cis-acting sequences of the rat troponin I slow gene confer tissue- and development-specific transcription in cultured muscle cells as well as fiber type specificity in transgenic mice.
Transcription of the genes coding for troponin I slow (TnIslow) and other contractile proteins is activated during skeletal muscle differentiation, and their expression is later restricted to specific fiber types during maturation. We have isolated and characterized the rat TnIslow gene in order to begin elucidating its regulation during myogenesis. Transcriptional regulatory regions were delineated by using constructs, containing TnIslow gene sequences driving the expression of the chloramphenicol acetyltransferase (CAT) reporter gene, that were transiently transfected into undifferentiated and differentiated C2C12 cells. TnIslow 5'-flanking sequences directed transcription specifically in differentiated cells. However, transcription rates were approximately 10-fold higher in myotubes transfected with constructs containing the 5'-flanking sequences plus the intragenic region residing upstream of the translation initiation site (introns 1 and 2), indicative of interactions between elements residing upstream and in the introns of the gene. Deletion analysis of the 5' region of the TnIslow gene showed that the 200 bp upstream of the transcription initiation site is sufficient to confer differentiation-specific transcription in C2C12 myocytes. MyoD consensus binding sites were found both in the upstream 200-bp region and in a region residing in the second intron that is highly homologous to the quail TnIfast enhancer. Transactivation experiments using transfected NIH 3T3 fibroblasts with TnI-CAT constructs containing intragenic and/or upstream sequences and with the myogenic factors MyoD, myogenin, and MRF4 showed different potentials of these factors to induce transcription. Transgenic mice harboring the rat TnI-CAT fusion gene expressed the reporter specifically in the skeletal muscle. Furthermore, CAT levels were approximately 50-fold higher in the soleus than in the extensor digitorum longus, gastrocnemius, or tibialis muscle, indicating that the regulatory elements that restrict TnI transcription to slow-twitch myofibers reside in the sequences we have analyzed.
Banerjee-Basu
S
S
Unit on Molecular Neurobiology, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Buonanno
A
A
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D005786:Gene Expression Regulation; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection; D014336:Troponin / Q000096:biosynthesis* / Q000235:genetics*; D019210:Troponin I
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014336:Troponin; D019210:Troponin I; D015500:Chloramphenicol O-Acetyltransferase