Activation of a muscle-specific actin gene promoter in serum-stimulated fibroblasts.
Treatment of AKR-2B mouse fibroblasts with serum growth factors or inhibitors of protein synthesis, such as cycloheximide, results in a stimulation of cytoskeletal beta-actin transcription but has no effect on transcription of muscle-specific isotypes, such as the vascular smooth muscle (VSM) alpha-actin gene. Deletion mapping and site-specific mutagenesis studies demonstrated that a single "CArG" element of the general form CC(A/T)6GG was necessary and possibly sufficient to impart serum and cycloheximide-inducibility to the beta-actin promoter. Although the VSM alpha-actin promoter exhibits at least three similar sequence elements, it remained refractory to serum and cycloheximide induction. However, deletion of a 33 base pair sequence between -191 and -224 relative to the transcription start site resulted in the transcriptional activation of this muscle-specific promoter in rapidly growing or serum-stimulated fibroblasts. Although the activity of this truncated promoter was potentiated by cycloheximide in a manner indistinguishable from that of the beta-actin promoter, this was dependent on a more complex array of interacting elements. These included at least one CArG box and a putative upstream activating element closely associated with the -191 to -224 inhibitory sequences. These results demonstrate that the expression of a muscle-specific actin gene in fibroblasts is suppressed by a cis-acting negative control element and that in the absence of this element, the promoter is responsive to growth factor-induced signal transduction pathways.
Stoflet
E S
ES
Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, Minnesota 55905.
Schmidt
L J
LJ
Elder
P K
PK
Korf
G M
GM
Foster
D N
DN
Strauch
A R
AR
Getz
M J
MJ
D000199:Actins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002874:Chromosome Mapping; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000235:genetics; D004742:Enhancer Elements, Genetic; D005347:Fibroblasts / Q000378:metabolism; D017353:Gene Deletion; D005786:Gene Expression Regulation; D051379:Mice; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000187:drug effects
D000199:Actins; D004247:DNA; D003513:Cycloheximide