2-Hydroxyisobutyrylation on histone H4K8 is regulated by glucose homeostasis in Saccharomyces cerevisiae

Abstract

New types of modifications of histones keep emerging. Recently, histone H4K8 2-hydroxyisobutyrylation (H4K8_hib) was identified as an evolutionarily conserved modification. However, how this modification is regulated within a cell is still elusive, and the enzymes adding and removing 2-hydroxyisobutyrylation have not been found. Here, we report that the amount of H4K8_hib fluctuates in response to the availability of carbon source in Saccharomyces cerevisiae and that low-glucose conditions lead to diminished modification. The removal of the 2-hydroxyisobutyryl group from H4K8 is mediated by the histone lysine deacetylase Rpd3p and Hos3p in vivo. In addition, eliminating modifications at this site by alanine substitution alters transcription in carbon transport/metabolism genes and results in a reduced chronological life span (CLS). Furthermore, consistent with the glucose-responsive H4K8_hib regulation, proteomic analysis revealed that a large set of proteins involved in glycolysis/gluconeogenesis are modified by lysine 2-hydroxyisobutyrylation. Cumulatively, these results established a functional and regulatory network among K_hib, glucose metabolism, and CLS.

Keywords: chronological life span; histone deacetylase; lysine 2-hydroxyisobutyrylation; lysine acetylation; protein posttranslational modifications.

Fig. 1.

H4K8_hib is dynamically affected by the glucose and glycolysis pathway. (A) H4K8_hib level under different stress conditions. The WT (BY4741) cells were first cultured in YPD medium to log phase, collected, washed three times with sterile water, and then transferred to each stress condition for 4 h. NaCl, methyl methanesulfonate (MMS), DTT, and Benomyl were added to the YPD medium at the indicated concentration. (B) The restoration of H4K8_hib level by glucose alone after water or SC-D treatment. WT (BY4741) cells at log phase were washed three times with sterile ddH₂O and then suspended in water or SC-D for 4 h, and harvested directly or after adding 2% glucose to the starved cells for 30 min. (C) The H4K8_hib level during water treatment and resuming process. WT (BY4741) cells were treated with water for a different time, and 2% glucose was added after treatment in water for 4 h. (D) Supplying cells with glucose and fructose, but not other carbon sources, can restore H4K8_hib level quickly. WT (BY4741) yeast cells were treated with SC-D for 4 h, and then different carbon sources were added to a final concentration of 2% to treat the cell for 30 min. Eth, ethanol; Fru, fructose; Gal, galactose; Glu, glucose; Gly, glycerol. (E) Deletion of PFK1 blocks restoration of the H4K8_hib level upon glucose replenishment. The WT (BY4741) and pfk1Δ cells were first cultured in YPD medium to log phase and then transferred into SC-D medium for 4 h; the glucose was added last and treated for 30 min. (F) The fba1-ts mutant failed to restore the H4K8hib level at restrictive temperature. The WT (BY4741) and fba1-ts mutant was grown at 25 °C in YPD medium and then shifted to 37 °C or washed with sterile ddH2O three times and suspended in water at 37 °C for 4 h. Glucose was added to starved cells, and cells were harvested after 30 min.

Fig. S1.

The fba1 ts mutant performed similarly to WT at permissive conditions. The fba1 ts mutant was grown at 25 °C and then washed with sterile dH₂O twice and finally resuspended in SC-D medium at 25 °C for 4 h. Glucose was added to the starved cell, and cells were harvested after 30 min.

Fig. 2.

Rpd3p and Hos3p are required for the decrease of H4K8_hib level during glucose starvation. (A) Water treatment did not decrease H4K8_hib in the rpd3∆hos3∆ strain. The indicated strains were first cultured in YPD medium to log phase and then treated with sterile ddH2O for 4 h. The yeast cells with or without water treatment were collected, and Western blot (WB) was performed. (B) Deletion of HOS3 combined with disrupting the Rpd3 complex by deleting SIN3 disabled cells to catalyze H4K8 de-2–hydroxyisobutyrylation. The experiment was done similarly to that in A. (C) The inactive form of Rpd3p (Rpd3p-H150A H151A) could not mediate H4K8 de-2–hydroxyisobutyrylation upon water treatment. The rpd3Δ hos3Δ strain was transformed with corresponding plasmids containing WT or an inactive form of Rpd3p. The vector was also transformed as a control.

Fig. S2.

Screening the de-2–hydroxyisobutyrylation enzyme for H4K8_hib. (A) Deletion of individual HDAC has no obvious effect on H4K8_hib in YPD medium. (B) Single HDAC deletion strains act similarly to WT after water treatment for 4 h. (C) Deleting other deacetylatases, except for HOS3, in rpd3Δ strain could not block the decrease of H4K8_hib level after carbon source starvation. rpd3Δ cells with other single HDAC deletions were grown to log phase in YPD, washed twice with sterile ddH₂O, and resuspended in sterile ddH₂O for 4 h. The cells before or after water treatment were harvested for WB. (D) The components in each Rpd3p-containing complex. The complex specific component used was labeled by red. (E) Different Rpd3 complexes act together to remove H4K8_hib during water treatment. Different subunits were deleted in the hos3Δ strain, and the double-mutant cells were harvested as described in B.

Fig. 3.

Modification of H4K8 is required for CLS. (A) The H4K8 mutation does not affect growth in different glucose concentrations. The log-phase cells were spotted onto different plates in a 10-fold series dilution. (B) The dynamics of H4K8_hib and glucose level in medium during normal culture condition. The overnight-cultured BY4741 cells were diluted to OD₆₀₀ = 0.1 using YPD medium, and cells were collected at each time point. Growth curve and glucose concentration data are represented as mean ± SEM. (C) Stationary-phase H4K8A mutant is more sensitive to H₂O₂ stress. Cells cultured in Sc medium for chronological aging assay at day 3 were washed with sterile water twice and then suspended in a 0.1-M K₃PO₄ (pH 6.0) buffer containing the indicated concentration of H₂O₂ for 1 h. Finally, the treated cells were spotted onto YPD plate in a 10-fold series dilution. (D) The H4K8A mutation leads to a shortened CLS. The survival rate is represented as mean ± SEM. (E) Transcriptome changes in the carbon transport and metabolism process in the H4K8A mutant. The down-regulated genes are in purple; up-regulated genes are in red. (F) A model for the actions of H4K8 site modifications.

Fig. S3.

The H4K8A mutation showed no difference in growth under several stress conditions. WT and H4K8A mutant were grown in YPD medium to log phase and then washed three times with sterile ddH2O and plated onto different plates. A 10-fold series dilution with a start concentration of 0.2 OD was conducted.

Fig. 4.

Landscape of the lysine 2-hydroxyisobutyrylation proteome. (A) Schematic representation of the workflow used for HPLC-MS/MS–based K_hib site identification in S. cerevisiae. The log-phase WT (BY4741) yeast cells cultured in the YPD medium were harvested and lysed mechanically. The total proteins extracted were then trypsin-digested, and the 2-hydroxyisobutyrylated peptides were enriched using K_hib pan-antibody–conjugated beads. The purified peptides were analyzed by HPLC-MS/MS. (B) Gene ontology (GO) enrichment analysis of K_hib proteins and all yeast proteins using Saccaromyces Genome Database (SGD) GO Term Finder. The top five enriched GO terms are shown with their corresponding P value. All of the enriched GO terms can be found in Dataset S2. (C) KEGG pathway enrichment analysis of the K_hib proteins using DAVID software. All pathways with a P value < 0.05 are shown. Detailed information can be found in Dataset S2. (D) Venn diagram showing the overlaps among K_ac, K_succi, and K_hib proteins. The K_ac and K_succi proteome data are from two previously published papers (19, 37). (E) KEGG pathway enrichment analysis of the common proteins with K_ac, K_succi, and K_hib using DAVID software. The top three enriched pathways are shown. Detailed information can be found in Dataset S3.

Fig. S4.

Lysine 2-hydroxyisobutyrylation at specific sites of the electron transfer chain proteins is important for normal growth. (A) Schematic presentation of the electron transfer chain proteins found with only 2-hydroxyisobutyrylation modification but not with acetylation or succinylation (generated by KEGG). (B) Growth of the strains overexpressing these six proteins with or without mutations. The 2-hydroxyisobutyrylated lysine identified was mutated to alanine to remove the possible modification. All of the ORFs here were driven by a GAL-S promoter and carried by a pRS416 plasmid. A 10-fold series dilution was conducted at 30 °C.

Fig. S5.

The lysine 2-hydroxyisobutyrylation proteome is closely related to glucose metabolism. (A) 2-Hydroxyisobutyrylation of glycolysis enzymes in S. cerevisiae. Nearly all of the glycolysis enzymes were identified to be 2-hydroxyisobutyrylated (labeled by dashed red box). (B) WB result of total K_hib proteins under normal (2%) and low (0.2%) glucose concentration conditions using the K_hib pan-antibody. The H3 WB and Coomassie Brilliant Blue staining are shown as loading control. (C) Distribution of the SILAC ratio of lysine 2-hydroxyisobutyrylated peptides. Scatter plot was used to show the peptide intensities of the quantifiable lysine 2-hydroxyisobutyrylated peptides in relation to their dynamic changes in response to glucose concentration. The proteins of 2% glucose cultured cells were labeled by ¹³C, and the proteins of 0.2% glucose cultured cells were labeled by ¹²C.