Molecular Dissection of DNA Sequences and Factors Involved in Slow Muscle-Specific Transcription

Abstract

Transcription is a major regulatory mechanism for the generation of slow-and fast-twitch myofibers. We previously identified an upstream region of the slow TnI gene (slow upstream regulatory element [SURE]) and an intronic region of the fast TnI gene (fast intronic regulatory element [FIRE]) that are sufficient to direct fiber type-specific transcription in transgenic mice. Here we demonstrate that the downstream half of TnI SURE, containing E box, NFAT, MEF-2, and CACC motifs, is sufficient to confer pan-skeletal muscle-specific expression in transgenic mice. However, upstream regions of SURE and FIRE are required for slow and fast fiber type specificity, respectively. By adding back upstream SURE sequences to the pan-muscle-specific enhancer, we delineated a 15-bp region necessary for slow muscle specificity. Using this sequence in a yeast one-hybrid screen, we isolated cDNAs for general transcription factor 3 (GTF3)/muscle TFII-I repeat domaincontaining protein 1 (MusTRD1). GTF3 is a multidomain nuclear protein related to initiator element-binding transcription factor TF II-I; the genes for both proteins are deleted in persons with Williams-Beuren syndrome, who often manifest muscle weakness. Gel retardation assays revealed that full-length GTF3, as well as its carboxy-terminal half, specifically bind the bicoid-like motif of SURE (GTTAATCCG). GTF3 expression is neither muscle nor fiber type specific. Its levels are highest during a period of fetal development that coincides with the emergence of specific fiber types and transiently increases in regenerating muscles damaged by bupivacaine. We further show that transcription from TnI SURE is repressed by GTF3 when overexpressed in electroporated adult soleus muscles. These results suggest a role for GTF3 as a regulator of slow TnI expression during early stages of muscle development and suggest how it could contribute to Williams-Beuren syndrome.

Transcriptional Regulation of Acetylcholinesterase-associated Collagen ColQ

“…To understand the regulation of collagen-tailed AChE at the nmjs, we analyzed the factors that control transcription of the COLQ gene in slow and fast muscles; we show that the two independent promoters preferentially drive the expression of ColQ-1 transcript in slow muscle and of ColQ-1a in fast muscle. We find that the fiber type-specific expression patterns of ColQ-1 and ColQ-1a are controlled by specific transcription elements, which strongly resemble those previously identified in promoters of other muscle proteins (23)(24)(25); that is, a slow upsteam regulatory element (SURE) and a fast intronic regulatory element (FIRE), which are active, respectively, in slow and fast twitch muscles. In addition, we identified elements that control the synapse-specific expression of ColQ.…”

“…Finally, the synapse-specific expression pattern of ColQ transcripts is mediated by N-box element, as previously demonstrated for the synaptic expression of AChR subunits (42) and AChE (43) at nmjs; both ColQ promoters contain N-box elements and are activated by neuregulin in cultured chick myotubes. SURE and FIRE motifs consist of closely grouped DNA regulatory elements, CAGG, CCAC, MEF2, and E-box (23)(24)(25); in addition, the SURE motif contains an NFAT element between MEF2 and E-box (23). These unique arrangements of DNA regulatory elements are sufficient to determine muscle fiber type-specific expression patterns (23,30).…”

TnIfast IRE enhancer: Multistep developmental regulation during skeletal muscle fiber type differentiation

“…(TnIfast mRNA began to accumulate to detectable levels during the time of secondary fiber formation and increased thereafter.) However, in situ hybridization analysis of developing mouse muscle indicated the presence of TnIfast mRNA during the period of primary myogenesis, at E12.5 (Zhu et al, 1995) and E15 (Calvo et al, 2001). Moreover, TnIfast immunoreactivity was detected in prenatal rat slow fibers (Dhoot and Perry, 1980), many of which are derived from primary fibers.…”

“…Transgenic mouse studies, including direct analysis at the cellular level, have established that the TnIslow upstream enhancer drives slow fiber-type-specific expression (Corin et al, 1995;Nakayama et al, 1996); however, the fiber-type regulatory capabilities of the TnIfast IRE enhancer have not been characterized at the cellular level. In elegant transgenic mouse experiments, Nakayama et al (1996) and Calvo et al (2001) coupled the TnIfast enhancer, or a chimeric TnIfast/TnIslow enhancer containing the downstream half of the TnIfast IRE, to a minimal TnIslow promoter and found preferential expression in glycolytic, fast-fiber-enriched muscles compared with the soleus, an oxidative mixed fast/slow muscle. This finding indicates a role for the IRE enhancer in muscle type specificity, but also immediately raises the question of whether IRE-driven differential expression among muscles reflects fast vs. slow fiber-type specificity at the cellular level, or whether other anatomic/functional differences between muscles, e.g., oxidative vs. glycolytic metabolism, determine or contribute to the pattern.…”

Activity-dependent repression of muscle genes by NFAT

“…To this end, we chose to study the troponin I slow (TnIs) and troponin I fast (TnIf) genes because their expression is restricted to slow-or fast-twitch myofibers in adult muscle. TnIs and TnIf mRNAs are initially coexpressed in all muscle groups during embryonic development (15,20) and in differentiating cultured myocytes (21,22). Later, as developing myofibers are innervated and depolarized by motoneurons, their …”