Abstract

The glycoprotein (GP) Ib-V-IX multisubunit complex binds to von Willebrand factor and mediates the adhesion of platelets to the subendothelium of damaged blood vessels. Expression of the GPIX subunit is required for stability of the complex, and its absence in platelets is associated with the rare bleeding disorder Bernard-Soulier syndrome. Comparative analyses indicate that the four GPIb-V-IX subunits are members of the leucine-rich repeat family and suggest that GPIX resembles a possible primitive progenitor of this group. To characterize GPIX transcriptional regulation, a series of 5' deletion constructs was made linking the GPIX upstream flanking sequence to the luciferase marker gene, and promoter activity was measured in transiently transfected human erythroleukemia cells. This analysis identified two negative regulatory domains between -686 to -423 and -311 to -203 and two positive regulatory domains at -323 to -311 and -151 to -100 relative to the GPIX transcription start site. In addition, site-directed mutagenesis experiments and in vitro gel retardation assays identified Ets and GATA elements at -42 and -65, which positively regulate GPIX promoter activity and specifically bind nuclear factors derived from human erythroleukemia cells. DNase I protection experiments identified a protein-dependent "footprint" and hypersensitive site within the GPIX Ets sequence. These results provide a framework for comparison of the GPIX promoter with others of the GPIb-V-IX system, other megakaryocyte-specific genes, and other members of the leucine-rich repeat family.

Highlights

  • Retardation assays identified Ets and GATA elements at ؊42 and ؊65, which positively regulate GPIX promoter activity and bind nuclear factors derived from human erythroleukemia cells

  • 1 The abbreviations used are: GP, glycoprotein; HEL, human erythroleukemia; PCR, polymerase chain reaction; PF-4, platelet factor 4; comparative studies of transcriptional regulation of the four subunits of the GPIb-V-IX complex can potentially identify which subunit is rate-limiting during assembly of the complex

  • GPIX5Ј-686Luc showed detectable luciferase activity only in the hematopoietic HEL cell line, which indicates that the GPIX upstream flanking sequence contains cell-specific promoter activity and suggests that this activity is selective for hematopoietic cells

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Summary

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol 271, No 31, Issue of August 2, pp. 18554 –18560, 1996 Printed in U.S.A. Analysis of the Megakaryocyte Glycoprotein IX Promoter Identifies Positive and Negative Regulatory Domains and Functional GATA and Ets Sites*. To characterize GPIX transcriptional regulation, a series of 5؅ deletion constructs was made linking the GPIX upstream flanking sequence to the luciferase marker gene, and promoter activity was measured in transiently transfected human erythroleukemia cells This analysis identified two negative regulatory domains between ؊686 to ؊423 and ؊311 to ؊203 and two positive regulatory domains at ؊323 to ؊311 and ؊151 to ؊100 tightly, but noncovalently [8], bound, and GPV is loosely associated with the complex [9]. DNase I protection experiments identified a protein-dependent “footprint” and hypersensitive site within the GPIX Ets sequence These results provide a framework for comparison of the GPIX promoter with others of the GPIb-V-IX system, other megakaryocyte-specific genes, and other members of the leucine-rich repeat family. Positive regulatory Ets and GATA sites were shown to function in vivo and bind factors in vitro

TABLE I Comparison of GPIX promoter activity in different cell types
EXPERIMENTAL PROCEDURES
RESULTS
GPIX Promoter Regulation
DISCUSSION

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