif, which binds GTF-3, is required for confining transcription to slow muscle (15,27).…”

“…Interestingly, these enhancers share four homologous cis-acting regulatory motifs: an E-box and MEF-2, CACC and CAGG sites (26,27). The TnIs SURE is bipartite, with the half that contains the first three elements conferring general muscle specificity, whereas the other half harboring the CAGG site and a bicoid-like motif, which binds GTF-3, is required for confining transcription to slow muscle (15,27). Although the TnIf FIRE is sufficient to confer developmental and fiber-type-specific transcription (20,26), the cis elements conferring this specificity are not known.…”