Figure 6. Rbf1 binds to the promoter of the Rbf1-dependently expressed dik6 gene.

(A) Overview of the dik6 promoter region investigated by qChIP in strain AB31 rbf1:3xHA. Shown are the positions of the amplicons used for qChIP (numbered from 1 to 10) and the promoter truncations and internal deletions assayed in the GFP-reporter assay. (B) qChIP analysis of Rbf1-binding to the dik6-promoter in strains AB31 and AB31rbf1:3xHA 5h after induction of the bE1/bW2-heterodimer in CMA. AB31rbf1:3xHA expresses an HA-tagged Rbf1 protein used for immunoprecipitation with anti-HA-antibody. Numbers give the enrichment in % of the input-DNA of the PCR amplicons in DNA co-immunoprecipitated with HA-antibody Start and end of the amplicons are given in nucleotides (nt) relative to the start codon of dik6. The relative positions of the amplicons are given in (A). As additional control for qChIP, a region from the eIF2b gene (um04869) was used. Given are the mean values of two technical replicates of three independent experiments each, and the standard deviation (SD). Significance of the difference to values obtained for the control-amplicon located within the dik6 open reading frame (amplicon 11) was calculated using Students t-test; the respective p-values are given for AB31rbf1:3xHA. Highest enrichment was observed for amplicons 3, 4, 5 and 6. No significant enrichment was observed in control strain AB31. (C) The dik6 promoter fragments outlined in (A) were fused to GFP as a reporter and integrated in single copy into the ip-locus of U. maydis strain CP27 (a2 Δb::P_crg1:rbf1). GFP-expression was visualized microscopically 5 hours after induction of rbf1 expression in CMA medium. GFP expression declined when the promoter was truncated from 816 bp to 638 bp, and was abolished when a 298 bp promoter fragment was used. Similarly, the internal deletion Δ3 led to reduced GFP expression, while no GFP signal was detectable in the Δ 5 deletion. Scale bar = 20 µm.