Abstract
Sporulation in Dictyostelium fruiting bodies evolved from amoebozoan encystation with both being induced by cAMP acting on PKA, but with downstream components still being unknown. Using tagged mutagenesis to find missing pathway components, we identified a sporeless mutant defective in a nuclear protein, SpaA. Expression of prespore genes was strongly reduced in spaA- cells, while expression of many spore stage genes was absent. Chromatin immunoprecipitation (ChIP) of a SpaA-YFP gene fusion showed that (pre)spore gene promoters bind directly to SpaA, identifying SpaA as a transcriptional regulator. SpaA dependent spore gene expression required PKA in vivo and was stimulated in vitro by the membrane-permeant PKA agonist 8Br-cAMP. The PKA agonist also promoted SpaA binding to (pre)spore promoters, placing SpaA downstream of PKA. Sequencing of SpaA-YFP ChIPed DNA fragments revealed that SpaA binds at least 117 (pre)spore promoters, including those of other transcription factors that activate some spore genes. These factors are not in turn required for spaA expression, identifying SpaA as the major trancriptional inducer of sporulation.
Highlights
Most free-living protists differentiate individually into dormant cysts or spores when challenged by environmental stress
CotC protein accumulates in Golgi-derived prespore vesicles (PSVs) in slugs and is exocytosed at spore maturation to become incorporated in the spore coat[15] and this was the case for cotC-mRFP (Fig. 1B)
A sporulation deficient mutant was isolated from a REMI screen for mutants defective in prespore gene expression
Summary
Most free-living protists differentiate individually into dormant cysts or spores when challenged by environmental stress. CAMP crucially regulates this process both as a secreted signal inducing chemotactic aggregation of starving amoebas and expression of aggregation genes and prespore genes, and as an intracellular messenger acting on PKA to induce maturation of spore and stalk cells. In D. discoideum the sensor histidine kinases detect signals within the fruiting body that regulate the transition from motile amoebas into encapsulated spore and stalk cells at the correct time and place. SrfA null mutants have defects in morphogenesis and form spherical instead of elliptical spores, with diminished viability[8,9]. The expression of both BzpF and SrfA is upregulated by PKA, but they are not targets for phosphorylation by PKA7,10. Sequencing of immuno-precipitated chromatin fragments cross-linked to SpaA-YFP revealed over 300 putative target genes for SpaA
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