on August 22, 2002
Published online before print August 22, 2002, doi: 10.1161/01.ATV.0000034493.76465.FF
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Submitted on July 10, 2002
Accepted on July 31, 2002
Characterization of Human Glycoprotein VI Gene 5' Regulatory and Promoter Regions
Kenichi Furihata and Thomas J. Kunicki *
From the Roon Research Center for Arteriosclerosis and Thrombosis, Division of Experimental Hemostasis and Thrombosis, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, Calif.
* To whom correspondence should be addressed. E-mail: tomk{at}scripps.edu.
Objective—Platelet glycoprotein VI is a collagen receptor belonging to the immunoglobulin-like protein family that is essential for platelet interactions with collagen and is exclusively expressed in the megakaryocytic lineage. The objective of this study was to characterize the human glycoprotein VI gene (GP6) 5' regulatory and promoter regions.
Methods and Results—We first used 5' RACE to establish experimentally that the major transcription start site lies 28 bp upstream from the start codon. We next subcloned the 5' regulatory region of GP6 into pGL3-basic [pGL3(9-1576)] and used deletion mutagenesis to identify important regulatory regions, comparing the activity of transiently expressed promoter-luciferase constructs in Dami and HeLa cells. We found that megakaryocyte lineage-specific transcription is largely controlled within the segment -191/-39. By site-directed mutagenesis, we confirmed that a GATA-1 site at -176 and an Ets-1 site at -45 play important roles in the regulation of GP6 transcriptional activity.
Conclusions—We have determined that the GP6 sequence -191 to -39 represents the core promoter and that transcription is driven largely by GATA-1 (-176) and c-Ets-1 (-45) sites within this segment.
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