IDENTIFICATION AND CHARACTERIZATION OF AN ESSENTIAL, ACTIVATING REGULATORY ELEMENT OF THE HUMAN SIS PDGFB PROMOTER IN HUMAN MEGAKARYOCYTES/

The SIS/PDGFB gene, encoding the B polypeptide of platelet-derived gro wth factor (PDGF-B), is transcriptionally activated (>50 fold) in huma n K562 erythroleukemia cells when they are induced to differentiate in to megakaryocytic cells by treatment with phorbol 12-myristate 13-acet ate (''tetradecanoylphorbol acetate,'' TPA). A 250-bp PDGF-B gene prom oter attached to a reporter gene was shown to reproduce this TPA-induc ed activation. In a series of mutants that we constructed, a 10-bp lin ker sequence was systematically moved across the 250-bp PDGF-B promote r sequence, and the effect upon luciferase reporter activity was measu red to identify a site through which this TPA-induced transcriptional activation occurred. We identified a site, which we named the SIS prox imal element (SPE), at positions -58 to -39 relative to the PDGF-B mRN A initiation site that was essential for the TPA-induced activation. T he SPE site contains two repeated sequences (TCTC and CACC) arranged i n an ABBA configuration. The SPE sequence was not found in the existin g list of consensus sequences for transcription factor binding sites. Gel mobility-shift assays using an SPE oligonucleotide and K562 cell n uclear extracts showed three shifted complexes, one of which was forme d only following TPA treatment of K562 cells. In a time-course study, TPA induction of the endogenous PDGF-B mRNA and formation of the TPA-i nducible complex occurred over the same time frame, and both events we re specifically blocked by the addition of cycloheximide. The 20-bp SP E sequence was highly conserved (19/20) in both the cat and the mouse PDGF-B promoter, and conserved portions of the SPE sequence were also found at two sites within the human PDGF-A promoter. The role of the S PE in regulating the concurrent expression of the PDGF-B and PDGF-A ge nes in megakaryocytes, as well as various human tumor cells, is consid ered.