GATA and NF-Y participate in transcriptional regulation of FcgammaRIIA in megakaryocytic cells.
Human FcgammaRIIA, expressed on platelets, neutrophils, and macrophages, plays a major role in platelet activation and immune clearance. Clinical observations indicate that regulation of expression of this receptor is an important factor influencing the course of immune thrombocytopenia. We used both transient transfection with FcgammaRIIA promoter constructs and electrophoretic mobility shift assays (EMSA) to study the regulation of FcgammaRIIA transcription. In HEL (erythromegakaryocytic) cells, the 200 bp immediately 5' of the ATG start codon accounted for the majority of the activity 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 supershift 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 activity (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 additively with GATA factors to regulate transcription. Identification of other factors that must be operational for FcgammaRIIA transcription in myelomonocytic cells which lack GATA factors will bolster our ongoing efforts to dissect the function of these Fc receptors in megakaryocytic and myelomonocytic cells in vivo.
Cassel
D L
DL
Department of Pediatrics, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.
Subudhi
S K
SK
Surrey
S
S
McKenzie
S E
SE
D020121:5' Untranslated Regions / Q000235:genetics; D015703:Antigens, CD / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D001665:Binding Sites; D023081:CCAAT-Binding Factor / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D050989:GATA2 Transcription Factor; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D008533:Megakaryocytes / Q000187:drug effects / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017452:Receptors, IgG / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D014157:Transcription Factors / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D014407:Tumor Cells, Cultured
D020121:5' Untranslated Regions; D015703:Antigens, CD; D023081:CCAAT-Binding Factor; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; C080568:Fc gamma receptor IIA; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D050989:GATA2 Transcription Factor; C494711:GATA2 protein, human; D017452:Receptors, IgG; D014157:Transcription Factors