Fig. 3: Fitness values predict the transcriptional activation and cell cycle regulatory functions of Hcm1.

a Transcriptional activation assay of LexA-DBD fused to the Hcm1 TAD with the indicated phosphosite mutations, and WT or empty vector (EV) controls. Black represents EV, gray represents WT, color gradient from blue (hcm1-8A) to red (hcm1-8E) corresponds to increasing number of phosphomimetic mutations. Error bars represent standard deviation, shown is an average of n = 3 replicates. Significance was tested using repeated measures one-way ANOVA with Dunnett’s multiple comparisons test. Asterisks indicate significantly different from WT, ****P < 0.0001, *P < 0.05, ns = non-significant. EV, AAAAAAAA, AAAEAAAA, AAAAEAAA P < 0.0001; AAAEEAAA P = 0.9922; EEEAAEEE P = 0.0484; EEEAEEEE, EEEEAEEE, EEEEEEEE, P < 0.0001. Supplementary Fig. 4a confirms that the fusion proteins are expressed at similar levels. b Western blot of the indicated Hcm1 proteins expressed from the endogenous HCM1 locus. Hcm1 was detected using an antibody against the C-terminal 3V5 tag, PSTAIRE shown as a loading control. Representative blots from n = 3 replicates are shown. c, d Doubling times of strains from (b) growing at 30 °C in rich medium (c) or in rich medium with 5 μg/mL nocodazole (d). Gray represents WT, color gradient from blue (hcm1-8A) to red (hcm1-8E) corresponds to increasing numbers of phosphomimetic mutations. Error bars represent standard deviation, shown is an average of n = 3 replicates. Significance was tested using repeated measures one-way ANOVA with Dunnett’s multiple comparisons test. Asterisks indicate significantly different from WT, ****P < 0.0001, ***P < 0.0005, ns = non-significant. d AAAAAAAA P < 0.0001; AAAEAAAA P = 0.0002; AAAAEAAA P = 0.0003; AAAEEAAA P = 0.1666; EEEAAEEE P = 0.7932; EEEAEEEE P = 0.9971; EEEEAEEE P = 0.9997; EEEEEEEE P = 0.9886.