Fig. 2: Decoding Hcm1 phosphoregulation with Phosphosite Scanning.
a Schematic of phosphomutant library design. Each S/T-P site within the TAD was mutated to either unphosphorylatable A-P or phosphomimetic E-E in all 256 possible combinations. b Schematic of the Phosphosite Scanning approach, see text for details. Created with Biorender.com. c Results from the A/E Phosphosite Scanning screen. Each row represents a mutant, shown is the log2 fold change in normalized read counts with respect to time zero for each mutant, all mutants have been normalized to WT. Blue indicates depletion, red indicates enrichment. Shown is an average of n = 3 biological replicates. See Supplementary Figs. 2b–d and 3a for additional analyses and correlation between replicates. d Graph of select mutants from (c). For each mutant, the log2 fraction of normalized reads, normalized to WT, is plotted over time. The slope of a linear regression represents the selection coefficient (SC). Data points are an average of n = 3 biological replicates. e, f Box and whisker plots showing the selection coefficients of groups of mutants with the indicated number of phosphomimetic mutations (e), or with specific substitutions at the indicated positions (f). The black center line indicates the median, boxes indicate the 25th−75th percentiles, black lines represent 1.5 interquartile range (IQR) of the 25th and 75th percentile, black circles represent outliers. Data from n = 3 biological replicates are included. g Mean selection coefficients of indicated mutants. Error bars show standard deviation from the mean in n = 3 biological replicates. Significance was tested using repeated measures one-way ANOVA with Dunnett’s multiple comparisons test. Asterisks indicate significantly different from WT (SC = 0), ****P < 0.0001, ***P < 0.0005, ns non-significant. AAAAAAAA, AAAEAAAA, AAAAEAAA P < 0.0001; AAAEEAAA P = 0.3153; EEEAAEEE P = 0.0010; EEEAEEEE P = 0.9436; EEEEAEEE, EEEEEEEE P < 0.0001. In all panels, the color gradient from blue (hcm1-8A) to red (hcm1-8E) corresponds to increasing numbers of phosphomimetic mutations.
