Functional and regulatory analysis of the Dictyostelium G-box binding factor

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 abse nce of GBF-targeted gene transcription. GBF-mediated gene expression is act ivated by stimulation of cell-surface, seven-span cAMP receptors, but this activation is independent of heterotrimeric G-proteins. To further characte rize 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. Ln 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 in tact zinc finger. In addition, expression of some GBF mutants results in a partial complementation phenotype, suggesting that these mutants are hypomo rphic alleles. We used a 2.4-kb GBF-promoter fragment to examine the regula tion of GBF expression. GBF promoter-reporter studies confirmed the previou s finding that GBF transcription is induced by continuous, micromolar extra cellular cAMP. We also show that, like the activation of GBF-regulated tran scription, the induction of GBF expression requires cell-surface cAMP recep tors, but not heterotrimeric G-proteins. Finally, reporter studies demonstr ated that induction of GBF-promoter-regulated expression does not require t he presence of GBF protein, indicating that GBF expression is not regulated by a positive autoregulatory loop. (C) 2001 Academic Press.