Fig. 6: Tpk2-catalyzed Jhd2 phosphorylation reduces the interaction between Jhd2 and Rpd3.

a Western blot analysis of histone modifications in WT, ras2∆, and gpa2∆ mutants. b Analysis of histone modifications in WT, Jhd2-S321A, S340A, tpk2∆, and tpk2∆ Jhd2-S321A, S340A mutants. c Analysis of histone modifications in WT, Jhd2-S321A, S340A, tpk2∆, not4Δ, Jhd2-S321A, S340A not4Δ, and tpk2Δ not4Δ mutants. d LC-MS analysis of proteins co-purified with WT Jhd2 (Jhd2-FLAG) and Jhd2-S321A, S340A (Jhd2-S321A, S340A-FLAG). e, f In vivo co-IP analysis of the interaction between Jhd2 and Rpd3 in WT, Jhd2-S321A, S340A and tpk2Δ mutants. g In vitro co-IP showing Tpk2-catalyzed Jhd2 phosphorylation reduced the interaction between Rpd3 and Jhd2. h In vitro histone deacetylase assay showing Jhd2 complex purified from Jhd2-S321A, S340A and tpk2Δ mutants interacted with more Rpd3 and had higher deacetylase activity. i Effects of Jhd2-S321A, S340A on total Rpd3, cytoplasmic Rpd3 and chromatin-bound Rpd3. j Heatmap showing the genome-wide occupancy of Rpd3 in WT Jhd2 and Jhd2-S321A, S340A mutant. k The genome-wide occupancy of Rpd3 was higher in Jhd2-S321A, S340A mutant than WT Jhd2. l Venn diagram showing the overlap binding sites of Jhd2 and Rpd3 in Jhd2-S321A, S340A mutant. m ChIP-seq tracks of Jhd2 and Rpd3 occupancy in WT Jhd2 and Jhd2-S321A, S340A mutant. n Boxplots showing Jhd2-S321A, S340A mutation increased the genome-wide occupancy of Rpd3. o ChIP-qPCR analysis of Rpd3 binding at NCS2 and EGR28 in WT, Jhd2-S321A, S340A and tpk2Δ mutants. p Analysis of histone modifications in WT, Jhd2-S321A, S340A, tpk2∆, rpd3Δ, Jhd2-S321A, S340A rpd3Δ, and tpk2Δ rpd3Δ mutants. q Analysis of histone modifications in WT, Jhd2-S321A, S340A, tpk2∆, rpd3Δ Jhd2-S321A, S340A, and rpd3Δ tpk2Δ mutants when grown in 0.05%, 0.5 and 1% glucose. For o, data represent the mean ± SEM of three biological independent experiments. For n, centre lines denote medians; box limits denote 25th and 75th percentiles; whiskers denote maxima and minima. Two-sided Wilcoxon test in R (package ggpval) was used for statistical analysis. The center in ChIP-seq data of Jhd2 with Rpd3 is coincident with the center of Jhd2-S321A, S340A with Rpd3. Source data are provided as a Source data file.