IN-VIVO FOOTPRINTING AND FUNCTIONAL-ANALYSIS OF THE HUMAN C-SIS PDGF-B GENE PROMOTER PROVIDES EVIDENCE FOR 2 BINDING-SITES FOR TRANSCRIPTIONAL ACTIVATORS

By in vivo DMS footprint and reporter gene analyses we identified two transcription factor binding sites in the human c-sis/PDGF B gene prom oter, The low basal activity of the PDGF B promoter in HeLa and undiff erentiated K562 cells, which express low PDGF B mRNA levels, and in PC 3 cells, which express a high PDGF B mRNA level, results from binding of a weak transcriptional activator between positions -64 and -61 rela tive to the transcription start site, Cytotrophoblast-tike JEG-3 cells , which do not express the 3.5 kb PDGF B mRNA, contain a transcription al activator directed at the -64/-61 sequence, but DNA methylation may render the endogenous promoter inaccessible to this activator. A CCAC CCAC element at position -61/-54 was identified as the in vivo binding site for a strong transcriptional activator in phorbol ester-treated megakaryocytic K562 cells, which express a high PDGF B mRNA level, Pri mary human fibroblasts, which do not transcribe the PDGF B gene, conta in a transcriptional activator that recognizes an element between posi tions -60 and -45 but does not bind to the endogenous unmethylated pro moter. Our results show that the complex expression pattern of the hum an PDGF B gene involves the cell type-specific expression of weak and strong transcriptional activators and regulation of promoter accessibi lity to these factors.