© 1999 American Heart Association, Inc.
Rapid Communication |
Regulation of Smooth Muscle 
-Actin Expression In Vivo Is Dependent on CArG Elements Within the 5' and First Intron Promoter Regions
From the Department of Molecular Physiology and Biological Physics, University of Virginia Medical School, Charlottesville, Va.
Correspondence to Gary K. Owens, PhD, Box 449 Health Sciences Center, University of Virginia, Charlottesville, VA 22908.
Abstract—The aims of the
present studies were to define sufficient promoter sequences
required to drive endogenous expression of smooth muscle
(SM) 
-actin and to determine whether regulation of SM -actin
expression in vivo is dependent on CArG (CC(A/T)6GG)
cis elements. Promoter deletions and site directed
mutagenesis techniques were used to study gene regulation in transgenic
mice as well as in smooth muscle cell (SMC) cultures. Results
demonstrated that a Lac Z transgene that contained 547 bp of the 5' rat
SM -actin promoter was sufficient to drive embryonic expression of
SM -actin in the heart and in skeletal muscle but not in SMCs.
Transient transfections into SMC cultures demonstrated that the
conserved CArG element in the first intron had significant positive
activity, and gel shift analyses demonstrated that the intronic
CArG bound serum response factor. A transgene construct from -2600
through the first intron (p2600Int/Lac Z) was expressed in embryos and
adults in a pattern that closely mimicked endogenous SM
-actin expression. Expression in adult mice was completely
restricted to SMCs and was detected in esophagus, stomach, intestine,
lung, and nearly all blood vessels, including coronary,
mesenteric, and renal vascular beds. Mutation of CArG B completely
inhibited expression in all cell types, whereas mutation of the
intronic CArG selectively abolished expression in SMCs, which suggests
that it may act as an SMC-specific enhancer-like element. Taken
together, these results provide the first in vivo evidence for the
importance of multiple CArG cis elements in the
regulation of SM -actin expression.
Key Words: muscle, smooth • transgenic mice • transfection • gene expression
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