Fig. 1

The TTT subunit Tti2 contributes to Tra1- and Tra2-dependent gene expression. a, b RNA-seq analyses of control creER, inducible tti2 (tti2-CKO) and tra2 knockouts (tra2-CKO), tra1Δ and wild-type (WT) strains (n = 3 independent biological samples). creER, tti2-CKO and tra2-CKO cultures were supplemented with either DMSO (+Tti2 or +Tra2) or β-estradiol (-Tti2 or -Tra2), for either 21 h (creER and tra2-CKO) or 18 h (tti2-CKO). a Density scatter plots comparing Tti2- with Tra2-depleted cells (left, r = 0.46, P < 0.001), Tti2-depleted cells with Tra1 deletion mutants (middle, r = 0.36, P < 0.001), and Tra2-depleted cells with Tra1 deletion mutants (right, r = −0.07, P < 0.001). Statistical significance and correlation were analysed by computing the Pearson correlation coefficient. Differential gene expression analyses were performed comparing cells treated with either DMSO (control) or β-estradiol (KO), while tra1Δ mutants were compared with isogenic WT cells. Genes whose expression is regulated by β-estradiol treatment (Supplementary Fig. 3a) were filtered out. b Venn diagrams showing the overlap of differentially expressed genes (DEGs) between all mutants. Using FC ≥ 1.5, P ≤ 0.01 thresholds, 184 DEGs were identified in Tti2-depleted cells, 153 in Tra2-depleted cells and 59 in tra1Δ mutants. Statistical significance was calculated using a hypergeometric test: P = 8.66e-115 comparing tti2-CKO, tra2-CKO and tra1Δ; P = 3.67e-72 comparing tti2-CKO with tra2-CKO; P = 2.37e-15 comparing tti2-CKO with tra1Δ; P = 0.004 comparing tra2-CKO with tra1Δ. c, d mRNA levels of the Tra1-dependent genes SPCC569.05c and gst2 (c) and the Tra2-dependent genes SPCC1884.01 and SPAC977.12 (d) were measured using RT-qPCR of RNA extracted from tti2-CKO, tra2-CKO and tra1Δ strains grown as described in panels a, b. Each value represents mean mRNA levels from three independent experiments with the SEM. act1 served as a control for normalisation across samples. Values from one control experiment were set to 1, allowing comparisons across culture conditions and strains. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison tests (asterisk: P < 0.05). e ChIP-qPCR was performed using tti2-CKO cells treated with either DMSO (+) or β-estradiol (−) for 18 h. ChIP of Spt7-MYC at the pho84 and mei2 promoters and of Epl1-MYC at the ssa2 promoter serve as proxies for SAGA and NuA4 binding, respectively. A non-tagged strain was used as control for background IP signal (MYC: no). Ratios of MYC ChIP to input (IP/IN) from three independent experiments are shown as individual points, overlaid with the mean and SEM. Statistical significance was determined as in (c, d). f Anti-MYC and -HA western blotting of Tti2-HA, Spt7-MYC and Epl1-MYC in a fraction of the chromatin samples used for the ChIP-qPCR experiments shown in panel e. Equal loading was controlled using an anti-tubulin antibody. Source data are provided as a Source Data file