Cloning of the murine Drm gene (Cktsf1b1) and characterization of its oncogene suppressible promoter.
The Drm gene, first identified in rat cells in our laboratory, appears to play a significant role in early embryo patterning and limb bud development. We have now isolated mouse Drm (mDrm) cDNA as well as genomic DNA clones and have mapped the Drm gene (Cktsf1b1) to murine chromosome 2. Cktsf1b1 is regulated in a tissue specific fashion and is expressed only in nontransformed mouse cells or primary fibroblasts in culture, but not in established transformed or tumor-derived mouse cell lines. The major transcription start sites map to within 69 bp upstream of the initiating ATG. A promoter was contained in the -214 to +1 bp 5' flanking region, and promoter/reporter constructs showed 10-fold higher activity than control in REF-1 (rat), A31 (mouse) and CHO (hamster) cells. The region contains a TATA sequence and multiple potential transcription factor binding sites. Promoter activity was dose-dependently inhibited by cotransfection with either ras or mos oncogenes, but oncogene inhibition was reversed and the overall activity increased when cells were treated with the MAP kinase kinase (MKK) inhibitor PD98059. An NF-1 and Yi-like site, identified in the minimal promoter region, showed different mobility shift patterns when normal and transformed cell nuclear extracts are compared. Mutation of the NF-1 site reduced Cktsf1b1 promoter activity 25%, while mutation of the Yi-like site destroyed all the activity. Our results indicate that the expression of Cktsf1b1, a gene associated with early development and cell transformation, is sensitive to MKK levels and may be regulated via multiple transcription factor complexes.
Zhang
Q
Q
Basic Research Laboratory, National Cancer Institute, Frederick, MD, USA.
Topol
L Z
LZ
Athanasiou
M
M
Copeland
N G
NG
Gilbert
D J
DJ
Jenkins
N A
NA
Blair
D G
DG
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D019485:Bone Morphogenetic Proteins; D016466:CHO Cells; D002460:Cell Line; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D006224:Cricetinae; D016207:Cytokines; D004247:DNA / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005260:Female; D015870:Gene Expression; D005786:Gene Expression Regulation; D011905:Genes, ras / Q000235:genetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009115:Muridae; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014018:Tissue Distribution; D014158:Transcription, Genetic
D019485:Bone Morphogenetic Proteins; D016207:Cytokines; D018076:DNA, Complementary; C107276:Grem1 protein, rat; D009687:Nuclear Proteins; D011506:Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D004247:DNA; D008156:Luciferases