Transcriptional analyses presented in this study demonstrate that multiple sequence elements present within SURE determine the early developmental as well as the slow-fiber-type-specific activity of the TnIs promoter. Of these elements, four sites, an E box, an AT-rich element, a CACC sequence, and a CAGG motif, are also conserved in the enhancer element of the quail TnIf gene (39
). In addition, SURE contains a second AT-rich sequence and a bicoid consensus site in its 5′ end that are not present in FIRE. Mutations in any of the conserved sites, with the exception of CACC, result in the loss of muscle-specific SURE activity in adult mice, suggesting that interactions among the factors binding to these sites might be essential for its enhancer function. Moreover, since some of these elements are present in both SURE and FIRE, the fiber-specific activity is possibly controlled by the existence of distinct factors that interact with the nonconserved regions within these enhancers.
The importance of the combinatorial involvement of some of these sites in the regulation of many other skeletal muscle genes has been well documented. In the quail TnIf IRE, which we refer to as FIRE, mutations in any single element among the E-box, AT-rich, and CACC sequences abrogate MyoD-induced enhancer activity in myofiber cell cultures (39
). The presence of CCAC and AT-rich elements was shown to be essential for expression of the human myoglobin promoter in Sol8 myotubes (26
). Similarly, the requirement for a novel site, referred to as MEF-3, along with AT-rich MEF-2 and CACC elements, for transcription of the slow/cardiac TnC gene (44
) and of MEF-3 and MEF-2 for the regulation of rat aldolase distal promoter (pM) activity (29
) has been demonstrated in muscle cells. However, evidence for the involvement of these elements in the regulation of fiber-type-specific gene expression in the adult muscle has not been established, although MEF-3 in conjunction with NFI was implicated in the activity of rat aldolase pM in a subset of fast-twitch muscles (54
In order to characterize the discrete elements responsible for both the myocyte-specific and the slow-twitch-fiber-specific activity of the TnIs promoter in its chromatin context, we performed mutational analyses of conserved and non-conserved regions of SURE in stably transfected Sol8 myotubes and transgenic mice. Three elements that are present in the 5′ half of SURE, which we refer to as the A/T1, bicoid-like, and CAGG motifs, were also shown to be necessary for the muscle-specific activity of the enhancer (Fig. and ). Moreover, since the deletion of these sites, along with CACC, from SURE diminished enhancer activity only in stably transfected myocytes and not in the transiently expressing cells (Fig. ), this region might play a critical role in establishing the open chromatin structure needed for active transcription. In addition, the deleterious effect of the A/T1 mutation in transgenic mice (Fig. ) suggests that this site and the sequences flanking it might be important for TnIs promoter activity during early development. Within this region, A/T1 and an adjacent bicoid consensus site (GTTAATCCG) bind nuclear proteins from Sol8 myotubes (see above and reference 9
). Although the identity of the factor(s) binding to these sequences is not established, recently a similar AT-rich element in the murine IIB myosin heavy chain gene promoter, named mAT2 site, has been shown to bind Oct-1, a protein that contains a POU binding domain (36
). Similarly, sequences related to the GTTAATCCG site, mutation of which results in the loss of SURE activity in the stably transfected Sol8 myotubes (Fig. ), have been demonstrated to bind the bicoid subfamily of homeobox proteins in Drosophila
and mice (37
). Oct-1 and bicoid-related proteins are suggested to play an important role in cell lineage specification and pattern formation, respectively, during the embryonic stages of development (11
). Experiments involving the analysis of transgenes carrying bicoid site mutations should help in the evaluation of the role of this sequence in the fiber-type-specific expression of the TnIs promoter.
We could not detect any direct interaction between the CAGG sequence and the nuclear extracts from myocytes, although this site is essential for SURE activity in both myocytes and transgenic mice. It is possible that this site interacts with complexes formed on other motifs in the TnIs promoter, and thus the complexes bound to this sequence might not be detected in the absence of those elements. Another possibility is that the phosphorylation status of the protein(s) in the extract might dictate its interaction with the CAGG element. Alternatively, this site and the sequences flanking it might play an important role in the chromatin remodeling mediated by the MyoD family of transcription factors during myogenic lineage determination (22
), a hypothesis that is not inconsistent with the observation that E-box mutations result in a complete loss of SURE activity (Fig. and ). A possible requirement for interaction between the proximal sequences (which include E-box and A/T2 elements) and the distal sequences (which contain A/T1, bicoid consensus, and CAGG motifs) of SURE within the chromosomal context is also indicated by the fact that the SURE construct which includes only the proximal sequences is insufficient to support TnIs promoter activity in the stably transfected Sol8 myotubes (Fig. B).
Since the interactions between the factors binding to multiple elements within SURE are necessary for its function, it was important that the order and spacing among these sites were preserved for further characterization of fiber-type-specific regulatory elements in the TnIs enhancer. Using a SURE-FIRE fusion construct, we have examined the role that the E boxes present in SURE and FIRE play in the determination of TnI expression in slow and fast muscles, respectively. Functional differences among various E-box elements, based on variations both within the consensus binding site and the flanking sequences, have been demonstrated in the context of many other muscle-specific gene promoters (1
). Differential expression of myogenic factors that interact with E-box sequences has been demonstrated for slow and fast muscles (32
). In addition, simultaneous mutation of three E boxes in the muscle creatine kinase promoter reduce transcription in slow-twitch SOL but not in fast-twitch skeletal muscles (53
). Based on these findings, it has been proposed that the bHLH factors may regulate the slow- and fast-fiber-type-specific expression of the contractile genes in the muscle (32
). However, several lines of evidence that indicated a negative correlation between the transcriptional activation of these genes and the expression of bHLH factors in the skeletal muscle have also been presented (8
). Thus, in order to study if the E-box elements of slow and fast TnI enhancers determined the fiber type specificity of their respective promoters by virtue of their ability to bind different bHLH factors, we analyzed the expression of an E-box chimera in transgenic mice (Fig. ). This construct, in which the E box within SURE was replaced with the equivalent sequence from FIRE, was still transcriptionally active only in the slow-muscle fibers, similar to the wild-type TnI SURE, indicating that the FIRE E box is able to functionally substitute for the SURE E element and that the E box on its own cannot determine the fiber-type-specific activity of the TnI enhancer sequences. These results are in agreement with the observations that mutations of E-box sequences in the MLC1f/3f (47
) and aldolase A (50
) promoters do not affect fiber-type-specific expression in transgenic mice.
Recently, Chin et al. (10
) have shown that expression of the human TnIs and myoglobin promoters in transfected cells is controlled by the calcineurin- and NFAT-mediated signaling pathway. Based on their results, it was proposed that the NFAT transcription factors regulate slow-fiber-type specificity of muscle gene expression. However, as we have shown in this study, a SURE-FIRE E-box chimera construct, in which the NFAT site of SURE (−760/−753) is removed, continues to exhibit slow-fiber-specific expression. In addition, FIRE, which contains at least two consensus NFAT sites (+770/+763 and +756/+749), directs fast-fiber-specific transcription and is not expressed in slow muscles. The various analyses we have presented in this study, performed with both myocytes and transgenic mice, provide strong evidence for the conclusion that the interaction between factors binding to multiple cis
elements within SURE are essential for conferring slow-fiber-type specificity to the TnIs promoter and that the fiber-specific expression of SURE does not require the NFAT binding site.