Characterization of human glycoprotein VI gene 5' regulatory and promoter regions.
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.


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(-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.
Furihata
Kenichi
K
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 92037, USA.
Kunicki
Thomas J
TJ
D020121:5' Untranslated Regions / Q000235:genetics*; D000818:Animals; D019677:Artificial Gene Fusion / Q000379:methods; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D001792:Blood Platelets / Q000737:chemistry; D018983:DNA Footprinting / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology; D017930:Genes, Reporter / Q000235:genetics / Q000502:physiology; D006239:Haplotypes / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000502:physiology; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology; D050783:Proto-Oncogene Proteins c-ets; D012313:RNA / Q000097:blood / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D024363:Transcription Initiation Site; D014407:Tumor Cells, Cultured
D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; C494041:ETS1 protein, human; D050981:Erythroid-Specific DNA-Binding Factors; C494042:Ets1 protein, mouse; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; C494689:Gata1 protein, mouse; D009687:Nuclear Proteins; D010980:Platelet Membrane Glycoproteins; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D014157:Transcription Factors; C098341:platelet membrane glycoprotein VI; D012313:RNA; D008156:Luciferases; D012194:RNA-Directed DNA Polymerase