The Role of the Yap5 Transcription Factor in Remodeling Gene Expression in Response to Fe Bioavailability
Figure 4
GRX4 gene expression is dependent on Yap5.
(A) Exponentially growing cells from wild-type (WT) and yap5 mutant (yap5) strains were upshifted to high-Fe medium, by supplementation of SC medium with the indicated FeSO4 concentrations, and harvested at the indicated time-points. The expression of GRX4 was assessed by qRT-PCR as described in Materials and Methods. Values are the mean of biological triplicates ± s.d. (B) WT and yap5 strains were transformed with a plasmid carrying GRX4-HA, treated with 5 and 15 mM of FeSO4 for 20 min and analyzed by Western blot with an anti-HA antibody. Pgk1 protein levels were used as loading control. (C) Representation of the GRX4 constructs without (a) or with (b,c,d) mutations in the YREs. (D) Mutant grx4 cells expressing the constructs depicted in (C) were grown under Fe-adequate or Fe overload (5 mM FeSO4) conditions, and the expression of GRX4 was assessed by qRT-PCR as described in Materials and Methods. Values are the mean of biological triplicates ± s.d. (E) yap5 cells were transformed with HA-tagged Yap5 and grown in SD medium not supplemented (−Fe) or supplemented (+Fe) for 15 min with 2 mM of FeSO4, before being processed for ChIP. ChIP analysis was performed using probes specific for GRX4. (F) ChIP analyses combined with qRT-PCR, were used to determine the fold enrichment of GRX4, SCR1 and ARN2. The sequence enrichment in the ChIP (i.e. IP/IN) was normalized using the ACT gene as a reference.