EXPRESSION OF THE SMOOTH-MUSCLE MYOSIN HEAVY-CHAIN GENE IS REGULATED BY A NEGATIVE-ACTING GC-RICH ELEMENT LOCATED BETWEEN 2 POSITIVE-ACTINGSERUM RESPONSE FACTOR-BINDING ELEMENTS
Cs. Madsen et al., EXPRESSION OF THE SMOOTH-MUSCLE MYOSIN HEAVY-CHAIN GENE IS REGULATED BY A NEGATIVE-ACTING GC-RICH ELEMENT LOCATED BETWEEN 2 POSITIVE-ACTINGSERUM RESPONSE FACTOR-BINDING ELEMENTS, The Journal of biological chemistry, 272(10), 1997, pp. 6332-6340
To identify cis- and trans-acting factors that regulate smooth muscle-
specific gene expression, we studied the smooth muscle myosin heavy ch
ain gene, a rigorous marker of differentiated smooth muscle. A compari
son of smooth muscle myosin heavy chain promoter sequences from multip
le species revealed the presence of a highly conserved 227-base pair d
omain (nucleotides -1321 to -1095 in rat). Results of a deletion analy
sis of a 4.3-kilobase pair segment of the rat promoter (nucleotides -4
220 to +88) demonstrated that this domain was necessary for maximal tr
anscriptional activity in smooth muscle cells. Gel-shift analysis and
site-directed mutagenesis demonstrated that one true CArG and another
CArG-like element contained within this domain were both recognized by
the serum response factor and were both required for the positive act
ivity attributable to this domain. Additional studies demonstrated tha
t mutation of a GC-rich sequence within the 227-base pair conserved do
main resulted in a nearly 100% increase in transcriptional activity. G
el-shift analysis showed that this GC rich repressor element was recog
nized by both Spl and Sp3. These data demonstrate that transcriptional
control of the smooth muscle myosin heavy chain gene is highly comple
x, involving both negative and positive regulatory elements, including
CArG sequences found in the promoters of multiple smooth muscle diffe
rentiation marker genes.