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. 1993 Aug 15;90(16):7563–7567. doi: 10.1073/pnas.90.16.7563

Identification and characterization of an essential, activating regulatory element of the human SIS/PDGFB promoter in human megakaryocytes.

H M Jin 1, M L Brady 1, W E Fahl 1
PMCID: PMC47182  PMID: 8356057

Abstract

The SIS/PDGFB gene, encoding the B polypeptide of platelet-derived growth factor (PDGF-B), is transcriptionally activated (> 50 fold) in human K562 erythroleukemia cells when they are induced to differentiate into megakaryocytic cells by treatment with phorbol 12-myristate 13-acetate ("tetradecanoylphorbol acetate," TPA). A 250-bp PDGF-B gene promoter attached to a reporter gene was shown to reproduce this TPA-induced activation. In a series of mutants that we constructed, a 10-bp linker sequence was systematically moved across the 250-bp PDGF-B promoter sequence, and the effect upon luciferase reporter activity was measured to identify a site through which this TPA-induced transcriptional activation occurred. We identified a site, which we named the SIS proximal element (SPE), at positions -58 to -39 relative to the PDGF-B mRNA initiation site that was essential for the TPA-induced activation. The SPE site contains two repeated sequences (TCTC and CACC) arranged in an ABBA configuration. The SPE sequence was not found in the existing list of consensus sequences for transcription factor binding sites. Gel mobility-shift assays using an SPE oligonucleotide and K562 cell nuclear extracts showed three shifted complexes, one of which was formed 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-inducible complex occurred over the same time frame, and both events were specifically blocked by the addition of cycloheximide. The 20-bp SPE 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 SPE in regulating the concurrent expression of the PDGF-B and PDGF-A genes in megakaryocytes, as well as various human tumor cells, is considered.

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