Isolation of genes involved in the commitment of eosinophils and basophils

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Granule major basic protein (MBP), is expressed exclusively in eosinophils, basophils, and placental trophoblasts. To identify the cis-elements and transcription factors involved in regulating MBP expression, we subcloned 3.2kb of MBP upstream sequence and serial 5' deletions into the pXP2 luciferase reporter vector. An 80% decrement in promoter activity was obtained when MBP sequences between bp -117 and -67 were deleted. To identify transcription factors that bind to and transactivate through the bp -117 to -67 region, we first compared the upstream genomic sequences of human and murine MBP ; a potential GATA binding consensus site was conserved in the 50bp region between the two genes. To determine which GATA proteins bind this consensus site, we performed electrophoretic mobility shift assays (EMSAs) which demonstrated that both GATA-1 and GATA-2 can bind to this consensus site. To determine the functionality of this site, we tested whether GATA-1 and GATA-2, either individually or in combinantion, can transactivate the MBP promoter in the Jurkat T cell line. Co-transfection with a GATA-1 expression vector produced 20-fold augmentation of MBP promoter activity, whereas GATA-2 had no activity. In contrast, combined co-transfection of GATA-1 and GATA-2, decreased the ability of GATA-1 to transactivate the MBP promoter by approximately 50%. Our results provide the first evidence for a GATA-1 target gene in eosinophils, and a negative regulatory role for GATA-2 in MBP expression, and possibly eosinophil gene transcription in general during myelopoiesis.