Flanking sequences modulate the cell specificity of M-CAT elements.

M-CAT elements mediate both muscle-specific and non-muscle-specific transcription. We used artificial promoters to dissect M-CAT elements derived from the cardiac troponin T promoter, whose regulation is highly striated muscle specific. We show that muscle-specific M-CAT-dependent expression requires two distinct components: the core heptameric M-CAT motif (5'-CATTCCT-3'), which constitutes the canonical binding site for TEF-1-related proteins, and specific sequences immediately flanking the core motif that bind an additional factor(s). These factors are found in higher-order M-CAT DNA-protein complexes with TEF-1 proteins. Non-muscle-specific promoters are produced when the sequences flanking the M-CAT motif are removed or modified to match those of non-muscle-specific promoters such as the simian virus 40 promoter. Moreover, a mutation of the 5'-flanking region of the cardiac troponin T M-CAT-1 element upregulated expression in nonmuscle cells. That mutation also disrupts a potential E box that apparently does not bind myogenic basic helix-loop-helix proteins. We propose a model in which M-CAT motifs are potentially active in many cell types but are modulated through protein binding to specific flanking sequences. In nonmuscle cells, these flanking sequences bind a factor(s) that represses M-CAT-dependent activity. In muscle cells, on the other hand, the factor(s) binding to these flanking sequences contributes to both the cell specificity and the overall transcriptional strength of M-CAT-dependent promoters.

DTEF-1, a novel member of the transcription enhancer factor-1 (TEF-1) multigene family.

M-CAT motifs mediate muscle-specific transcriptional activity via interaction with binding factors that are antigenically and biochemically related to vertebrate transcription enhancer factor-1 (TEF-1), a member of the TEA/ATTS domain family of transcription factors. M-CAT binding activities present in cardiac and skeletal muscle tissues cannot be fully accounted for by existing cloned isoforms of TEF-1. TEF-1-related cDNAs isolated from heart libraries indicate that at least three classes of TEF-1-related cDNAs are expressed in these and other tissues. One class are homologues of the human TEF-1 originally cloned from HeLa cells (Xiao, J. H., Davidson, I., Matthes, H., Garnier, J. M., and Chambon, P. (1991) Cell 65, 551-568). A second class represents homologues of the avian TEF-1-related gene previously isolated (Stewart, A. F., Larkin, S. B., Farrance, I. K., Mar, J. H., Hall, D. E., and Ordahl, C. P. (1994) J. Biol. Chem. 269, 3147-3150). The third class consists of a novel, divergent TEF-1 cDNA, named DTEF-1, and its preliminary characterization is described here. Two isoforms of DTEF-1 (DTEF-1A and DTEF-1B) were isolated as 1.9-kilobase pair clones with putative open reading frames of 433 and 432 amino acids whose differences are attributable to alternative splicing at the C terminus of the TEA DNA binding domain. Cardiac muscle contains high levels of DTEF-1 transcripts, but unexpectedly low levels are detected in skeletal muscle. DTEF-1 transcripts are present at intermediate levels in gizzard and lung, and at low levels in kidney. DTEF-1A is a sequence-specific M-CAT-binding factor. The distinct spatial pattern of expression, and unusual amino acid sequence in its DNA binding domain, may indicate a particular role for DTEF-1 in cell-specific gene regulation. Recent work also suggests that at least one more TEF-1-related gene exists in vertebrates. We propose a naming system for the four TEF-1 gene family members identified to date that preserves existing nomenclature and provides a means for extending that nomenclature as additional family members may be identified.

Muscle-enriched TEF-1 isoforms bind M-CAT elements from muscle-specific promoters and differentially activate transcription.

M-CAT elements mediate cardiac- and embryonic skeletal muscle-specific expression of the cardiac troponin T gene and a number of other cardiac-specific genes. M-CAT binding factor was shown to be related to cloned human TEF-1, a transcriptional regulator of the SV40 viral enhancer. Here we describe the cloning of TEF-1 from chick heart and the identification of several novel isoforms. We show that TEF-1 mRNA is considerably enriched in cardiac and skeletal muscle, consistent with a proposed role in muscle gene transcription. The predominant TEF-1 isoforms, TEF-1A and a novel isoform TEF-1B, bind M-CAT elements with high affinity and in a sequence-specific manner. We further demonstrate that the C-terminal portion of TEF-1B, which contains the 13-amino acid exon that distinguishes this isoform, can activate transcription when linked to a heterologous DNA binding domain, while the same domain of TEF-1A cannot. Therefore, isoforms of TEF-1 may play different roles in the regulation of M-CAT-dependent promoters in striated muscle cells.