Fiber-type-specific transcription of the troponin I slow gene is regulatedby multiple elements

The regulatory elements that restrict transcription of genes encoding contr actile proteins specifically to either slow- or fast-twitch skeletal muscle s are unknown. As an initial step towards understanding the mechanisms that generate muscle diversity during development, we have identified a 128-bp troponin I slow upstream element (SURE) and a 144-bp troponin I fast intron ic element (FIRE) that confer fiber type specificity in transgenic mice (M. Nakayama et al., Mel. Cell. Biol. 16:2408-2417, 1996). SURE and FIRE have maintained the spatial organization of four conserved motifs (3' to 5'): an E box, an AT-rich site (A/T2) that binds MEF-2, a CACC site, and a novel C AGG motif. Troponin I slow (TnIs) constructs harboring mutations in these m otifs were analyzed in transiently and stably transfected Sol8 myocytes and in transgenic mice to assess their function. Mutations of the E-box, A/T2, and CAGG motifs completely abolish transcription from the TnI SURE. In con trast, mutation of the CACC motif had no significant effect in transfected myocytes or on the slow-specific transcription of the TnI SURE in transgeni c mice. To assess the role of E boxes in fiber type specificity, a chimeric enhancer was constructed in which the E box of SURE was replaced with the E box from FIRE. This TnI E box chimera, which lacks the SURE NFAT site, co nfers essentially the same levels of transcription in transgenic mice as th ose conferred by wild-type SURE and is specifically expressed in slow-twitc h muscles, indicating that the E box an its own cannot determine the fiber- type-specific expression of the TnI promoter. The importance of the 5' half of SURE, which bears little homology to the TnI FIRE, in muscle-specific e xpression was analyzed by deletion and linker scanning analyses. Removal of the 5' half of SURE (-846 to -811) results in the loss of expression in st ably transfected but not in transiently expressing myocytes. Linker scannin g mutations identified sequences in this region that are necessary for the function of SURE when integrated into chromatin. One of these sites (GTTAAT CCG), which is highly homologous to a bicoid consensus site, binds to nucle ar proteins from several mesodermal cells. These results show that multiple elements are involved in the muscle-specific activity of the TnIs promoter and that interactions between upstream and downstream regions of SURE are important for transcription in the context of native chromatin.