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. 2004 Sep 7;101(40):14315–14322. doi: 10.1073/pnas.0405353101

Fig. 4.

Fig. 4.

Sfp1 is a direct regulator of RP genes and is required for appropriate down-regulation of RP gene expression in response to stress. (A) Microarray analysis comparing expression of RP genes in the following lanes: a, wild-type strain treated with rapamycin (Cy5 red) versus an untreated wild-type strain (Cy3 green) (1 h after addition of 100 nM rapamycin); b, sfp1Δ strain treated with rapamycin (Cy5 red) versus untreated sfp1Δ (Cy3 green) (1 h); c, sfp1Δ (Cy5 red) versus wild type (Cy3 green); d, sfp1Δ treated with rapamycin (Cy5 red) versus wild type treated with rapamycin (Cy3 green) (1 h). Data presented are the average of three independent microarray experiments. When examining the behavior of genes from various functional groups (47, 48) in the microarray experiments, RPs were the most significantly regulated functional group in lanes a, b, and c with P < 7.19 × 10-106, 1.13 × 10-25, and 5.9 × 10-104, respectively. (B) Quantitative RT-PCR validation of microarray data. Expression levels of several RP mRNAs relative to ACT1 transcript levels were measured in wild-type and sfp1Δ strains, untreated or treated with 100 nM rapamycin (rap) for 1 h. (Upper) The fold repression of RP genes in response to rapamycin in both wild-type and sfp1Δ strains. (Lower) The comparison of RP gene expression in the sfp1Δ strain versus wild type, either under optimal growth conditions or after treatment with rapamycin. Values are the averages of three independent experiments; error bars show SEM. (C) Yeast cells lacking Sfp1 are defective in RP regulation in response to oxidative stress. Expression levels of several RP mRNAs relative to the ACT1 transcript were measured by quantitative RT-PCR in wild-type and sfp1Δ strains untreated or treated with 0.4 mM H2O2 for 60 min. (Upper) The fold repression of RP genes in response to H2O2 in both wild-type and sfp1Δ strains. (Lower) The comparison of RP gene expression in the sfp1Δ strain versus wild type, either under optimal growth conditions or after treatment with H2O2. Values are the averages of three independent experiments; error bars show SEM. (D) Chromatin immunoprecipitation analysis of Sfp1-HA3 in cells treated or untreated with 100 nM rapamycin. Sfp1 binding for the indicated promoter DNA relative to ACT1 DNA is represented by (immunoprecipitated DNA/input DNA)/(ACT1 immunoprecipitated DNA/ACT1 input DNA). Values are the averages of three independent experiments; error bars show standard deviations.