The TTGA-binding factor is a transcriptional regulator activated by DIF, the chlorinated hexaphenone that induces prestalk cell differentiation in Dictyostelium. The same activity also functions as a repressor, controlling stalk cell differentiation. We show that the TTGA-binding factor is a STAT protein. Like the metazoan STATs, it functions via the reciprocal interaction of a phosphotyrosine residue on one molecule with an SH2 domain on a dimerizing partner. Furthermore, it will bind specifically to a mammalian interferon-stimulated response element. In Saccharomyces cerevisiae, where the entire genomic sequence is known, SH2 domains have not been identified. It would seem, therefore, that SH2 signaling pathways arose very early in the evolution of multicellular organisms, perhaps to facilitate intercellular comunication.

The ecmA gene is specifically expressed in prestalk cells and its transcription is induced by the chlorinated hexaphenone DIF-1. We have purified a novel bZIP transcription factor, DimB, by affinity chromatography on two spatially separated ecmA promoter fragments. Mutagenesis of the cap-site proximal DimB-binding site (the -510 site) greatly decreases ecmA expression in the pstO cells, which comprise the rear half of the prestalk zone, and also in the Anterior-Like Cells, which lie scattered throughout the prespore region. However, DimB is not essential for normal expression of the ecmA gene, instead it spatially limits its expression; ecmA is relatively highly expressed in the subset of prestalk cells that coats the prestalk zone, but in slugs of a DimB-null strain, ecmA is highly expressed throughout the prestalk zone. Because the -510 site is required for correct ecmA expression, we posit a separate activator protein that competes with DimB for binding to the -510 site. DimB rapidly accumulates in the nucleus when cells are exposed to DIF-1, and ChIP analysis shows that, in the presence of extracellular cAMP, DIF-1 causes DimB to associate with the ecmA promoter in vivo. Thus, DIF-1 regulates DimB activity to generate a gradient of ecmA expression in the prestalk zone of the slug.

DNA double-strand breaks (DSBs) can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). In vertebrates, the first step in NHEJ is recruitment of the DNA-dependent protein kinase (DNA-PK) to DNA termini. DNA-PK consists of a catalytic subunit (DNA-PKcs) that is recruited to DNA ends by the Ku70/Ku80 heterodimer. Although Ku has been identified in a wide variety of organisms, to date DNA-PKcs has only been identified experimentally in vertebrates. Here, we report the identification of DNA-PK in the nonvertebrate Dictyostelium. Dictyostelium Ku80 contains a conserved domain previously implicated in recruiting DNA-PKcs to DNA and consistent with this observation, we have identified DNA-PKcs in the Dictyostelium genome. Disruption of the gene encoding Dictyostelium DNA-PKcs results in sensitivity to DNA DSBs and defective H2AX phosphorylation in response to this form of DNA damage. However, these phenotypes are only apparent when DNA damage is administered in G(1) phase of the cell cycle. These data illustrate a cell cycle-dependent requirement for Dictyostelium DNA-PK in signaling and combating DNA DSBs and represent the first experimental verification of DNA-PKcs in a nonvertebrate organism.