Regulatory effect of GAGA element-related protein on the Drosophila GAGA-dependent promoter activity in Jurkat Cells

Abstract

To explore the regulatory effect of the GAGA element-related protein (GRP) on the Drosophila GAGA-dependent promoter activity in Jurkat cells. Drosophila GAGA (dGAGA) factor (dGAF), GRP, and either the chloramphenicol acetyltransferase (CAT) reporter plasmid driven by the GAGA element-containing promoter of ftz gene or its mutant GAGA control were selectively co-transfected into Jurkat cells. Promoter activity analyses were performed by analyzing the RNA expression of the CAT in a real-time-RT PCR system, with pRC-CMV-betaGal co-transfected with the CAT reporter as a transfection efficiency control. Electrophoretic mobility shift assays (EMSA) were carried out to examine the binding profile of Jurkat nuclear extracts with a biotin labeled probe-containing dGAGA. In Jurkat cells, GRP, either singly or combined with dGAF, elevated the activity of wild type dGAGA-containing ftz promoter dose-dependently in certain range. However, when the level of GRP was excessively high, it reduced or even fully inhibited the promoter activity of the ftz gene. On the contrary, either GRP or dGAF could not activate the ftz promoter with mutant GAGA. EMSA profile showed a specific band composed of the GAGA element and its binding proteins from Jurkat cells. Human GAGA element-binding proteins exist in Jurkat cells. Its may either directly regulate the gene via GAGA elements or mediate the biphasic regulation of relevant gene in a GRP dose-dependent way.