GATA and NF-Y participate in transcriptional regulation of Fc gamma RIIA in megakaryocytic cells

Human Fc gamma RIIA, expressed on platelets, neutrophils, and macrophages, plays a major role in platelet activation and immune clearance. Clinical ob servations indicate that regulation of expression of this receptor is an im portant factor influencing the course of immune thrombocytopenia. We used b oth transient transfection with Fc gamma RIIA promoter constructs and elect rophoretic mobility shift assays (EMSA) to study the regulation of Fc gamma RIIA. transcription. In HEL (erythromegakaryocytic) cells, the 200 bp imme diately 5' of the ATG start codon accounted for the majority of the activit y of a 3.6-kb promoter fragment. Putative GATA (-161) and NF-Y (-119) sites are present. EMSA analyses demonstrate specific binding of both GATA-1 and GATA-2 to labeled oligonucleotides containing the putative GATA site with HEL but not U937 (myelomonocytic) nuclear extracts. Antibodies to NF-Y supe rshift the specific -119 NF-Y complex with HEL, U937, Jurkat (T-lymphocytic ), and HeLa (nonhematopoietic) nuclear extracts. Comparison of the activity of GATA and NF-Y mutant constructs in HEL and U937 demonstrates that while either GATA or NF-Y mutation results in a large decrease in the promoter a ctivity (2.2- and 2.3-fold, respectively) in HEL cells, neither mutation is effective in reducing activity in U937 cells. This is the first example of a promoter active in the megakaryocyte lineage in which NF-Y cooperates ad ditively with GATA factors to regulate transcription. Identification of oth er factors that must be operational for Fc gamma RIIA transcription in myel omonocytic cells which lack GATA factors will bolster our ongoing efforts t o dissect the function of these Pc receptors in megakaryocytic and myelomon ocytic cells in vivo. (C) 2000 Academic Press.