Isolation of the embryonic form of smooth muscle myosin heavy chain (SMemb/NMHC-B) gene and characterization of its 5'-flanking region.
To examine the molecular mechanisms that regulate the expression of the SMemb/NMHC-B gene, a nonmuscle myosin heavy chain isoform predominantly expressed in fetal aorta, we have isolated and characterized the 5'-flanking region of the rabbit SMemb/NMHC-B gene. Transient transfection experiments demonstrated that 105 base pairs of 5'-flanking sequence was necessary to direct high level transcription in C2/2 cells, vascular smooth muscle cells derived from rabbit aorta. An essential cis-regulatory element was localized between -100 and -91 base pairs from the transcription start site based on the results that replacement mutagenesis within this region significantly reduced promoter activity. Sequence of this region is completely conserved between mouse and rabbit and fits no known DNA binding consensus. Gel mobility shift assays revealed that a specific DNA-protein complex was formed at this site with nuclear extracts from C2/2 cells, which can be competed by H-2Kb CCAAT box but not by Hsp70 CCAAT box or other CCAAT-containing sequences. We conclude that expression of the SMemb/NMHC-B gene is regulated through an interaction between a sequence element located at -100 and a distinct member of CCAAT-binding proteins.
Manabe
I
I
The Third Department of Internal Medicine, University of Tokyo, Japan.
Kurabayashi
M
M
Shimomura
Y
Y
Kuro-o
M
M
Watanabe
N
N
Watanabe
M
M
Aikawa
M
M
Suzuki
T
T
Yazaki
Y
Y
Nagai
R
R
D016475:3T3 Cells; D000818:Animals; D001011:Aorta; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism; D005333:Fetus; D005796:Genes; D017930:Genes, Reporter; D015698:Genomic Library; D051379:Mice; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000737:chemistry / Q000196:embryology*; D016297:Mutagenesis, Site-Directed; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D024501:Nonmuscle Myosin Type IIB; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011817:Rabbits; D012045:Regulatory Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D024501:Nonmuscle Myosin Type IIB; C103815:nonmuscle myosin type IIB heavy chain; D018995:Myosin Heavy Chains