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

Significance

DNA–histone complexes are packed into the eukaryotic genome and fit into the nucleus of the cell. One mechanism to access the genetic information is to disrupt the complexes through posttranslational modification of histones. Recently, histone H4K8 2-hydroxyisobutyrylation (H4K8_hib) was identified as an evolutionarily conserved active mark. However, how this modification is regulated and what are the enzymes to modulate this modification within a cell remain a mystery. In this study, we discover that this modification is regulated by the availability of a carbon source in Saccharomyces cerevisiae and identify the enzymes catalyzing the removal of this active mark in vivo. This discovery provides insight into the function and regulation of the histone mark H4K8_hib.

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.

Posttranslational modifications (PTMs) of proteins influence their properties, such as cellular localization, stability, interaction, and enzymatic activity (1–5). Over the past decade, several new types of PTMs have been identified on lysine residues, including propionylation, butyrylation, crotonylation, succinylation, malonylation, glutarylation, and 2-hydroxyisobutyrylation, which, collectively, are termed as lysine acylation (6–11). The possible donors of these acylations are, presumably, their corresponding acyl-CoAs, the intermediates of many cellular metabolic processes (12). Several recent studies indicated that these new acylations occur on thousands of proteins in various cellular metabolic processes and play important roles in metabolic regulation (11, 13–20).

Acylations were also found on histones (6, 8, 10, 21), the major protein components of chromatin, which, together with a fragment of DNA, form the basic building block of a eukaryotic genome (22). Modifications on histones have important functions in transcriptional regulation, DNA repair, replication, and chromatin condensation (23). Two major mechanisms have been proposed. One mechanism is to alter the charge state of the modified residues, which might interfere with the histone–histone and/or histone–DNA electrostatic interactions, leading to a chromatin state transition. The other mechanism is to act as “bait” to recruit effector proteins to chromatin (23). A previous study of histone crotonylation suggested that it is functionally distinct from lysine acetylation (K_ac) and marks active testis-specific genes in postmeiotic cells (8). Furthermore, histone crotonylation was proved to stimulate transcription to a greater extent than histone acetylation in a cell-free transcription system (24).

Although quite a few new modifications have been identified, identifying the enzymes that write or erase the modification has largely lagged behind. Some histone acetyltransferases (HATs) and histone deacetylases (HDACs) have been reported to be able to catalyze additional acylation or deacylation reactions in addition to acetylation or deacetylation (6, 24–26), hinting that the regulation and function of these new histone acylations may also be similar to or perhaps redundant with histone acetylation. However, until now, little about the regulation and function of these new acylations on histones is known.

Lysine 2-hydroxyisobutyrylation (K_hib) is a newly identified histone mark conserved from yeast to humans; specifically, H4K8 2-hydroxyisobutyrylation (H4K8_hib) has been detected in actively transcribed genes in mouse meiotic and postmeiotic cells (10). Here we report the discovery of a carbon stress-related function and regulation of H4K8_hib in Saccharomyces cerevisiae. We found that H4K8_hib was a stress-responsive modification and identified its modifying enzymes. In addition, we showed that the nonmodifiable substrate mutant, H4K8A, leads to a reduced chronological life span (CLS). Finally, we performed K_hib proteomics analysis in Saccharomyces cerevisiae and identified 1,458 modified sites on 369 proteins, revealing an enrichment of this modification in the glycolysis/gluconeogenesis pathway.

Results

H4K8_hib Is Dynamically Regulated by the Availability of a Carbon Source.

Histone K_hib has been reported as a dynamic mark enriched in active chromatin, but its regulatory mechanism remains elusive (10). To identify potential modulators and dissect its functions, we monitored changes of this modification under different stress conditions using the H4K8_hib-specific antibody that was developed, characterized, and reported in a previous study (10). We found that H4K8_hib exhibited no or little response upon most treatments such as DNA damage, temperature, and redox stresses (Fig. 1A). In contrast, we detected significant reduction of H4K8_hib after cells were incubated in water (Fig. 1 A and B). During water treatment, cells were challenged by multiple stresses including osmotic pressure and severe nutrient starvation. Since 1 M sodium chloride (NaCl, osmotic stress) and synthetic defined medium lacking nitrogen (SD-N, nitrogen starvation) treatment had little effect on H4K8_hib (Fig. 1A), we asked whether glucose deprivation might be the cause. As shown in Fig. 1B, treating the cells in synthetic complete medium lacking glucose (SC-D) resulted in a decreased H4K8_hib level comparable to that after water treatment. In addition, supplementing glucose in both water and SC-D could restore the amount of H4K8_hib (Fig. 1B). Together, these results strongly argue that the presence of glucose in the medium is the major factor required to maintain a normal level of H4K8_hib.

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.

To study the dynamics of this modification, we monitored the change of the H4K8_hib level during water treatment. As shown in Fig. 1C, the amount of H4K8_hib decreased gradually and was largely eliminated after 4 h, suggesting that H4K8_hib is a relatively stable mark. On the other hand, the H4K8_hib level recovered quickly in response to glucose supply, and it took only 30 min to restore H4K8_hib completely (Fig. 1C). This observation correlates well with the glucose level in the cell, since it has been shown that the cellular glucose level decreases gradually during glucose starvation, but adding glucose back into the medium could lead to a faster increase of cellular glucose level (27). Therefore, we proposed that H4K8_hib, as a histone mark, orchestrates glucose level within the cells with chromatin epigenetic state regulation.

S. cerevisiae preferentially uses fermentable sugars (such as glucose and fructose), but it can also use nonfermentable substrates (such as glycerol and ethanol) as sole energy and carbon sources. To test whether other carbon sources can also regulate H4K8_hib, we supplied different sugars back into the SC-D medium and monitored the recovery of H4K8_hib, respectively. As shown in Fig. 1D, not only glucose, but also fructose, is able to restore the H4K8_hib level rapidly after glucose starvation. In contrast, glycerol, ethanol, and even galactose that is fermentable but a “secondary” carbon source failed to rescue H4K8_hib, suggesting that a preferred fermentable sugar is required for this modification. Taken together, these data indicate that H4K8_hib is a dynamic modification directly regulated by glucose/fructose availability and establishes a link between histone modification and carbon metabolism.

Glycolysis Is Required to Restore H4K8_hib but Dispensable for Its Establishment and Maintenance.

Given that H4K8_hib is a type of glucose-regulated modification, we next asked how glucose is able to modulate H4K8_hib. Glucose and fructose are preferentially used by many unicellular organisms since they can directly enter the glycolytic pathway. Therefore, we hypothesized that the presence of an intact glycolysis pathway is essential for this modification. Mutations of genes encoding two key enzymes, PFK1 and FBA1, in the pathway were used to test whether the rapid recovery of this modification is impaired. PFK1 encodes a subunit of phosphofructokinase that catalyzes the formation of fructose 1,6-biphosphate from fructose 6-phosphate (28). FBA1 encodes the enzyme converting fructose 1,6-biphosphate into two 3-carbon molecules in glycolysis and is essential for survival of yeast (29). Consistent with our hypothesis, deletion of PFK1 completely blocked regeneration of H4K8_hib after replenishment of glucose for 30 min (Fig. 1E). It should be noted that a similar amount of H4K8_hib could be identified in both pfk1Δ and WT strains when they are cultured in YPD medium (Fig. 1E). This observation is consistent with the previous observation that the pfk1 mutant strain still can grow on the glucose-containing medium, depending on the residual fermentative activity of Pfk2p in yeast (28, 30), indicating that a fully functional glycolysis pathway is required for quick recovery of H4K8_hib after adding glucose. Similarly, using a temperature-sensitive mutant (fba1-ts), glucose failed to restore H4K8_hib modification when cells were shifted to the restrictive temperature (37 °C) simultaneously (Fig. 1F). The loss of the H4K8_hib signal at 37 °C results from the inactivation of Fba1p since under permissive temperature (25 °C) the signal could be restored as that in the WT strain (Fig. S1). Therefore, we concluded that the fast regeneration of H4K8_hib modification after glucose starvation depends on the glycolysis pathway.

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.

Rpd3p and Hos3p Are Required for the Removal of H4K8_hib During Glucose Starvation.

To identify enzyme(s) that could remove the 2-hydroxyisobutyryl group from H4K8, we reasoned that the loss of the de-2–hydroxyisobutyryation enzyme would lead to an increase of H4K8_hib. However, and unfortunately, after screening the 10 HDAC deletion mutants in the Yeast Knockout (YKO) Collection, none of them was able to increase the H4K8_hib signal, suggesting that either they were not able to remove this modification or the increased modification is not detectable (Fig. S2A). As shown above, treating the cells with water led to a fast decrease of the H4K8_hib signal, indicating that the de-2–hydroxyisobutyryation enzyme(s) must be required during this process. Therefore, we treated the HDAC deletion strains with water, looking for failure to decrease H4K8_hib signal. However, none of the HDAC mutants alone could completely block the removal of H4K8_hib (Fig. S2B). However, among these mutants, we found that the signal was reduced slightly but reproducibly in rpd3Δ (Fig. 2A). Therefore, we hypothesized that multiple enzymes might be functioning redundantly to remove this modification, one of which may be Rpd3p. To test this hypothesis, we deleted RPD3 in each HDAC knockout strain, respectively, to generate the double mutants. Among these mutants, we found that most of them—except the rpd3Δ hos3Δ double mutant—still failed to prevent the decrease of the H4K8_hib signal (Fig. 2A and Fig. S2C). In this strain, compared with that of each single mutant, the amount of H4K8_hib remained constant upon water treatment (Fig. 2A). These data strongly suggest that Rpd3 and Hos3p orchestrate de-2–hydroxyisobutyryation in H4K8. In addition, based on the H4K8_hib signal from each single mutant, it is likely that Rpd3 is the major player during this process.

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. 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.

There are three Rpd3p-containing complexes, Rpd3μ, Rpd3L, and Rpd3S, in budding yeast. To further test which Rpd3 complex is specifically responsible for this modification, we deleted several complex-specific genes in the hos3Δ strain, respectively, including Rco1p for the Rpd3S complex, Sds3p for the Rpd3L complex, and Snt2p for the Rpd3μ complex (Fig. S2D) and then monitored changes in H4K8_hib level (31, 32). We found that H4K8_hib decreased in all three strains upon water treatment, similar to that of WT (Fig. S2E), suggesting that deletion of a single Rpd3 complex (in combination with hos3Δ) is not enough to prevent the removal of H4K8_hib modification.

In addition to these complex-specific proteins, Sin3p is a core subunit of both Rpd3L and Rpd3S complexes (31). We deleted SIN3 in the hos3Δ strain and found that the H4K8_hib level was resistant to glucose deprivation in the double mutant (Fig. 2B), similar to what was observed in the rpd3Δ hos3Δ strain. This result indicates that either Rpd3L or Rpd3S are sufficient to remove H4K8_hib and that inactivation of both complexes simultaneously is required to prevent the removal of H4K8_hib. In addition, we found that an Rpd3p mutant (H150A, H151A), which compromises HDAC activity (33), could not remove 2-hydroxyisobutrylation from H4K8 when the cells were under glucose deprivation (Fig. 2C). This result not only indicated that a functional Rpd3 is required to remove 2-hydroxyisobutyrylation but also suggested that the deacetylase and de-2–hydroxyisobutyrylase activities share a common active center. In summary, our data reveal that both Rpd3p and Hos3p are required for the decrease of the H4K8_hib level during glucose starvation and that they orchestrate the catalyzation of histone de-2–hydroxyisobutyrylation reaction in vivo.

H4K8A Alters Transcription of Carbon Transport/Metabolism Genes and Reduces the CLS in S. cerevisiae.

To dissect the function of H4K8_hib, we first mutated the lysine to alanine to eliminate 2-hydroxyisobutyrylation at this site. Phenotypic analysis of H4K8A showed almost no difference between WT and the H4K8A mutant under several conditions (Fig. S3), consistent with a previous report (34). Since glucose deprivation could affect the H4K8_hib level (Fig. 1 A and B), we tested whether this mutant would behave differently on media containing a different amount of glucose. To our surprise, no alteration in growth was observed between the mutant and WT (Fig. 3A). In addition, we found that the rate of glucose consumption during cultivation was also similar between WT and the H4K8A mutant, as shown in Fig. 3B. These data indicate that the H4K8_hib modification does not affect glucose utilization in yeast. On the other hand, we also traced the change in the H4K8_hib level during cultivation and found that the H4K8_hib level decreased gradually during the process and was lost entirely when entering the stationary phase (Fig. 3B). This prompted us to ask whether H4K8hib would affect the physiological state of cells in the stationary phase; the physiology state is known to be closely related to CLS in yeast (35).

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. 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.

Based on the close connection between CLS and oxidative resistance of stationary-phase cells (35), we at first tested the sensitivity of WT and H4K8A mutant cells in the stationary phase to oxidative stress and found that the H4K8A mutant was more sensitive (Fig. 3C). This result suggested that the H4K8A mutant might have a reduced CLS. Using a standard chronological aging assay (36), we confirmed that CLS in the H4K8A mutant was significantly shorter than WT (Fig. 3D), suggesting that the modification of the H4K8 site might have an important function in chronological aging.

To better understand the change in cellular metabolism and function for the H4K8A mutant, we performed a transcriptome analysis using RNA-seq. A total of 236 genes were identified as differentially expressed between H4K8A and WT (P < 0.05, Dataset S1). Of these genes, 26 were up-regulated over twofold in H4K8A versus WT, whereas 23 were down-regulated. Interestingly, we found that genes involved in the carbohydrate metabolic process (P = 0.00019) and carbohydrate transport process (P = 0.00098) were highly enriched. Particularly, genes in glucose transportation, trehalose metabolism, and gluconeogenesis were repressed, whereas genes in fatty acid β-oxidation and amino acid deamination were up-regulated. These data are consistent with cellular responses during glucose starvation, inferring that alternative pathways are used, presumably, to provide more intermediates for glycolysis and the trichloroacetic acid (TCA) cycle to ensure energy supply (Fig. 3E).

Putting all data together, we proposed a regulatory and functional linkage among glucose metabolism, H4K8_hib, and CLS as shown in Fig. 3F. When glucose is present, the 2-hydroxyisobutyryl-CoA is abundant and a high H4K8_hib level is maintained; when glucose is absent, the Rpd3p and Hos3p act together to reduce the H4K8_hib level to orchestrate the glucose level, which signals the cells to change their transcriptome and expedite chronological aging.

The Lysine 2-Hydroxyisobutyrylation Proteome Is Intricately Interlinked to Glucose Metabolism in S. cerevisiae.

The identification of H4K8_hib as a glucose-responsive epigenetic element prompts us to ask whether a relationship between K_hib and glucose metabolism exists at the proteome level. To map the K_hib proteome in S. cerevisiae, an enrichment-based method was applied (Fig. 4A), using K_hib pan-antibody–conjugated beads to collect the modified peptides, which were subsequently analyzed by HPLC-MS/MS. Using this method, we identified 1,458 K_hib sites on 369 proteins (Dataset S2). Further bioinformatics analysis of the modified proteins reveals a strong enrichment in the ribosome and glycolysis/glycogenesis pathways, suggesting a possible function of K_hib in regulating cellular glucose metabolism (Fig. 4 B and C). In addition, the modified proteins were also enriched in the aminoacyl-tRNA biosynthesis pathway and in some amino acid metabolism pathways, which indicates a possible function of this modification in coordinating carbon metabolism with nitrogen metabolism.

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.

Lysine acetylome and succinylome have been studied extensively in S. cerevisiae recently. These two types of acylation and 2-hydroxyisobutyrylation have many features in common. For example, they are evolutionarily conserved and can use their corresponding acyl-CoAs as cofactors for modifying lysine (16, 37). By comparing the published data with ours, we found that 206 proteins were modified by all three acylations, suggesting a comprehensive cross-talk among them (Fig. 4D and Dataset S3). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the 206 proteins reveals a close relationship among the three acylations with ribosome and glycolysis/gluconeogenesis pathways, which further enhances the correlation of these three acylations with glucose metabolism (Fig. 4E) (38, 39). Although many proteins are modified by these three modifications, there are some proteins that are modified by only one or two types of acylations (Fig. 4D). Interestingly, we found that each part of the electron transfer chain complex I–V contains at least one protein that is 2-hydroxyisobutyrylated specifically (Fig. S4A). Site-directed mutagenesis analysis showed that these sites are critical for normal yeast growth (Fig. S4B).

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.

Given that histone H4K8_hib correlates with the availability of glucose, we tested if the entire 2-hydroxyisobutyrylation proteome was affected similarly using Western blot and stable isotope labeling by amino acid in cell culture (SILAC). We found that nearly all enzymes involved in the glycolysis pathway were 2-hydroxyisobutyrylated (Fig. S5A), and the K_hib level was differentially regulated under different glucose concentrations (Fig. S5 B and C and Dataset S4). Therefore, by analyzing the lysine 2-hydroxyisobutyrylation proteome, we revealed an intricate link between K_hib and glucose metabolism.

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.

Discussion

Protein posttranslational modification is a key regulatory mechanism used by the cell to fine-tune protein functions. Recently, many new acylation forms have been identified on histones (6, 8, 10, 21). Compared with the functions of histone N-tail acetylation, very little is known about the function and regulation of these new modifications. Using S. cerevisiae as a model organism, we demonstrated that the newly identified histone H4K8_hib is a histone mark responsive to carbon starvation. We further showed that only the preferred carbon sources (glucose and fructose) could restore the modification rapidly through a pathway depending on glycolysis. And interestingly, the fully active glycolysis pathway is not required for its maintenance in YPD medium. The selectivity of carbon source and glycolysis-dependent rapid restoration of this modification further strengthen the previously proposed link between protein acylations and metabolism (3, 12, 40, 41), suggesting a delicate active regulation of these new acylation forms by cells.

Previous findings have shown that glucose availability affects histone acetylation (42, 43), which very likely affects the production and cellular concentration of acetyl-CoA and, therefore, influences histone acetylation and cell proliferation or differentiation (44–46). Similarly, it is possible that the decreased H4K8_hib level as glucose deprivation may result from the reduced production of 2-hydroxyisobutyl-CoA, the potential donor for K_hib. Given that glucose metabolism is the center of carbon and energy metabolism in a cell and the closed relationship between acy-CoA and energy metabolism (12), other acylation may also be influenced in a similar manner. However, other possibilities, such as regulation of the activities of acyltransferase or deacylase, cannot be ruled out, and more efforts are needed to detail the regulation mechanism. Overall, the stress response of H4K8_hib gives us a good model to explore the regulation and function details of these new histone acylations.

HPLC-MS/MS–based proteome analysis is a widely used method for dissecting the possible functions of protein modification. The proteome study of lysine acetylation and succinylation (K_succ) suggests a broad function in cellular metabolism and signaling (7–9, 11). Since all types of acylation require the corresponding acyl-CoAs as donors, it is not surprising that a strong linkage existed between these new acylations and metabolism (3, 12, 40, 41). However, many of the metabolic enzymes are heavily acylated, making it difficult to study the exact functions of individual modification. Given that the H4K8_hib is also regulated by glucose availability and the glycolysis pathway, we speculate that the link between K_hib and glucose metabolism exists at both epigenetic and proteome levels. A similar link may also exist for other types of lysine acylations, such as succinylation and crotonylation.

Our study shows that the removal of H4K8_hib during glucose starvation requires both Rpd3p (class I HDACs) and Hos3p (class II HDACs) in budding yeast. Consistently, the mammalian HDAC3 (class I HDACs) was capable of carrying out the de-2–hydroxyisobutyrylation reaction in vitro (10). In addition, several studies have demonstrated that Sirt5 (class III) is a de-succinylase, de-malonylase, and de-glutarylase both in vitro and in vivo (7, 11, 26). Furthermore, at least one HAT, p300, in human cells has been reported as a propionylase, butyrylase, and crotonylase (6, 24). Together, these results suggest that many HATs and HDACs might function promiscuously and catalyze different acylations.

How cells decide to add or remove a particular type of acyl group onto or from the same residue and differentiate them will be of great interest. A possible explanation is that this will help a delicate regulation of gene expression in response to the complex metabolic state of the cell and environmental nutrient change. Since the different acyl groups have distinct structures and charge states, a differential transcription response is expected based on the known epigenetic models (23). As reported previously, the H4K8_hib may be a better indicator of high transcriptional activity, and an additive effect is seen between H4K8_hib and H4K8_ac. The cellular crotonyl-CoA regulates histone crotonylation through p300, and the histone crotonylation up-regulates transcription to a greater extent than histone acetylation (10, 24). It will also be important to evaluate whether some specific “readers” exist to distinguish different acylation forms.

Our data clearly show a correlation between H4K8A and reduced CLS. However, we cannot attribute the effect to a particular modification since H4K8 is known to be acetylated as well. Acetylation on H4K8 (H4K8_ac) recruits the SWI/SNF complex, a chromatin-remodeling complex involved in gene transcription, and correlates with opening the chromatin domain (47, 48). Similarly, H4K8_hib is also reported as an active marker (10), suggesting a general role of H4K8 modification in transcription activation. In addition, both H4K8_ac and H4K8_hib decreased after entering the stationary phase (42) and are closely related to glucose metabolism, which implies that the calorie restriction affecting CLS may be partly through modifying the modifications on H4K8. Identifying the “readers” that distinguish these acylation forms will help to dissect the functions of these histone acylations.

Materials and Methods

Strains and Antibodies Used in This Study.

Strains used in this study are listed in Table S1, except those from the yeast YKO library (49). Standard methods for gene disruption and transformation were applied. Both H4K8_hib and pan-K_hib antibodies were purchased from PTM Biolabs (catalog no. PTM-805 for H4K8_hib antibody, catalog no. PTM-804 for the pan-K_hib antibody). The H4K8_Ac antibody was purchased from Abcam (catalog no. ab15823). All antibodies were used according to the product manuals.

Table S1.

Strains used in this study

Strain	Gene Name	Genotype	Ref.
BY4741	BY4741	MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0
JHY131	fba1 ts	MATa ura3Δ0, leu2Δ0, his3Δ1, lys2Δ0(or LYS2), met15Δ0 (or MET15), can1Δ::LEU2-MFA1pr::His3, yfeg-ts::URA3
JHY110	sin3∆ hos3∆	MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, hos3::LEU2, sin3::kanMX
H4WT	H4 WT	his3∆200 leu2∆0 lys2∆0 trp1∆63 ura3∆0 met15∆0 hht1-hhf1::NatMX4 can1::MFA1pr-HIS3 hht2-hhf2::HHT2-HHFS2	34
H4K8A	H4 K8A	his3∆200 leu2∆0 lys2∆0 trp1∆63 ura3∆0 met15∆0 hht1-hhf1::NatMX4 can1::MFA1pr-HIS3 hht2-hhf2::HHT2-HHFS2(K8A)	34
JHY022	rpd3∆ hda1∆	MATα,Δleu2,Δura3,Δhis3,Δlys2, hda1::kanMX, rpd3::URA3
JHY023	rpd3∆ hos1∆	MATα,Δleu2,Δura3,Δhis3,Δlys2, hos1::kanMX, rpd3::URA3
JHY024	rpd3∆ hos2∆	MATα,Δleu2,Δura3,Δhis3,Δlys2, hos2::kanMX, rpd3::URA3
JHY025	rpd3∆ hos3∆	MATα,Δleu2,Δura3,Δhis3,Δlys2, hos3::kanMX, rpd3::URA3
JHY026	rpd3∆ sir2∆	MATα,Δleu2,Δura3,Δhis3,Δlys2, sir2::kanMX, rpd3::URA3
JHY027	rpd3∆ hst1∆	MATα,Δleu2,Δura3,Δhis3,Δlys2, hst1::kanMX, rpd3::URA3
JHY028	rpd3∆ hst2∆	MATα,Δleu2,Δura3,Δhis3,Δlys2, hst2::kanMX, rpd3::URA3
JHY029	rpd3∆ hst3∆	MATα,Δleu2,Δura3,Δhis3,Δlys2, hst3::kanMX, rpd3::URA3
JHY030	rpd3∆ hst4∆	MATα,Δleu2,Δura3,Δhis3,Δlys2, hst4::kanMX, rpd3::URA3
JHY072	hos3∆ dep1∆	MATα,Δleu2,Δura3,Δhis3,Δlys2, dep1::kanMX, hos3::LEU2
JHY073	hos3∆ sds3∆	MATα,Δleu2,Δura3,Δhis3,Δlys2, sds3::kanMX, hos3::LEU2
JHY075	hos3∆ rco1∆	MATα,Δleu2,Δura3,Δhis3,Δlys2, rco1::kanMX, hos3::LEU2
JHY076	hos3∆ snt2∆	MATα,Δleu2,Δura3,Δhis3,Δlys2, snt2::kanMX, hos3::LEU2
JHY077	hos3∆ ecm5∆	MATα,Δleu2,Δura3,Δhis3,Δlys2, ecm5::kanMX, hos3::LEU2
ZLY018	arg4∆	arg4::kanMX4 leu2Δ0 lys2Δ0 ura3Δ0 his3Δ1
JHY113	RPD3 WT	JHYO25+ pHJ032 (LEU2 HIS3 RPD3-3FLAG-10HIS CEN)
JHY132	RPD3(H150,151A)	JHYO25+ pHJ035 [LEU2 HIS3 RPD3(H150A,H151A)-3FLAG-10HIS CEN]
JHY112	Vector	JHYO25+ pRS415 (LEU2 CEN)

Stress Resistance Assay and Chronological Life Span Assay.

Both assays were conducted by following the protocols in previously reports (36). A detailed description of the assays is included in SI Materials and Methods.

Stable Isotope Labeling and Mass Spectrometry Analysis.

The yeast strain ZLY018 (arg4::kanMX4 leu2Δ0 lys2Δ0 ura3Δ0 his3Δ1) was inoculated in synthetic complete medium containing 2% glucose plus arginine (arg) (¹³C₆) and lysine (lys) (¹³C₆) or in synthetic complete medium containing 0.2% glucose plus arg (¹²C₆) and lys (¹²C₆). After 24 h, the cells were reinoculated into the same medium and grown until log phase before harvesting and subsequently disrupted with glass beads. The total proteins were isolated by 20% TCA and digested by trypsin. The 2-Hydroxyisobutyrylated peptides were enriched with pan-anti-K_hib–conjugated resin (PTM BioLabs, catalog no. PTM-804) and subjected to HPLC-MS/MS analysis.

SI Materials and Methods

Strains and Plasmids Used in This Study.

Single-deletion strains except for the ones listed in Table S1 are from the yeast YKO library (49). Other strains used in this study are listed below. Gene disruption and yeast transformation were the standard methods used.

Stress Resistance Assay.

The oxidative stress resistance assay was conducted as reported in a previous study (36). Briefly, cells cultured in SC medium at different growth times were treated with different concentrations of H₂O₂ in 0.1 M K₃PO₄ (pH 6.0) with a final cell concentration of 1 OD at 30 °C. The treated cell was 10-fold serially diluted and spotted onto YPD plates.

Chronological Life Span Assay.

Chronological life span was measured as previous study reported (36). Briefly, a single colony was cultured overnight in aging medium composed of SC medium with a fourfold excess of tryptophan, leucine, lysine, and methionine. The overnight culture was diluted 1:100 into 50 mL fresh aging medium in a 250-mL flask (cultured at 30 °C, 220 × g), and this day was regarded as day 0. At day 3, aliquots from cultures were diluted and then plated onto YPD plates. The YPD plates were culture at 30 °C for 2–3 d, and then the colony-forming units (cfu’s) were counted. The viability at day 3 was considered as 100% survival. Every 2 d, the aliquots were sampled, diluted, and plated onto YPD plates, and cfu’s were counted to calculate the survival rate.

RNA-seq Analysis.

WT and H4K8A mutant cells were grown to log phase and then harvested. Total RNA was extracted using the hot phenol method. In brief, 10 OD₆₀₀ units of yeast were collected and suspended in 400 μL of AE buffer (50 mM NaOAc, 10 mM EDTA, pH 5.0). Forty microliters of 10% SDS were added and vortexed briefly. This was immediately mixed with 500 μL hot phenol (equilibrated with AE buffer and preheated to 65 °C), incubated at 65 °C for 5 min, and cooled to room temperature (RT) by placing the tube on ice for ∼30 s. This was then centrifuged for 10 min at 3,500 × g at RT and the aqueous supernatant was transferred to a fresh 1.5-mL tube. An equal volume of phenol:CHCl₃ (50% phenol + 50% CHCl₃) and, subsequently, CHCl₃:isoamyl alcohol (24:1 vol/vol) was extracted. After centrifugation, the aqueous supernatant was transferred into a fresh 1.5-mL tube. RNA was precipitated and washed with 75% EtOH, and dissolved in 10 μL double-distilled water. All agents used to extract RNA were RNase free. Three independent biological replicates for each strain were subjected to RNA-seq analysis.

Yeast Culture, Protein Extraction, and Digestion for HPLC-MS/MS Analysis.

For detecting the landscape of the lysine 2-hydroxyisobutyrylation proteome, BY4741 was cultured in the YPD medium containing 2% glucose in the log phase, and cells were collected by centrifugation for further experiments. For the SILAC experiment treated with different glucose concentration, three independent colonies from the yeast strain ZLY018 (arg4::kanMX4 leu2Δ0 lys2Δ0 ura3Δ0 his3Δ1) were grown in synthetic complete medium until they reached log phase. An equal amount cells from each colony were added to the synthetic complete medium containing 2% glucose plus arg (¹³C₆) and lys (¹³C₆) or synthetic complete medium containing 0.2% glucose plus arg (¹²C₆) and lys (¹²C₆). After 24 h of culturing, the cells were refreshed with the corresponding medium to log phase and harvested. Before protein isolation, the 0.2% glucose-cultured yeast cells were mixed with the 2% glucose-cultured group with equal ODs, respectively. Yeast cells were centrifuged (1,000 × g for 5 min), and the supernatant was discarded. The acid-washed glass beads (Sigma) were added in an equal volume. Yeast cells and glass beads were suspended in 1 mL precooled lysis buffer (20 mM Tris⋅HCl, pH 8, 0.5% Nonidet P-40, 100 mM NaCl, 1 mM EDTA, 0.5 mM DTT, 1.5 μM Trichostatin A (TSA), 15 mM nicotilamide, and 1% proteinase inhibitor mixture). The sample tubes were placed in a high-throughput tissue extractor with oscillation (70 Hz) for 30 s and intermittently for 1 min on ice. This cycle was repeated five times. After cell breaking, the sample tubes were centrifuged at 14,000 × g for 2 min to remove the foam, and the sample was further broken down with an ultrasonic cell-disruption system. Sonication was carried out in the mode of 3 s on followed with 3 s off, for a total of 60 times. After centrifugation at 14,000 × g under 4 °C for 20 min, the supernatant was collected and added with TCA to a final concentration of 20%. The samples were centrifuged (1,000 × g for 5 min), and the protein pellets were collected and resuspended with 0.1 M NH₄HCO₃. The protein concentration was determined by a BCA Protein Assay Kit (Thermo), and the digestion followed the procedure described in ref. 50. In brief, ∼1 mg of protein was added with trypsin at an enzyme-to-substrate ratio of 1:50 (wt/wt). After incubating at 37 °C for 16 h, the tryptic peptides were denatured with 5 mM DTT under 56 °C for 1 h and alkylated with 15 mM iodoacetamide (IAM) for 45 min. The excessive IAM was quenched with 30 mM cysteine for 30 min. Additional trypsin (1:100 wt/wt) was added, and the mixture was incubated at 37 °C for 3 h to ensure complete digestion. Peptide mixtures were lyophilized in a SpeedVac.C.

2-Hydroxyisobutyrylated Peptide Isolation.

The pan–anti-K_hib conjugated resin (PTM BioLabs) was used to isolate the 2-hydroxyisobutyrylated peptides. Briefly, the ∼1-mg tryptic peptides were resuspended with NETN buffer (50 mM Tris⋅HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) and incubated with 50 μL antibody-immobilized beads at 4 °C overnight with gentle shaking. After incubation, the beads were washed three times with NETN buffer, twice with ETN buffer (50 mM Tris⋅HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA), and once with water. The bound peptides were eluted by washing three times with 100 μL of 0.1 M glycine solution (pH 2.5). The elution was combined and dried in a SpeedVac.

LC-MS Setup.

Yeast 2-hydroxyisobutyrylated peptides were analyzed on a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo) equipped with a nanoelectrospray ionization source and an Easy-nLC 1000 high-performance liquid chromatography system (Thermo). The peptides were resuspended with Mobile phase A [99.9% water/0.1% formic acid (FA)] and separated on a capillary column packed with C18 at a flow rate of 300 nL/min. The elution gradient used was from 8 to 50% mobile phase B (99.9% ACN/0.1% FA) for 78 min. Data acquisition was performed using the data-dependent mode. The m/z range for the full-scan survey was from 300 to 1,400 (automatic gain control target value, 3e6), with a resolution of 70,000 at 400 m/z and a maximum ion injection time of 60 ms. The 20 most abundant ions detected in the full scan underwent MS2 analysis (automatic gain control target value, 5e4) with a resolution of 70,000 under the collision energy of NCE 27.

Protein Sequence Database Searching.

The RAW files generated from Q Exactive MS were searched against the Uniprot yeast protein sequence database (Release_201311, 6652 entries) using Maxquant (ver 1.5) software (51). Multiplicity of 2 was selected with lys6 and arg6 modification. Trypsin was selected with 2 max missed cleavage. Carbamidomethylation was specified as fixed modification, and M_oxidation, K_hib (+86.0368 Da), and K_ac on protein N-terminal were specified as the variable modification. The first search peptide tolerance was set to 20 ppm; the main search peptide tolerance was 4.5 ppm.