Identification of a novel slow-muscle-fiber enhancer binding protein, MusTRD1.

The molecular mechanisms which are responsible for restricting skeletal muscle gene expression to specific fiber types, either slow or fast twitch, are unknown. As a first step toward defining the components which direct slow-fiber-specific gene expression, we identified the sequence elements of the human troponin I slow upstream enhancer (USE) that bind muscle nuclear proteins. These include an E-box, a MEF2 element, and two other elements, USE B1 and USE C1. In vivo analysis of a mutation that disrupts USE B1 binding activity suggested that the USE B1 element is essential for high-level expression in slow-twitch muscles. This mutation does not, however, abolish slow-fiber specificity. A similar analysis indicated that the USE C1 element may play only a minor role. We report the cloning of a novel human USE B1 binding protein, MusTRD1 (muscle TFII-I repeat domain-containing protein 1), which is expressed predominantly in skeletal muscle. Significantly, MusTRD1 contains two repeat domains which show remarkable homology to the six repeat domains of the recently cloned transcription factor TFII-I. Furthermore, both TFII-I and MusTRD1 bind to similar but distinct sequences, which happen to conform with the initiator (Inr) consensus sequence. Given the roles of MEF2 and basic helix-loop-helix (bHLH) proteins in muscle gene expression, the similarity of TFII-I and MusTRD1 is intriguing, as TFII-I is believed to coordinate the interaction of MADS-box proteins, bHLH proteins, and the general transcription machinery.

Transcription occurs in pulses in muscle fibers.

We report a novel mechanism of gene regulation in skeletal muscle fibers. Within an individual myofiber nucleus, not all muscle loci are transcriptionally active at a given time and loci are regulated independently. This phenomenon is particularly remarkable because the nuclei within a myofiber share a common cytoplasm. Both endogenous muscle-specific and housekeeping genes and transgenes are regulated in this manner. Therefore, despite the uniform protein composition of the contractile apparatus along the length of the fiber, the loci that encode this structure are not transcribed continuously. The total number of active loci for a particular gene is dynamic, changing during fetal development, regeneration, and in the adult, and potentially reflects the growth status of the fiber. The data reveal that transcription in particular stages of muscle fiber maturation occurs in pulses and is defined by a stochastic mechanism.

The human troponin I slow promoter directs slow fiber-specific expression in transgenic mice.

Troponin I (TnI) is a muscle-specific protein involved in the calcium-mediated contraction of striated muscle. Three TnI isoforms have been identified, each encoded by a separate gene and expressed in specific striated muscles in the adult. The slow isoform gene (TnIs) is transcriptionally regulated during skeletal muscle development such that its expression in the adult is restricted to muscle fibers innervated by a slow nerve. To delineate regions of this gene that are responsive to information imparted by the slow nerve, we generated transgenic mice carrying -4,200 to +12 bp of the human TnIs gene linked to the bacterial chloramphenicol acetyltransferase (CAT) coding region. By Northern blot analysis, we detected transgene transcripts only in muscles containing slow-twitch fibers. CAT histochemical analysis revealed that expression of the transgene is restricted solely to slow-twitch fibers as characterized by type I myosin heavy-chain (MyHC) expression. Using regeneration as a model for neural influenced expression, we show that this gene construct also contains sequences necessary to respond to cues from the central nervous system.

Expression vectors encoding human growth hormone (hGH) controlled by human muscle-specific promoters: prospects for regulated production of hGH delivered by myoblast transfer or intravenous injection.

We report here the construction of vectors that produce and secrete human growth hormone (hGH) in a muscle-specific manner. The promoter regions of the genes encoding human skeletal alpha-actin (HSA) and troponin I slow (HTnIs) were linked to the hGH-encoding gene. These vectors were designated pHSA2000GH and pHTnIs4200GH, respectively. The HSA and HTnIs promoters linked to the cat gene have previously been shown to be necessary and sufficient for developmentally regulated muscle-specific expression. Furthermore, these promoters function in a fibre-type-specific manner in transgenic animals. Transient and stable transfection analyses with pHSA2000GH and pHTnIs4200GH indicated that: (i) these vectors efficiently synthesized hGH in a muscle-specific manner; (ii) the myogenic master regulatory gene, myoD, a determinant of cell fate, trans-activated expression of hGH in pluripotential non-muscle cells; and (iii) these hGH expression vectors were developmentally regulated during myogenic differentiation. These regulated tissue/fibre-type-specific hGH-containing plasmids are suitable vectors for the delivery and stable production of GH in livestock and GH-deficient hosts by either transgenesis, myoblast transfer or liposome-mediated intravenous injection.

cDNA sequence of a human skeletal muscle ADP/ATP translocator: lack of a leader peptide, divergence from a fibroblast translocator cDNA, and coevolution with mitochondrial DNA genes.

We have characterized a 1400-nucleotide cDNA for the human skeletal muscle ADP/ATP translocator. The deduced amino acid sequence is 94% homologous to the beef heart ADP/ATP translocator protein and contains only a single additional amino-terminal methionine. This implies that the human translocator lacks an amino-terminal targeting peptide, a conclusion substantiated by measuring the molecular weight of the protein synthesized in vitro. A 1400-nucleotide transcript encoding the skeletal muscle translocator was detected on blots of total RNA from human heart, kidney, skeletal muscle, and HeLa cells by hybridization with oligonucleotide probes homologous to the coding region and 3' noncoding region of the cDNA. However, the level of this mRNA varied substantially among tissues. Comparison of our skeletal muscle translocator sequence with that of a recently published human fibroblast translocator cognate revealed that the two proteins are 88% identical and diverged about 275 million years ago. Hence, tissues vary both in the level of expression of individual translocator genes and in differential expression of cognate translocator genes. Comparison of the base substitution rates of the ADP/ATP translocator and the oxidative phosphorylation genes encoded by mitochondrial DNA revealed that the mitochondrial DNA genes fix 10 times more synonymous substitutions and 12 times more replacement substitutions; yet, these nuclear and cytoplasmic respiration genes experience comparable evolutionary constraints. This suggests that the mitochondrial DNA genes are highly prone to deleterious mutations.

Alternative splicing generates variants in important functional domains of human slow skeletal troponin T.

We provide the first nucleotide sequence information for the slow isoform of troponin T (TnT). Sequence and hybridization analyses revealed that a single slow TnT gene present in the human genome gives rise to at least two different slow TnT variants by alternative splicing. The observed variations in slow TnT splicing generated major structural differences between the two corresponding slow TnT proteins in a domain that is likely to be involved in critical interactions with troponin C, troponin I, and tropomyosin in the thin filament. Corresponding variations have not been found for fast or for cardiac TnT. The comparison of splicing patterns for fast, cardiac, and slow TnT reveals that the splicing pattern for each isoform is unique. These features raise important questions of why and how all the individual members of the closely related TnT gene family developed such complex but different schemes of alternative splicing to create sets of variant proteins. This unusual familial trait is not known in any other muscle or nonmuscle multigene family.

The 10 kb Drosophila virilis 28S rDNA intervening sequence is flanked by a direct repeat of 14 base pairs of coding sequence.

Most repeat units of rDNA in Drosophila virilis are interrupted in the 28S rRNA coding region by an intervening sequence about 10 kb in length; uninterrupted repeats have a length of about 11 kb. We have sequenced the coding/intervening sequence junctions and flanking regions in two independent clones of interrupted rDNA, and the corresponding 28S rRNA coding region in a clone of uninterrupted rDNA. The intervening sequence is terminated at both ends by a direct repeat of a fourteen nucleotide sequence that is present once in the corresponding region of an intact gene. This is a phenomenon associated with transposable elements in other eukaryotes and in prokaryotes, and the Drosophila rDNA intervening sequence is discussed in this context. We have compared more than 200 nucleotides of the D. virilis 28S rRNA gene with sequences of homologous regions of rDNA in Tetrahymena pigmentosa (Wild and Sommer, 1980) and Xenopus laevis (Gourse and Gerbi, 1980): There is 93% sequence homology among the diverse species, so that the rDNA region in question (about two-thirds of the way into the 28S rRNA coding sequence) has been very highly conserved in eukaryote evolution. The intervening sequence in T. pigmentosa is at a site 79 nucleotides upstream from the insertion site of the Drosophila intervening sequence.