Molecular cloning and functional analysis of the promoter region of rat nonmuscle myosin heavy chain-B gene.
Rat nonmuscle myosin heavy chain-B (r-nmMHC-B) mRNA was previously found downregulated in Rat 6 fibroblasts transformed by mutant p53(val135) [J. W. P. Yam, J. Y. Zheng, and W. L. W. Hsiao (1987) Biochem. Biophys. Res. Commun. 266, 472-480]. Overexpression of exogenous r-nmMHC-B could partially reverse the transforming phenotypes both in vitro and in vivo. The downregulation of r-nmMHC-B was also observed in Rat 6 transformed by c-H-ras and v-myc oncogenes. We cloned a 5.2-kb r-nmMHC-B promoter region. Sequence analysis of -1248 to +1 revealed no TATA box, but did show that it contained CAAT boxes, E12/E47, MyoD, MEF, E2F, CREB, and SP1 binding sites. Based on transient reporter assays, the promoter/enhancer activities were unusually extended to the entire 5.2 kb region in normal Rat 6 cultures, but markedly suppressed in p53(val135)-, and c-H-ras-transformed cells. The activity detected by the reporter assay corresponded to levels of mRNA as analyzed previously by Northern blots in each respective cell line. Thus, the switch-off of the r-nmMHC-B in the transformed cells is very likely controlled by upstream transcriptional factors, which might have been altered in the course of neoplastic transformation.
Yam
J W
JW
Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, Kowloon, China.
Chan
K W
KW
Li
N
N
Hsiao
W L
WL
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D016259:Genes, myc / Q000235:genetics; D016158:Genes, p53 / Q000235:genetics; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D018995:Myosin Heavy Chains / Q000235:genetics*; D024501:Nonmuscle Myosin Type IIB; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D051381:Rats; D020218:Response Elements / Q000235:genetics; D016415:Sequence Alignment; D014158:Transcription, Genetic / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D024501:Nonmuscle Myosin Type IIB; C103815:nonmuscle myosin type IIB heavy chain; D018995:Myosin Heavy Chains