CHARACTERIZATION OF REGULATORY ELEMENTS IN THE 5'-FLANKING REGION OF THE RAT GPIIB GENE BY STUDIES IN A PRIMARY RAT MARROW CULTURE SYSTEM

Glycoprotein (GP)IIb/IIIa, an integrin complex found on the surface of platelets, is a receptor for fibrinogen and other ligands, and is inv olved in platelet aggregation. Because GPIIb is specifically expressed in megakaryocytes, we have studied the 5'-flanking region of the rat (r) GPIIb gene as a model of a megakaryocyte-specific gene. The studie s presented here used a rat marrow expression system, which allows the study of primary cells undergoing terminal differentiation into megak aryocytes. The determination of megakaryocyte-specific expression of D NA constructs was possible by immunomagnetically separating megakaryoc ytes from total bone marrow cells. Transient expression constructs, co ntaining varying lengths of the 5'-flanking region from -39 to -912 bp , localized a regulatory element between -460 and -439 bp upstream of the transcriptional start site. This region contains a GATA consensus binding element between -457 and -454 (GATA(454)). Further constructs demonstrated that this GATA binding element was indeed essential for e xpression. A 25-bp substitution, covering the region -450 to -426 imme diately downstream of the GATA(454), demonstrated that this region was essential for full expression, which suggests that this region may in teract with the GATA(454) site in promoting high-level lineage-specifi c expression. To define regulatory elements between the GATA(454) and the transcriptional start site further, we tested additional construct s derived from the original -912 construct; each of which contained th e GATA(454) but had different 50-bp deletions from -450 to the start s ite. Virtually all of these constructs continued the GATA(454), demons trated that this region was essential for full expression, which sugge sts that this region may interact with the GATA(454) site in promoting high-level lineage-specific expression. To define regulatory elements between the GATA(454) and the transcriptional start site further, we tested additional constructs derived from the original -912 construct; each of which contained the GATA(454) but had different 50-bp deletio ns from -450 to the start site. Virtually all of these constructs cont inued to show high-level tissue-specific expression. The deleted -150 to -101 construct had twice the level of expression of the full-length wild-type construct; therefore, this region may contain a negative re gulatory element. Comparison of our data with expression studies perfo rmed with the 5'-region of the human GPIIb gene using HEL cells, a cel l line with some megakaryocytic properties, demonstrates significant d ifferences, which may reflect our use of primary rat bone marrow cells . In particular, our study points to the importance of the GATA(454) f or high levels of GPIIb expression in developing megakaryocytes. (C) 1 994 by The American Society of Hematology.