We have characterized the specific DNA regulatory elements responsible for the 
function of the human cardiac troponin C gene (cTnC) muscle-specific enhancer in 
myogenic cells. We used functional transient transfection assays with deletional 
and site-specific mutagenesis to evaluate the role of the conserved sequence 
elements. Gel electrophoresis mobility shift assays (EMSA) demonstrated the 
ability of the functional sites to interact with nuclear proteins. We 
demonstrate that three distinct transcription activator binding sites commonly 
found in muscle-specific enhancers (a MEF-2 site, a MEF-3 site, and at least 
four redundant E-box sites) all contribute to full enhancer activity but a CArG 
box does not. Mutation of either the MEF-2 or MEF-3 sites or deletion of the 
E-boxes reduces expression by 70% or more. Furthermore, the MEF-2 site and the 
E-boxes specifically bind, respectively, to MEF-2 and myogenic determination 
factors derived from nuclear extracts. EMSA assays using a MEF-3 containing 
oligonucleotide revealed indistinguishable separation patterns with extracts 
from myogenic cells and nonmyogenic cells. These data suggest that expression of 
the cTnC gene in slow-twitch skeletal muscle is sustained through complex 
interactions at the 3'Ile enhancer between muscle-specific and 
nontissue-specific transcription factors: either a myogenic bHLH complex or 
MEF-2 can activate transcription but only in the presence of a third 
transcriptional activator that appears not to be muscle specific. We conclude 
from these observations that the cTnC 3'Ile element is a composite enhancer that 
functions through the combined interactions of at least five regulatory elements 
and their cognate binding factors: three or four E-boxes, a MEF-2 site, and a 
MEF-3 site. The data support the notion that all of these sites contribute to 
enhancer function in cell systems in an additive way but that none are 
absolutely required for enhancer activity. The data imply that the levels of 
transcription of cTnC in myogenic tissues in which the activities of one of the 
transcriptional factors is lacking would be partially but not wholly suppressed. 
Our data support the critical role of E-box sites in conjunction with the 
adjacent elements. Hence, we assign CTnC gene regulation to the "ordinary" 
rather than to the "novel" category of transcriptional regulation during 
skeletal myogenesis.