A developmentally regulated trans-acting factor recognizes dissimilar G/C-rich elements controlling a class of cAMP-inducible Dictyostelium genes.

Abstract

Transcriptional response elements involved in the cAMP-inducible and developmentally regulated expression of the Dictyostelium aggregate-stage gene pst-cath/CP2 have been shown to include a G/C-rich sequence element [G-box regulatory element {GBRE1]. We have recently identified a trans-acting factor, GBF (GBRE binding factor), that specifically interacts with this sequence and have shown that the binding activity of GBF to GBRE is developmentally regulated and inducible by cAMP. Here, we examine further the possible role of GBF in the regulation of pst-cath/CP2 and three other coordinately regulated, cAMP-inducible aggregate-stage genes. We show that GBF itself {or other closely related factorsl recognizes dissimilar G/C-rich elements present in the 5'-flanking regions of these genes and that the ability of the individual, distinct G/C-rich elements to confer regulated expression on a promoter deletion mutant of the pst-cath/CP2 gene is correlated with the relative affinity for GBF. G/C-rich elements carrying point mutations that prevent in vitro binding of GBF to two of the G/C-rich elements fail to activate expression in vivo. An analysis of major points of contact between the GBF protein and two distinctly different binding sites suggests that binding of GBF to these sequence elements involves a considerable degree of flexibility in DNA-protein interactions. These results suggest that the regulated expression of a class of aggregate-stage cAMP-inducible genes involves the interaction of GBF or homologous factors with dissimilar G/C-rich sequence elements and that induction of GBF activity or that of homologous factors by cAMP may thus be a limiting step in the induction of this temporally coordinate set of genes during Dictyostelium development. Dictyostelium discoideum propagates as a vegetative amoeba in the presence of nutrients and initiates, upon starvation, a multicellular differentiation process that results in the formation of a mature fruiting body containing two distinctly different cell types--stalk cells and spores. Within several hours of starvation, Dictyostelium cells aggregate in response to pulsatile cAMP signals. Binding of cAMP to cell-surface receptors activates adenylate cyclase, and repeated cycles of cAMP synthesis and secretion serve to transmit, or relay, the chemotactic signal outward through the population of aggregating cells (see Janssens and van Haastert 1987;Firtel et al. 1989). After formation of tightly associated multicellular aggregates, a migrating pseudoplasmodium (slug) is formed in which precursor cells to the 3Corresponding author.

A spatial gradient of expression of a cAMP-regulated prespore cell-type-specific gene in Dictyostelium.

“…It is possible that the CAE boxes represent a prespore cell-type-specific element or an element required for cAMP regulation. Moreover, this sequence is homologous to one from DG17, which has been shown to confer cAMP and temporal regulation to a heterologous gene (Hjorth et al 1990). Although the three CAEs are quite homologous, there are nucleotide sequence differences, the significance of which is not presently known.…”

“…Molecular analysis has identified the cis-acting elements regulating cAMP induction and developmental expression, as well as a developmentally regulated trans-acting factor controlling their expression of the cAMP-inducible early prestalk genes Firtel 1987, 1988;Williams 1987, 1988;Hjorth et al 1989Hjorth et al , 1990. However, no information is available on the molecular requirements for prespore gene expression, nor is the spatial expression of these genes within the multicellular aggregate understood.…”

A GBF-binding site and a novel AT element define the minimal sequences sufficient to direct prespore-specific expression in Dictyostelium discoideum.

“…6A). Similarities in developmental and cAMP regulation, relative mobility, and binding-site sequence prompted us to examine whether this activity was identical to GBF, which has been shown to mediate the developmental induction of some prestalk genes (3,6,18,20,35). GBF binding requires two (19,20) During the course of these studies, GBF was purified and the gene encoding it was cloned (39).…”

“…GBF had been described previously as a developmentally regulated, cAMP-induced activity that interacts with cis-elements required for the developmental induction of the prestalk-enriched genes CP2/pst-cathepsin and ecmB (3,20). Since one GBF binding site from DG17 (20) fits the SP60cotC CAE consensus well, and since GBF has a competition profile similar to that of the CAE-binding activity (see Results), we hypothesized that the CAE-binding activity and GBF were identical. GBF has recently been purified, and the gene encoding it has been cloned (39).…”

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 interact specifically with the trans-acting factor GBF (41), with the element CAE-1 of cotC displaying the highest affinity (39). Competition experiments showed that a 300-fold excess of CAE-1 was about as effective a competitor as 30-fold box B (Fig.…”