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 chemoattractant, is composed of two subunits, A and B, which are synthesized by normal megakaryocytes. We have studied the transcriptional regulation of the c-sis/PDGF-B gene in human K562 erythroleukemia cells that have been induced to undergo megakaryoblastic differentiation by treatment with 12-O-tetradecanoylphorbol-13-acetate. Upon differentiation of these cells, c-sis/PDGF-B transcription is increased 50-100-fold. We show here that a minimal c-sis/PDGF-B promoter region, spanning nucleotides -64 to +6, retains full inducibility. Linker scanning mutagenesis within this minimal region identified four segments that were important for expression in differentiating K562 cells: a previously defined sis proximal element (SPE; -64 to -45), the TATA box, the 10 bp immediately 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 mutation 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 induction beyond that displayed by the least inducible single mutants. The combination of the SPE and the TNS was sufficient to confer wild-type levels of inducibility to a heterologous promoter. Both the SPE and the TNS were sensitive to alterations in the helical spacing between these elements and the TATA box. Using the electrophoretic mobility shift assay, we demonstrated binding of Sp family members and of two additional unidentified nuclear factors to the TNS in both 12-O-tetradecanoylphorbol-13-acetate-treated and untreated cells. The TNS, therefore, appears to represent a target for a constitutively bound factor(s) that 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.
Kujoth
G C
GC
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706, USA.
Fahl
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WE
D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics* / Q000473:pathology; D008533:Megakaryocytes / Q000187:drug effects* / Q000473:pathology; D008969:Molecular Sequence Data; D010982:Platelet-Derived Growth Factor / Q000096:biosynthesis* / Q000187:drug effects* / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000187:drug effects* / Q000235:genetics; D020574:Proto-Oncogene Proteins c-sis; D017384:Sequence Deletion; D016385:TATA Box / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014157:Transcription Factors / Q000187:drug effects / Q000378:metabolism; D014407:Tumor Cells, Cultured
D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; D014157:Transcription Factors; D011505:Protein-Tyrosine Kinases; D013755:Tetradecanoylphorbol Acetate