C-SIS PLATELET-DERIVED GROWTH FACTOR-B PROMOTER REQUIREMENTS FOR INDUCTION DURING THE 12-O-TETRADECANOYLPHORBOL-13-ACETATE-MEDIATED MEGAKARYOBLASTIC DIFFERENTIATION OF K562 HUMAN ERYTHROLEUKEMIA-CELLS

Platelet-derived growth factor (PDGF), a powerful mitogen and chemoatt ractant, is composed of two subunits, A and B, which are synthesized b y normal megakaryocytes. We have studied the transcriptional regulatio n of the c-sis/PDGF-B gene in human K562 erythroleukemia cells that ha ve been induced to undergo megakaryoblastic differentiation by treatme nt with 12-O-tetradecanoylphorbol-13-acetate. Upon differentiation of these cells, c-sis/PDGF-B transcription is increased 50-100-fold. We s how here that a minimal c-sis/PDGF-B promoter region, spanning nucleot ides -64 to +6, retains full inducibility. Linker scanning mutagenesis within this minimal region identified four segments that were importa nt for expression in differentiating K562 cells: a previously defined sis proximal element (SPE; -64 to -45), the TATA box, the 10 bp immedi ately downstream of the TATA box [TATA neighboring sequence (TNS); -24 to -15], and the mRNA start site region. Combined mutation of the SPE and TNS resulted in a greater impairment of induction than did mutati on of either sequence alone. In contrast, combined mutation of the SPE and the start site or of the TNS and the start site did not lower ind uction beyond that displayed by the least inducible single mutants. Th e combination of the SPE and the TNS was sufficient to confer wild-typ e levels of inducibility to a heterologous promoter. Both the SPE and the TNS were sensitive to alterations in the helical spacing between t hese elements and the TATA box. Using the electrophoretic mobility shi ft assay, we demonstrated binding of Sp family members and of two addi tional unidentified nuclear factors to the TNS in both 12-O-tetradecan oylphorbol-13-acetate-treated and untreated cells. The TNS, therefore, appears to represent a target for a constitutively bound factor(s) th at is required for cooperation with a differentiation-specific factor bound at the SPE to drive efficient c-sis/PDGF-B transcription in TPA- treated K562 cells.