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 do wnregulated 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 reve rse the transforming phenotypes both in vitro and in vivo. The downregulati on 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 b oxes, E12/E47, MyoD, MEF, E2F, CREB, and SP1 binding sites. Based on transi ent reporter assays, the promoter/enhancer activities were unusually extend ed to the entire 5.2 kb region in normal Rat 6 cultures, but markedly suppr essed in p53(val135)-, and c-H-ras-transformed cells. The activity detected by the reporter assay corresponded to levels of mRNA as analyzed previousl y by Northern blots in each respective cell line. Thus, the switch-off of t he r-nmMHC-B in the transformed cells is very likely controlled by upstream transcriptional factors, which might have been altered in the course of ne oplastic transformation. (C) 2000 Academic Press.