Figure 6

Validation of SpaA targets identified by ChIPseq. (A) SpaA target immunoprecipitation. Total chromatin and immuno-precipitates prepared with or without αGFP antibody from spaA-/spaA-YFP culminants for the ChIP-seq experiments (see Methods) were subjected to qPCR with promoter specific primers (Supplementary Table S2) to determine the presence of promoter regions of the psvA, cotC, DDB_G0286055, pspE and sigF genes. Amounts of amplified products are expressed relative to amounts obtained from diluted total chromatin. Means and SD of 3 independent experiments. (B) Developmental expression. mRNA levels of putative SpaA targets were measured by qRT-PCR during the final 12 h of Ax2 and spaA- development as in Fig. 2. Data are expressed relative to the highest expression obtained in Ax2, and data from three experiments are plotted separately with blue symbols for Ax2 and red symbols for spaA-. Blue and red lines connect the mean values of the three experiments for Ax2 and spaA-, respectively. (C) Expression profiles and prespore specificity. Heat maps of standardized expression profiles (read counts expressed as fraction of the read count sum of all developmental time points) and prespore/prestalk cell-type enrichment (²log fold-change) of the genes investigated in panels A and B. Data are retrieved from two high-throughput RNA sequencing experiments of D.discoideum AX4 developmental time courses and purified prestalk and prespore cells²¹. (D) SpaA expression in cudA- and srfA-. Null mutants in cudA⁶ and srfA⁸ and their respective parents Ax2 and Ax4, were developed for 16 hr in the dark untill standing slugs had formed. RNA was isolated and expression of spaA was determined by qRT-PCR. Expression in mutant cells was normalised to expression in the parental strain. Mean and SD of 2 experiments.