Identification of cis-acting elements in the DDI2 promoter.

(A) A series of promoter truncations were generated in plasmid YEpDDI2-lacZ and transformed into BY4741 cells. The left panel illustrates DNA sequence remaining after the truncation with marked truncation sites. The right panel shows the β-Gal activity of BY4741 cells carrying the indicated PDDI2-lacZ derivatives with CY, MMS or mock treatment. (B) Graphic illustration of basal level β-Gal activity of BY4741 cells carrying the indicated PDDI2-lacZ 5’ deletions. (C) Graphic illustration of fold induction of BY4741 cells carrying the indicated PDDI2-lacZ 5’ deletions in response to 20 mM CY (blue) or 0.1% MMS (red) treatment for 2 hours. Results of all β-Gal assays were from at least three independent experiments with standard deviations as shown in brackets.

Effects of CY and MMS treatments on cellular histone levels.

(A) Absolute nucleosome occupancy maps at the DDI2 (upper panel) and DDI3 (lower panel) promoter (up to 1 kb) and coding regions. Data were extracted from the Saccharomyces NucMap database (http://bigd.big.ac.cn/nucmap). RPM coverage provides a straightforward and rapid means to visualize overall nucleosome occupancy, DANPOS offers a robust framework for identifying and comparing dynamic chromatin features, and iNPS enables high-resolution nucleosome positioning. These three nucleosome mapping methods are included because NucMap database contains sufficient data for these approaches across a range of strains and experimental treatments. All datasets shown here are derived from strain BY4741, serving as the reference for comparisons with deletion strains. (B) Western blot analysis of histone H3 protein levels in BY4741 and its fzf1Δ mutant cells after 2-hour treatment with 20 mM CY or 0.1% MMS. H3 was detected using an anti-H3 antibody, with Pgk1 as an internal control. Numbers underneath the anti-H3 blot indicate relative histone H3 levels. (C) RT-qPCR analysis of genes encoding histone H3 (HHT1 and HHT2) and H4 (HHF1 and HHF2) after 2-hour treatment of BY4741 cells with 20 mM CY or 0.1% MMS. UBC6 was used as the internal reference gene. Values represent the average of at least three biological replicates, with standard deviations shown as error bars. The statistical analysis was performed using two-way ANOVA multiple comparison via GraphPad Prism. ****, P<0.0001; ***, P< 0.001; **, P<0.01. NT, no chemical treatment.

Effects of SPT10 deletion on histone and DDI2/3 gene expression.

(A) RT-qPCR analysis of genes encoding histone H3 (HHT1 and HHT2) and H4 (HHF1 and HHF2) after 2-hour treatment of BY4741 (WT) or BY4741 spt10Δ cells with 20 mM CY. UBC6 was used as the internal reference gene. (B) Western blot analysis of histone H3 protein levels in BY4741 and its spt10Δ mutant cells after 2-hour treatment with 20 mM CY or 0.1% MMS. H3 was detected using an anti-H3 antibody, with Pgk1 as an internal control. Numbers underneath the anti-H3 blot indicate relative histone H3 levels. (C) RT-qPCR analysis of DDI2/3 transcript levels in spt10Δ cells with or without chemical treatment. The DDI2/3 transcript were measured in the same sets of samples as in (A). Values in both (A) and (C) represent the average of at least three biological replicates, with standard deviations shown as error bars. The statistical analysis was performed using two-way ANOVA multiple comparison via GraphPad Prism. ****, P<0.0001; ***, P< 0.001; *, P<0.05. NT, no chemical treatment.

Effects of CY and MMS treatments on yeast cell growth and survival.

(A) Cell growth as measured by cell density. (B) Cell survival as measured by % survival. BY4741 cells were cultured to mid-log phase (OD₆₀₀ nm = 0.4 - 0.5) and subsequently treated with 5, 10, 20 or 40 mM CY, or with 0.0125%, 0.025%, 0.05% or 0.1% MMS for 2 hours. After treatment, cell density was measured by spectrophotometry at OD₆₀₀ nm, and aliquots of the cultures were serially diluted and plated on YPD agar plates. Survival rates were calculated by comparing the number of colony-forming units (CFUs) from treated samples to those from untreated controls. Values in both (A) and (B) represent the average of at least three biological replicates, with standard deviations shown as error bars.

Histone depletion and its effects on the expression of Fzf1-regulated genes.

(A) Schematic diagram illustrating the histone depletion strain RMY102 and the experimental plan. Both copies of genes encoding histone H3 and H4 are deleted, and RMY102 cells are maintained by a plasmid carrying HHT2 and HHF2 under the control of galactose-inducible promoters PGAL10 and PGAL1, respectively, and incubation in YPGal medium. Once transferring to a YPD medium, the PGAL promoters are repressed, leading to cessation of de novo H3 and H4 synthesis and creation of a histone depletion status. (B,C) RT-qPCR analysis of cellular HHT2 and HHF2 levels in RMY102 (B) and RMY102 fzf1Δ (C) cells under H3/H4 depletion conditions. (D, E) Western blot analysis of cellular histone H3 levels in histone depleted RMY102 (D) and RMY102 fzf1Δ (E) cells over time. Whole-cell lysates from yeast cells grown in either YPGal or YPD medium for the indicated times were subjected to immunoblotting using anti-H3 antibodies (upper panel), and anti-Pgk1 (lower panel) served as an internal control. Numbers underneath the anti-H3 blot indicate histone H3 levels relative to cells grown in YPGal. (F) RT-qPCR analysis of transcript levels of Fzf1-regulated genes in RMY102 and RMY102 fzf1Δ under histone depletion conditions. Values represent the average of at least three biological replicates, with standard deviations shown as error bars. The statistical analysis was performed using two-way ANOVA multiple comparison via GraphPad Prism. ****, P<0.0001; ns, not statistically significant.

Joint effects of CY treatment and histone depletion on the expression of Fzf1-regulated genes.

(A) Relative mRNA levels of Fzf1-regulated genes in RMY102 cells treated with different concentrations of CY under histone depletion conditions. Gene expression was quantified by RT-qPCR. Data represent the mean values from at least three independent biological replicates, with standard deviations represented as error bars. (B) Comparative analysis of Fzf1-regulated gene expression in RMY102 cells with or without histone H3/H4 depletion by culturing in YPD and YPGal media, respectively, under various CY treatment conditions. (C) Relative mRNA levels of Fzf1-regulated genes in RMY102 fzf1Δ cells under histone H3/H4 depletion conditions by culturing in YPD and YPGal media, respectively, treated with different concentrations of CY.

Joint effects of MMS treatment and histone depletion on the expression of Fzf1-regulated genes.

(A) Relative mRNA levels of Fzf1-regulated genes in RMY102 cells treated with different concentrations of MMS under histone depletion conditions. Gene expression was quantified by RT-qPCR. Data represent the mean values from at least three independent biological replicates, with standard deviations represented as error bars. (B) Comparative analysis of Fzf1-regulated gene expression in RMY102 cells with or without histone H3/H4 depletion by culturing in YPD and YPGal media, respectively, under various MMS treatment conditions. (C) Relative mRNA levels of Fzf1-regulated genes in RMY102 fzf1Δ cells under histone H3/H4 depletion conditions by culturing in YPD and YPGal media, respectively, treated with different concentrations of MMS.

Nucleosome occupancy at the DDI2 and DDI3 loci under various experimental conditions.

(A, B) Nucleosome occupancy profiles of (A) DDI2 and (B) DDI3 coding regions and up to 1 kp from the translation start in BY4741 (WT) and BY4741 fzf1Δ cells with or without 20 mM CY treatment for 2 hours. Two independent replicates were performed for each treatment. Only reads that uniquely mapped to DDI2 or DDI3 were used in the analysis, and the data were normalized to the genomic mean (Reads Per Genomic Content, RPGC). (C) The occupancy of individual nucleosomes upstream of DDI2 and DDI3 based on (Weiner et al., 2015). The four upstream nucleosomes in DDI2 (blue) and DDI3 (red) were compared with the mean of all other upstream (−1, −2, and −3) nucleosomes (grey) under given experimental conditions.

A working model to illustrate dual regulation of DDI2/3 genes by nucleosome occupancy and Fzf1.

In the absence of environmental stress, Fzf1 loosely binds CS2 in an inactivated state. Upon chemical stresses induced by CY or MMS, Fzf1 undergoes conformational changes so that its zinc-finger 5 (ZF5) activation domain is no longer inhibited by ZF4, leading to interaction and recruitment of a chromatin remodeling complex to the DDI2/3 promoters to reduce the nucleosome occupancy and induce the DDI2/3 expression. TATA, the TATA box; TIC, transcription initiation complex. Figure was created by using BioRender with academic licence.

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