Regulation oftheHumanCardiac/Slow-Twitch Troponin C GenebyMultiple, Cooperative, Cell-Type-Specific, and MyoD-Responsive Elements

expression ofthehumancTnC(HcTnC) gene.Atleast fourseparate elements cooperate to confer tissue-specific expression ofthis geneindifferentiated myotubes; a basal promoter(between -61and -13)augmentstranscription 9-fold, upstreammajorregulatory sequences(between -68and-142and between -1319and-4500) augment transcription asmuchas39-fold, andatleast twoenhancer-like elements inthefirst intron (between +58and+1028andbetween +1029and+1523)independently augment transcription 4-to5-fold. Theseenhancers inthefirst intron increase myotube-specific chloramphenicol acetyltransferase activity whenlinked totheir own promoter elements ortotheheterologous simian virus 40 promoter, andtheeffects aremultiplicative rather thanadditive. Eachofthemajormyotube regulatory regions iscapable ofresponding directly orindirectly tothemyogenic determination factor, MyoD.AMyoDexpression vector in1OT1/2 cells induced constructs carrying either theupstream HcTnCpromoter elements orthefirst intron ofthegene300-to500-fold. Expression was inhibited bycotransfection withId, anegative regulator of basic helix-loop-helix transcription factors. Thebasalpromoter contains five tandemTGGGC repeats that interact withSplor an Spl-like factor innuclear extracts. Mutational analysis ofthis element demonstrated thattwoofthefive repeat sequenceswere sufficient tosupport basal level muscle cell-specific transcription. Whereasthebasal promoter isalsocritical forexpression incardiac myocytes, theelements upstream of-67 appeartoplaylittle or no role. Majoraugmentation ofexpression incardiomyocytes isalsoprovided by sequencesinthefirst intron, butthese areupstream (between +58and+1028). Thedownstream segment of thefirst intron hasno enhancer activity incardiomyocytes. A specific DNA-protein complex isformedbythis C2 cellenhancer withextracts fromC2 cells butnotcardiomyocytes. Theseobservations suggest that tissue-specific expression oftheHcTnCgeneiscooperatively regulated bythecomplex interactions ofmultiple regulatory elements andthatdifferent elements areusedtoregulate expression inmyogenic andcardiac cells. Development ofmuscle structure andfunction requires thecoordinated expression ofanarrayofmuscle-specific genes(for areview, seereference 5).Study ofthegenesfor striated muscle contractile proteins hasproven useful forthe elucidation ofmechanisms responsible fortissue-specific geneexpression (95). Because thetroponin family ofgenesis expressed onlyinstriated muscles (there areno known smooth muscle ornonmuscle isoforms (74)), they constitute anexcellent modelsystemforstudy ofhighly restricted regulation. Thethree troponins, troponin I(TnI), TnT,and TnC,aremembersofevolutionary distinct andunrelated genefamilies (14). Theyencode thefunctionally interacting subunits ofthecalcium regulatory troponin complex of vertebrate striated muscle(71). InthecaseofTnC,the calcium-binding subunit ofthecomplex, thereisa fast- twitch skeletal muscle gene(TnCf) andasecond gene(cTnC, previously designated sTnC(slow-twitch troponin C))ex- pressed inbothslow-twitch skeletal muscleandcardiac muscle (27, 34,72a,101). A considerable bodyofevidence