Transactivation of the GATA-1 promoter by a myb-ets-containing mouse retrovirus is mediated by CACCC elements

The myb-ets-containing ME26 virus causes erythroleukemia in mice by a novel mechanism involving the inappropriate activation of erythroid-specific genes in hematopoietic precursor cells. We have previously shown that the ME26 viral protein can transactivate the GATA-1 promoter in transient transactivation assays carried out in mouse fibroblasts. The mouse GATA-1 promoter, whose activity is regulated by the GATA-1 protein itself, contains a double GATA consensus sequence at its 5' end and two CACCC elements at its 3' end, both of which are crucial for promoter activity in erythroid cells, as well as a nonconsensus GATA sequence and several putative c-myb and c-ets binding sites. In order to determine which sequences in the GATA-1 promoter are crucial for activation by the ME26 viral protein, we made deletions of the promoter, cloned them into a luciferase expression vector and tested their activity in mouse fibroblasts, which do not express GATA-1. Our results indicate that sequences in the 3' end of the GATA-1 promoter, which include two CACCC elements, are essential for transactivation by ME26 virus, while other upstream sites contribute to full activation by the virus. Mutation of the CACCC sites abolishes ME26 viral transactivation. The interaction of cell extracts containing ME26 viral protein and the GATA-1 promoter fragment containing the two CACCC elements was examined by electrophoretic mobility shift analysis (EMSA) and the results showed no direct interaction between the two. However, we could detect the ubiquitous transcription factor Sp1 bound to this sequence. These data demonstrate that the CACCC element is necessary for GATA-1 promoter transactivation by ME26 virus and that the viral protein may indirectly transactivate the promoter by binding to Sp1.