Muscle-specific activity of the skeletal troponin I promoter requires interaction between upstream regulatory sequences and elements contained within the first transcribed exon.

Abstract

Expression of the skeletal troponin I (sTnI) gene is regulated transcriptionally in a muscle-specific fashion. We show here that the region of the sTnI gene between -160 and +61 (relative to the transcription initiation site) is able to direct expression of the bacterial chloramphenicol acetyltransferase (CAT) During development of skeletal muscle, specific isoforms of the contractile proteins accumulate in the cytoplasm, where they become assembled into myofilaments. The mRNAs which encode these muscle-specific isoforms of the contractile proteins begin to accumulate during terminal differentiation, when proliferating myoblasts withdraw from the cell cycle and fuse to form multinucleated myotubes (10,24). Thus, coexpression of many unlinked genes occurs during differentiation to produce the skeletal muscle phenotype. How expression of this gene set is regulated is a fundamental question in muscle development. One approach to this question is to identify and compare the cis elements responsible for muscle-specific activity within individual muscle genes.At least three different regulatory elements have been identified which appear to be important in governing the cell-specific expression of individual muscle-specific genes. One such motif, 5'-CC(A+T rich)6GG-3' (CArG/CBAR motif) is located in multiple copies upstream of the transcription initiation sites of sarcomeric actin genes (5, 41) and has been shown to be required for the muscle-specific expression of sarcomeric actin genes in a number of diverse species (5, 41-43). A second conserved, variable sequence motif, dubbed MEF-1, has been shown to be an essential component of the muscle-specific enhancer of the creatine kinase gene (7) and may be an essential component of the enhancers of other muscle-specific genes (11,58 (29,58). Here we show that the sTnI promoter also contains elements which restrict expression to skeletal muscle cells, and by using deletion, chimeric promoter, and footprint analyses we localized the essential regulatory elements to two components, one located upstream and one downstream of the transcription initiation site. Thus, our results show that, for the sTnI gene, musclespecific expression is governed by a complex interaction between multiple elements located both in the promoter and in the intragenic regions.

Regulation of the rat cardiac troponin I gene by the transcription factor GATA-4

“…These results are in contrast with the skeletal-muscle TnI genes, in which intronic regulatory elements are important in the regulation of reporter gene activity both in itro and in i o [12][13][14][15]. In addition, multiple DNA-binding sites for the GATA-4 transcription factor were found, and GATA-4 was noted to trans-activate this gene in i o.…”

“…In this regard, the cardiac TnI gene differs from the fast and slow skeletal-muscle family members, which like other muscle genes, require intronic regions to mediate high levels of expression [12][13][14][15]. The finding that the proximal upstream region supports high levels of reporter gene activity in cardiocytes is not unexpected given the reports of other investigators noting that the cardiac-specific regulation of genes, such as myosin light chain 2 (MLC-2) and troponin C (TnC), may be mediated by immediate upstream regions [33,41].…”

Tissue-specific transcription of the cardiac myosin light-chain 2 gene is regulated by an upstream repressor element.

“…Ultimately, however, the overall developmental program involves not only multiple regulatory regions but a multiplicity of regulatory protein-protein and protein-DNA interactions as well. A variety of positive and negative elements with disparate functions have been described in several muscle genes (6,12,40,48,67,74). However, myogenic cell-specific motifs critical for one gene (43) are not necessarily required for the transcription activity of others (12).…”

Muscle-specific expression of the troponin I gene requires interactions between helix-loop-helix muscle regulatory factors and ubiquitous transcription factors.

“…Mutations within the MCK muscle regulatory factor-binding site (MRF site) alter the ability of MyoD and myogenin to bind to this element and block the transcriptional activation of the MCK gene (8,25). The observations that MRF sites also are found in the genes coding for myosin light chain 1/3 (MLC) (16), the 8 subunit of the acetylcholine receptor (47), and Tnl (37,51) raise the possibility that all contractile protein genes are regulated developmentally through common MRF sites. Although this is an attractive model that could account for the coordinate regulation of this evolutionarily unrelated gene set, most eucaryotic enhancers utilize multiple protein-binding sites to produce a controlled transcription pattern (reviewed in reference 2).…”