Cloning and analysis of the thrombopoietin-induced megakaryocyte-specific glycoprotein VI promoter and its regulation by GATA-1, Fli-1, and Sp1.
The exposure of collagen fibers at sites of vascular injury results in the adherence of platelets and their subsequent activation. The platelet collagen receptor glycoprotein (GP)(1) VI plays a crucial role in platelet activation and thrombus formation and decreased levels or defective GPVI may lead to excessive bleeding. In addition, elevated levels of collagen receptors may predispose individuals to coronary heart disease or strokes. GPVI expression is restricted to platelets and their precursor cell, the megakaryocyte. In this study we investigate the regulation of GPVI expression and show that thrombopoietin induces its expression in the megakaryocytic cell line UT-7/TPO. A 5'-region flanking the transcription start point of the GPVI gene was cloned (-694 to +29) and we report that this putative GPVI promoter bestows megakaryocye-specific expression. Deletion analyses and site-directed mutagenesis identified Sp1(227), GATA(177), and Ets(48) sites as essential for GPVI expression. We show that transcription factors GATA-1, Fli-1, and Sp1 can bind to and activate this promoter. Finally, GPVI mRNA was detected only in megakaryocytic cell lines expressing both Fli-1 and GATA-1, and we show that overexpression of Fli-1 in a stable cell line (which expresses endogenous GATA-1 and Sp1) results in expression of the endogenous GPVI gene.
D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA; D004268:DNA-Binding Proteins / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D008533:Megakaryocytes / Q000378:metabolism*; D008969:Molecular Sequence Data; D010980:Platelet Membrane Glycoproteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051789:Proto-Oncogene Protein c-fli-1; D011518:Proto-Oncogene Proteins; D012045:Regulatory Sequences, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000502:physiology*; D013926:Thrombopoietin / Q000502:physiology*; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D010980:Platelet Membrane Glycoproteins; D051789:Proto-Oncogene Protein c-fli-1; D011518:Proto-Oncogene Proteins; D016329:Sp1 Transcription Factor; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA; D013926:Thrombopoietin