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 g ene is expressed in a large percentage of human mesenchymal tumor cell s establishing a growth-promoting, autocrine growth circuit. A 4-kb fr agment, containing the SIS/PDGF-B promoter, was isolated from a human genomic library, and a series of 5'-nested deletions and linker-scanni ng 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 SI S/PDGF-B mRNA initiation site (+1), retained full pro meter activity a nd positive regulatory elements at -228 to -219, -97 to -88 (SIS dista l element) and -58 to -39 (SIS proximal element, SPE) were identified. insertion of the 20-bp SPE into a heterologous, minimal promoter resu lted in >5-fold transcriptional activation which was ablated by mutati ons to the SPE. High resolution mutagenesis within the 20-bp SPE, indi cated the necessity of a CACCC motif for activity Gel shift analysis o f SPE-binding proteins in U2-OS nuclear extracts identified Sp1 and tw o additional binding factors that could be competed away from SPE bind ing by adding excess consensus Sp1 or CACCC oligonucleotides. The indi vidual and aggregate roles of the SPE and two weaker positive regulato ry elements in regulating SIS/PDGF-B transcription in these tumor cell s is considered.