Delineation of a slow-twitch-myofiber-specific transcriptional element by using in vivo somatic gene transfer.
Contractile proteins are encoded by multigene families, most of whose members are differentially expressed in fast- versus slow-twitch myofibers. This fiber-type-specific gene regulation occurs by unknown mechanisms and does not occur within cultured myocytes. We have developed a transient, whole-animal assay using somatic gene transfer to study this phenomenon and have identified a fiber-type-specific regulatory element within the promoter region of a slow myofiber-specific gene. A plasmid-borne luciferase reporter gene fused to various muscle-specific contractile gene promoters was differentially expressed when injected into slow- versus fast-twitch rat muscle: the luciferase gene was preferentially expressed in slow muscle when fused to a slow troponin I promoter, and conversely, was preferentially expressed in fast muscle when fused to a fast troponin C promoter. In contrast, the luciferase gene was equally well expressed by both muscle types when fused to a nonfiber-type-specific skeletal actin promoter. Deletion analysis of the troponin I promoter region revealed that a 157-bp enhancer conferred slow-muscle-preferential activity upon a minimal thymidine kinase promoter. Transgenic analysis confirmed the role of this enhancer in restricting gene expression to slow-twitch myofibers. Hence, somatic gene transfer may be used to rapidly define elements that direct myofiber-type-specific gene expression prior to the generation of transgenic mice.
Corin
S J
SJ
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201, USA.
Levitt
L K
LK
O'Mahoney
J V
JV
Joya
J E
JE
Hardeman
E C
EC
Wade
R
R
D000818:Animals; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis* / Q000096:biosynthesis; D004251:DNA Transposable Elements; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D008156:Luciferases / Q000032:analysis* / Q000096:biosynthesis; D051379:Mice; D008822:Mice, Transgenic; D018485:Muscle Fibers, Skeletal / Q000378:metabolism / Q000502:physiology*; D018482:Muscle, Skeletal / Q000378:metabolism / Q000502:physiology*; D009928:Organ Specificity; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic; D014162:Transfection
D004251:DNA Transposable Elements; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase