Cloning and characterization of the G-box binding factor, an essential component of the developmental switch between early and late development in Dictyostelium.

Abstract

During Dictyostelium development, the cAMP-regulated induction of cell-type-speciKc late genes marks a developmental switch from the initial formation of the multicellular organism to the differentiation of the various cell types that mediate morphogenesis and eventually give rise to the mature fruting body. The G-box binding factor (GBF) is a developmentally regulated Dictyostelium transcription factor whose affinity for a DNA sequence correlates with the ability of that sequence to confer inducibility to late gene promoters in response to high, continuous levels of extracellular cAMP. We report the purification of GBF and cloning of the gene that encodes it, as confirmed by in vitro production of GBF activity. The predicted protein is highly basic and contains two putative zinc fingers. Disruption of the GBF gene by homologous recombination results in the loss of all GBF DNA-binding activity, developmental arrest at the loose aggregate stage, and the loss of late gene induction during development or in response to extracellular cAMP. Constitutive expression of GBF complements the null phenotype and allows for the rapid activation of a class of late genes in response to cAMP. Our results indicate that GBF acts as an extracellular cAMP-responsive transcriptional activator regulating late gene expression and is an essential component of a developmental switch between aggregation and cellular morphogenesis.

Identification of cis-Regulating Elements andtrans-Acting Factors Regulating the Expression of the Gene Encoding the Small Subunit of Ribonucleotide Reductase inDictyostelium discoideum

“…The CAEs exhibit strong affinity for the developmentally regulated transcriptional factor GBF (24). Cells carrying a null mutation in the gene encoding GBF are arrested at the loose aggregate stage, before cell differentiation has occurred (24). The latter results provide further evidence that besides the interaction between GBF and CAEs, prespore gene expression requires the interaction with other factors and regulatory elements.…”

“…For the binding assays, 10 g of protein extract were incubated with 3000 cpm of end-labeled probe (0.1-0.5 ng), 500 ng of double-stranded poly(dI-dC) (Sigma), and 1 g of bovine serum albumin in a final volume of 20 l of 0.25ϫ storage buffer (24). The components were allowed to bind at room temperature for 30 min, immediately applied on a 4.5% acrylamide-TBE gel, and resolved at 4°C at 140 V until the unbound probe reached the bottom of the gel.…”

“…Electrophoretic Mobility Shift Assays-The cytosolic and nuclear extracts were prepared as described (24). The probes were the oligonucleotides reconstituting boxes A and B as well as CAE-1 (21).…”

“…However, expression in prespore cells can be stimulated when the CAEs and the A/T-rich element are placed together with a heterologous basal promoter (19,20). The CAEs exhibit strong affinity for the developmentally regulated transcriptional factor GBF (24). Cells carrying a null mutation in the gene encoding GBF are arrested at the loose aggregate stage, before cell differentiation has occurred (24).…”

Identification and functional analysis of a developmentally regulated extracellular signal-regulated kinase gene in Dictyostelium discoideum.

“…Biochemical and molecular genetic analyses over the last few years have identified components required for various aspects of these processes and have led to a further understanding of the regulatory pathways controlling development. Genes encoding membrane receptors, coupled GP and Got subunits, the effectors adenylyl cyclase and phospholipase C, and the transcription factor GBF have been cloned and analyzed at a molecular level (12,17,32,65,72,81,82 the membrane to those in the nucleus is cAMP-dependent protein kinase (PKA). PKA has been shown to be essential for aggregation, cell-type-specific expression of the ecmB gene, the induction of prespore cell differentiation, and the induction of sporulation and spore cell-specific gene expression (1, 27, 35, 36, 40, 56-58, 76, 77 (8, 21-23, 61, 79, 84).…”