Fig. 2: Excess histones suppress a subtelomeric starvation response.
a RNA-Seq analysis of carbon source switch in synthetic complete medium. EtOH-inducible genes were categorized as transient or fast/slow adaptive based on their expression at 1 h or 20 h after switching sml1Δ cells from glucose to ethanol. b Expression fold changes (log2) of switch-induced genes in sml1Δrad53Δ vs sml1Δ cells at 1 h or 20 h after carbon source switch. Each dot represents one gene. c Enrichment analysis by hypergeometric test of genes in telomere proximity that are up- or downregulated by carbon source switch (EtOH/D) in sml1Δ cells, or by deletion of RAD53 (sml1Δrad53Δ/sml1Δ). d Expression fold changes (log2) of subtelomeric repressed genes in sml1Δrad53Δhht2Δ vs. sml1Δrad53Δ cells. Each dot represents one gene. e RT-qPCR expression analysis of COS1 and COS8 genes in the indicated genotypes in glucose and 90 min after ethanol switch. TFC1 was used as normalization control. (n = 4 replicate cultures). Significances were calculated by Kruskal–Wallis rank sum test (pD_COS1 = 0.0097, pEtOH_COS1 = 0.013, pD_COS8 = 0.0045, pEtOH_COS8 = 0.013), followed by Mann–Whitney test (two-sided) with Benjamini–Hochberg correction (pD_COS1_sml1Δrad53Δ_vs_sml1Δ = 0.029, pD_COS1_sml1Δrad53Δsir2Δ_vs_sml1Δrad53Δ = 0.029, pEtOH_COS1_sml1Δrad53Δ_vs_sml1Δ = 0.029, pEtOH_COS1_sml1Δrad53Δsir2Δ_vs_sml1Δrad53Δ = 0.029, pD_COS8_sml1Δrad53Δ_vs_sml1Δ = 0.029, pD_COS8_sml1Δrad53Δsir2Δ_vs_sml1Δrad53Δ = 0.029, pEtOH_COS8_sml1Δrad53Δ_vs_sml1Δ = 0.029, pEtOH_COS8_sml1Δrad53Δsir2Δ_vs_sml1Δrad53Δ = 0.029). f Cells of the indicated genotypes expressing endogenous 13xMyc-tagged Sir3 were culture in YPD, and the level of Sir3 protein was analyzed by western blot. The bar chart (right panel) provides a quantification of Sir3-Myc levels normalized by loading control Pgk1. Significances were calculated with one-way ANOVA (p = 0.0104) with post hoc Tukey HSD test (psml1Δrad53Δ_vs_sml1Δ = 0.012, psml1Δrad53Δhht2Δ_vs_sml1Δrad53Δ = 0.018). n = 3 independent experiments. g sml1Δ cells expressing endogenous 13xMyc-tagged Sir3 were cultured in normal glucose and subjected to either 2 h glucose-to-ethanol switch or 1 h rapamycin treatment. Sir3 bandshift reflecting phosphorylation was analyzed by western blot. The red dashed line indicates un-phosphorylated Sir3. The scheme summarizes the Mpk1-Sir3 pathway. Bars plots with error bars represent mean values and standard deviation, D glucose; EtOH ethanol. *p < 0.05.
