Identification of Promoter Elements Involved in Cell-specific Regulation of Rat Smooth Muscle Myosin Heavy Chain Gene Transcription

Abstract

In order to identify cis-acting regulatory elements involved in smooth muscle cell-specific gene regulation, we have cloned a 4. 7-kilobase pair fragment of the promoter for the rat smooth muscle myosin heavy chain, a protein expressed in differentiated smooth muscle cells. Sequence analysis of a 1.7-kilobase pair portion of this clone reveals potential binding sites for known transcription factors. A comparison of the primary sequence between the rat and rabbit smooth muscle myosin heavy chain promoters reveals numerous conserved consensus binding sites. Transient transfection analysis of promoter deletion constructs in rat aorta and tracheal smooth muscle cells, L8 myoblast cells, and rat pulmonary aorta endothelial cells suggests that a region of the promoter located between -1,249 and -1,317 base pairs is important for the restriction of gene expression to smooth muscle cells. Electrophoretic mobility shift analysis of a highly conserved region located between -1,317 and -1, 085 base pairs reveals specific DNA-protein complexes formed in smooth muscle cell extracts, which can be competed with an oligonucleotide containing a nuclear factor 1 binding site.