Functional and Regulatory Analysis of the Dictyostelium G-Box Binding Factor

Abstract

The Dictyostelium discoidium G-box binding factor (GBF) is required for the induction of known postaggregative and cell-type-specific genes. gbf-null cells undergo developmental arrest at the loose-mound stage due to the absence of GBF-targeted gene transcription. GBF-mediated gene expression is activated by stimulation of cell-surface, seven-span cAMP receptors, but this activation is independent of heterotrimeric G-proteins. To further characterize GBF, we assayed a series of GBF mutants for their ability to bind a G-box in vitro and to complement the gbf-null phenotype. In vitro DNA-binding activity resides in the central portion of the protein, which contains two predicted zinc fingers. However, in vivo GBF function requires only one intact zinc finger. In addition, expression of some GBF mutants results in a partial complementation phenotype, suggesting that these mutants are hypomorphic alleles. We used a 2.4-kb GBF-promoter fragment to examine the regulation of GBF expression. GBF promoter-reporter studies confirmed the previous finding that GBF transcription is induced by continuous, micromolar extracellular cAMP. We also show that, like the activation of GBF-regulated transcription, the induction of GBF expression requires cell-surface cAMP receptors, but not heterotrimeric G-proteins. Finally, reporter studies demonstrated that induction of GBF-promoter-regulated expression does not require the presence of GBF protein, indicating that GBF expression is not regulated by a positive autoregulatory loop.