SIS/PDGF-B promoter isolation and characterization of regulatory elements necessary for basal expression of the SIS/PDGF-B gene in U2-OS osteosarcoma cells.
Platelet-derived growth factor BB, encoded by the SIS/PDGF-B gene, is a potent mitogen for cells of mesenchymal origin, and the SIS/PDGF-B gene is expressed in a large percentage of human mesenchymal tumor cells establishing a growth-promoting, autocrine growth circuit. A 4-kb fragment, containing the SIS/PDGF-B promoter, was isolated from a human genomic library, and a series of 5'-nested deletions and linker-scanning mutants were used to identify positive regulatory elements that are necessary for the constitutive expression of this gene in human U2-OS osteosarcoma cells. A 250-bp fragment, lying immediately 5' to the SIS/PDGF-B mRNA initiation site (+1), retained full promoter activity, and positive regulatory elements at -228 to -219, -97 to -88 (SIS distal element) and -58 to -39 (SIS proximal element, SPE) were identified. Insertion of the 20-bp SPE into a heterologous, minimal promoter resulted in >5-fold transcriptional activation which was ablated by mutations to the SPE. High resolution mutagenesis within the 20-bp SPE, indicated the necessity of a CACCC motif for activity. Gel shift analysis of SPE-binding proteins in U2-OS nuclear extracts identified Sp1 and two additional binding factors that could be competed away from SPE binding by adding excess consensus Sp1 or CACC oligonucleotides. The individual and aggregate roles of the SPE and two weaker positive regulatory elements in regulating SIS/PDGF-B transcription in these tumor cells is considered.
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, 53706.
H M
HM
Jin
D F
DF
Robinson
Y
Y
Liang
W E
WE
Fahl
D001483:Base Sequence; D000077214:Becaplermin; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000378:metabolism; D012516:Osteosarcoma; D010982:Platelet-Derived Growth Factor / Q000235:genetics*; D011401:Promoter Regions, Genetic; D020574:Proto-Oncogene Proteins c-sis; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D009687:Nuclear Proteins; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D000077214:Becaplermin