Functional conservation of platelet glycoprotein V promoter between mouse and human megakaryocytes

Objective. In an attempt to clarify the megakaryo-specific regulatory mecha nism of GPV gene transcription, we characterized the 5'-flanking region of the mouse GPV gene. Materials and Methods. The promotor activity of a -481/+22 5'-fragment of t he mouse GPV gene was examined in normal mouse bone marrow cells (BMC) and various human cell lines using two distinct reporter gene assay systems, lu ciferase and green fluorescence protein (GFP). Results. When a DNA construct consisting of this fragment and a GFP reporte r gene were transiently expressed in thrombopoietin-supported mouse BMC cul ture, GFP was identified only in megakaryocytes. The same construct express ed high levels of GFP in the human megakaryocytic Dami line. When assessed by dual luciferase assay, the full -481/+22 fragment could drive variable p romoter activity in human as well as mouse megakaryocytic lines but did not work in non-megakaryocytic cells. Sufficient transcriptional activation of this fragment was restricted to the cells expressing apparent GPV mRNA, A deletion and point mutation study indicated that GATA and Ets motifs, typic al cia-acting elements for platelet-specific genes, located of -75 and -46, respectively, mere essential for promoter function. Conclusion. The GPV promoter has the general characteristics found in plate let-specific genes, and the mechanism for megakaryocyte-specific, maturatio n-dependent regulation of GPV gene transcription is highly conserved betwee n mouse and human. Analysis of GPV transcription mechanism utilizing human lines as well as BMC should provide new information on the final maturation al process of megakaryocytes. (C) 2000 International Society for Experiment al Hematology. Published by Elsevier Science Inc.