2-Hydroxyisobutyrylation on Histone H4K8 Is Regulated by Glucose Homeostasis in Saccharomyces Cerevisiae

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

Jing Huanga,b,1, Zhouqing Luoa,b,1, Wantao Yingc,1, Qichen Caoc, He Huangd, Junkai Donga, Qingyu Wua, Yingming Zhaod, Xiaohong Qianc,2, and Junbiao Daia,b,2

aMinistry of Education Key Laboratory of Bioinformatics, Centre for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China; bCenter for Synthetic Biology Engineering Research, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China; cState Key Laboratory of Proteomics, National Protein Science Center, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China; and dBen May Department of Cancer Research, University of Chicago, Chicago, IL 60637

Edited by Jef D. Boeke, New York University Langone Medical Center, New York, NY, and approved July 10, 2017 (received for review February 3, 2017) New types of modifications of histones keep emerging. Recently, and histone deacetylases (HDACs) have been reported to be histone H4K8 2-hydroxyisobutyrylation (H4K8hib) was identified as able to catalyze additional acylation or deacylation reactions in an evolutionarily conserved modification. However, how this mod- addition to acetylation or deacetylation (6, 24–26), hinting that ification is regulated within a cell is still elusive, and the enzymes the regulation and function of these new histone acylations may adding and removing 2-hydroxyisobutyrylation have not been also be similar to or perhaps redundant with histone acetylation. found. Here, we report that the amount of H4K8hib fluctuates in However, until now, little about the regulation and function of response to the availability of carbon source in Saccharomyces cer- these new acylations on histones is known. evisiae and that low-glucose conditions lead to diminished modifi- Lysine 2-hydroxyisobutyrylation (Khib) is a newly identified cation. The removal of the 2-hydroxyisobutyryl group from H4K8 is histone mark conserved from yeast to humans; specifically, mediated by the histone lysine deacetylase Rpd3p and Hos3p H4K8 2-hydroxyisobutyrylation (H4K8hib) has been detected in in vivo. In addition, eliminating modifications at this site by alanine actively transcribed genes in mouse meiotic and postmeiotic cells substitution alters transcription in carbon transport/metabolism (10). Here we report the discovery of a carbon stress-related genes and results in a reduced chronological life span (CLS). Further- function and regulation of H4K8hib in Saccharomyces cerevisiae.

more, consistent with the glucose-responsive H4K8hib regulation, We found that H4K8hib was a stress-responsive modification and BIOCHEMISTRY proteomic analysis revealed that a large set of proteins involved identified its modifying enzymes. In addition, we showed that the in glycolysis/gluconeogenesis are modified by lysine 2-hydroxyiso- nonmodifiable substrate mutant, H4K8A, leads to a reduced butyrylation. Cumulatively, these results established a functional and chronological life span (CLS). Finally, we performed Khib pro- regulatory network among Khib, glucose metabolism, and CLS. teomics analysis in Saccharomyces cerevisiae and identified 1,458 modified sites on 369 proteins, revealing an enrichment of protein posttranslational modifications | lysine acetylation | lysine 2- this modification in the glycolysis/gluconeogenesis pathway. hydroxyisobutyrylation | chronological life span | histone deacetylase Results

osttranslational modifications (PTMs) of proteins influence H4K8hib Is Dynamically Regulated by the Availability of a Carbon Ptheir properties, such as cellular localization, stability, in- Source. Histone Khib has been reported as a dynamic mark teraction, and enzymatic activity (1–5). Over the past decade, enriched in active chromatin, but its regulatory mechanism re- several new types of PTMs have been identified on lysine residues, mains elusive (10). To identify potential modulators and dissect including propionylation, butyrylation, crotonylation, succinylation, its functions, we monitored changes of this modification under malonylation, glutarylation, and 2-hydroxyisobutyrylation, which, different stress conditions using the H4K8hib-specific antibody collectively, are termed as lysine acylation (6–11). The possible donors of these acylations are, presumably, their corresponding Significance acyl-CoAs, the intermediates of many cellular metabolic processes (12). Several recent studies indicated that these new acylations oc- DNA–histone complexes are packed into the eukaryotic genome cur on thousands of proteins in various cellular metabolic processes and fit into the nucleus of the cell. One mechanism to access the and play important roles in metabolic regulation (11, 13–20). genetic information is to disrupt the complexes through post- Acylations were also found on histones (6, 8, 10, 21), the major translational modification of histones. Recently, histone H4K8 protein components of chromatin, which, together with a fragment 2-hydroxyisobutyrylation (H4K8hib) was identified as an evolution- of DNA, form the basic building block of a eukaryotic genome arily conserved active mark. However, how this modification is (22). Modifications on histones have important functions in regulated and what are the enzymes to modulate this modification transcriptional regulation, DNA repair, replication, and chromatin within a cell remain a mystery. In this study, we discover that this condensation (23). Two major mechanisms have been proposed. modification is regulated by the availability of a carbon source in One mechanism is to alter the charge state of the modified resi- Saccharomyces cerevisiae and identify the enzymes catalyzing the dues, which might interfere with the histone–histone and/or his- removal of this active mark in vivo. This discovery provides insight tone–DNA electrostatic interactions, leading to a chromatin state into the function and regulation of the histone mark H4K8hib. transition. The other mechanism is to act as “bait” to recruit ef- Author contributions: Y.Z., X.Q., and J. Dai designed research; J.H., Z.L., W.Y., Q.C., H.H., fector proteins to chromatin (23). A previous study of histone and J. Dong performed research; J.H., Z.L., W.Y., H.H., Q.W., Y.Z., X.Q., and J. Dai analyzed crotonylation suggested that it is functionally distinct from lysine data; and J.H., Z.L., and J. Dai wrote the paper. acetylation (Kac) and marks active testis-specific genes in post- Conflict of interest statement: Y.Z. is on the science advisory board of PTM Biolabs. meiotic cells (8). Furthermore, histone crotonylation was proved This article is a PNAS Direct Submission. to stimulate transcription to a greater extent than histone acety- 1J.H., Z.L., and W.Y. contributed equally to this work. lation in a cell-free transcription system (24). 2To whom correspondence may be addressed. Email: [email protected] or Although quite a few new modifications have been identified, [email protected]. identifying the enzymes that write or erase the modification has This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. largely lagged behind. Some histone acetyltransferases (HATs) 1073/pnas.1700796114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1700796114 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 that was developed, characterized, and reported in a previous H4K8hib, we supplied different sugars back into the SC-D me- study (10). We found that H4K8hib exhibited no or little response dium and monitored the recovery of H4K8hib, respectively. As upon most treatments such as DNA damage, temperature, and shown in Fig. 1D, not only glucose, but also fructose, is able to redox stresses (Fig. 1A). In contrast, we detected significant re- restore the H4K8hib level rapidly after glucose starvation. In duction of H4K8 after cells were incubated in water (Fig. 1 A contrast, glycerol, ethanol, and even galactose that is ferment- hib “ ” and B). During water treatment, cells were challenged by mul- able but a secondary carbon source failed to rescue H4K8hib, tiple stresses including osmotic pressure and severe nutrient suggesting that a preferred fermentable sugar is required for this starvation. Since 1 M sodium chloride (NaCl, osmotic stress) and modification. Taken together, these data indicate that H4K8hib is synthetic defined medium lacking nitrogen (SD-N, nitrogen a dynamic modification directly regulated by glucose/fructose availability and establishes a link between histone modification starvation) treatment had little effect on H4K8hib (Fig. 1A), we asked whether glucose deprivation might be the cause. As shown and carbon metabolism. in Fig. 1B, treating the cells in synthetic complete medium lacking glucose (SC-D) resulted in a decreased H4K8 level Glycolysis Is Required to Restore H4K8hib but Dispensable for Its hib Establishment and Maintenance. Given that H4K8 is a type of comparable to that after water treatment. In addition, supple- hib glucose-regulated modification, we next asked how glucose is menting glucose in both water and SC-D could restore the able to modulate H4K8 Glucose and fructose are preferen- amount of H4K8 (Fig. 1B). Together, these results strongly hib. hib tially used by many unicellular organisms since they can directly argue that the presence of glucose in the medium is the major enter the glycolytic pathway. Therefore, we hypothesized that the factor required to maintain a normal level of H4K8hib. presence of an intact glycolysis pathway is essential for this To study the dynamics of this modification, we monitored the modification. Mutations of genes encoding two key enzymes, change of the H4K8hib level during water treatment. As shown in PFK1 FBA1 C and , in the pathway were used to test whether the Fig. 1 , the amount of H4K8hib decreased gradually and was rapid recovery of this modification is impaired. PFK1 encodes a largely eliminated after 4 h, suggesting that H4K8hib is a relatively subunit of phosphofructokinase that catalyzes the formation of stable mark. On the other hand, the H4K8hib level recovered fructose 1,6-biphosphate from fructose 6-phosphate (28). FBA1 quickly in response to glucose supply, and it took only 30 min to encodes the enzyme converting fructose 1,6-biphosphate into C restore H4K8hib completely (Fig. 1 ). This observation correlates two 3-carbon molecules in glycolysis and is essential for sur- well with the glucose level in the cell, since it has been shown that vival of yeast (29). Consistent with our hypothesis, deletion of the cellular glucose level decreases gradually during glucose star- PFK1 completely blocked regeneration of H4K8hib after re- vation, but adding glucose back into the medium could lead to a plenishment of glucose for 30 min (Fig. 1E). It should be noted faster increase of cellular glucose level (27). Therefore, we pro- that a similar amount of H4K8hib could be identified in both posed that H4K8hib, as a histone mark, orchestrates glucose level pfk1Δ and WT strains when they are cultured in YPD medium within the cells with chromatin epigenetic state regulation. (Fig. 1E). This observation is consistent with the previous ob- S. cerevisiae preferentially uses fermentable sugars (such as servation that the pfk1 mutant strain still can grow on the glucose and fructose), but it can also use nonfermentable sub- glucose-containing medium, depending on the residual fermen- strates (such as glycerol and ethanol) as sole energy and carbon tative activity of Pfk2p in yeast (28, 30), indicating that a fully sources. To test whether other carbon sources can also regulate functional glycolysis pathway is required for quick recovery of

Fig. 1. H4K8hib is dynamically affected by the glucose and glycolysis pathway. (A) H4K8hib 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 H4K8hib 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 H4K8hib 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 H4K8hib 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 H4K8hib 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.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1700796114 Huang et al. Downloaded by guest on September 26, 2021 H4K8hib after adding glucose. Similarly, using a temperature- A sensitive mutant (fba1-ts), glucose failed to restore H4K8hib modification when cells were shifted to the restrictive temperature (37 °C) simultaneously (Fig. 1F). The loss of the H4K8hib 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 H4K8hib modification after glucose starvation depends on the glycolysis pathway.

Rpd3p and Hos3p Are Required for the Removal of H4K8hib During B 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 H4K8hib. However, and unfortunately, after screening the 10 HDAC deletion mutants in the Yeast Knockout (YKO) Collection, none of them was able to increase the H4K8hib 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 H4K8hib signal, indicating that the de-2– C hydroxyisobutyryation enzyme(s) must be required during this process. Therefore, we treated the HDAC deletion strains with water, looking for failure to decrease H4K8hib signal. However, none of the HDAC mutants alone could completely block the removal of H4K8hib (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 en- BIOCHEMISTRY zymes might be functioning redundantly to remove this modifi- Fig. 2. Rpd3p and Hos3p are required for the decrease of H4K8hib level cation, one of which may be Rpd3p. To test this hypothesis, we during glucose starvation. (A) Water treatment did not decrease H4K8hib in deleted RPD3 in each HDAC knockout strain, respectively, to the rpd3Δhos3Δ strain. The indicated strains were first cultured in YPD me- generate the double mutants. Among these mutants, we found dium 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) that most of them—except the rpd3Δ hos3Δ double mutant—still A was performed. (B) Deletion of HOS3 combined with disrupting the failed to prevent the decrease of the H4K8hib signal (Fig. 2 and Rpd3 complex by deleting SIN3 disabled cells to catalyze H4K8 de-2– Fig. S2C). In this strain, compared with that of each single mu- hydroxyisobutyrylation. The experiment was done similarly to that in A. tant, the amount of H4K8hib remained constant upon water (C) The inactive form of Rpd3p (Rpd3p-H150A H151A) could not mediate treatment (Fig. 2A). These data strongly suggest that Rpd3 and H4K8 de-2–hydroxyisobutyrylation upon water treatment. The rpd3Δ hos3Δ Hos3p orchestrate de-2–hydroxyisobutyryation in H4K8. In ad- strain was transformed with corresponding plasmids containing WT or an inactive form of Rpd3p. The vector was also transformed as a control. dition, based on the H4K8hib signal from each single mutant, it is likely that Rpd3 is the major player during this process. There are three Rpd3p-containing complexes, Rpd3μ, Rpd3L, during glucose starvation and that they orchestrate the catalyzation and Rpd3S, in budding yeast. To further test which Rpd3 complex is of histone de-2–hydroxyisobutyrylation reaction in vivo. specifically responsible for this modification, we deleted several hos3Δ complex-specific genes in the strain, respectively, including H4K8A Alters Transcription of Carbon Transport/Metabolism Genes and Rco1p for the Rpd3S complex, Sds3p for the Rpd3L complex, and Reduces the CLS in S. cerevisiae. To dissect the function of H4K8 , μ D hib Snt2p for the Rpd3 complex (Fig. S2 ) and then monitored we first mutated the lysine to alanine to eliminate 2-hydrox- changes in H4K8hib level (31, 32). We found that H4K8hib de- yisobutyrylation at this site. Phenotypic analysis of H4K8A showed creased in all three strains upon water treatment, similar to that of almost no difference between WT and the H4K8A mutant under E WT (Fig. S2 ), suggesting that deletion of a single Rpd3 complex several conditions (Fig. S3), consistent with a previous report (34). hos3Δ (in combination with ) is not enough to prevent the removal Since glucose deprivation could affect the H4K8hib level (Fig. 1 A of H4K8hib modification. and B), we tested whether this mutant would behave differently on In addition to these complex-specific proteins, Sin3p is a core media containing a different amount of glucose. To our surprise, no subunit of both Rpd3L and Rpd3S complexes (31). We deleted alteration in growth was observed between the mutant and WT (Fig. SIN3 hos3Δ in the strain and found that the H4K8hib level was 3A). In addition, we found that the rate of glucose consumption resistant to glucose deprivation in the double mutant (Fig. 2B), during cultivation was also similar between WT and the H4K8A rpd3Δ hos3Δ similar to what was observed in the strain. This result mutant, as shown in Fig. 3B. These data indicate that the H4K8hib indicates that either Rpd3L or Rpd3S are sufficient to remove modification does not affect glucose utilization in yeast. On the other H4K8hib and that inactivation of both complexes simultaneously is hand,wealsotracedthechangeintheH4K8hib level during culti- required to prevent the removal of H4K8hib. In addition, we found vation and found that the H4K8hib level decreased gradually during that an Rpd3p mutant (H150A, H151A), which compromises the process and was lost entirely when entering the stationary HDAC activity (33), could not remove 2-hydroxyisobutrylation from phase (Fig. 3B). This prompted us to ask whether H4K8hib would H4K8 when the cells were under glucose deprivation (Fig. 2C). This affect the physiological state of cells in the stationary phase; the result not only indicated that a functional Rpd3 is required to physiology state is known to be closely related to CLS in yeast (35). remove 2-hydroxyisobutyrylation but also suggested that the Based on the close connection between CLS and oxidative deacetylase and de-2–hydroxyisobutyrylase activities share a com- resistance of stationary-phase cells (35), we at first tested the mon active center. In summary, our data reveal that both Rpd3p sensitivity of WT and H4K8A mutant cells in the stationary and Hos3p are required for the decrease of the H4K8hib level phase to oxidative stress and found that the H4K8A mutant was

Huang et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 A D

B E

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 H4K8hib 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.

more sensitive (Fig. 3C). This result suggested that the H4K8A whether a relationship between Khib and glucose metabolism exists mutant might have a reduced CLS. Using a standard chrono- at the proteome level. To map the Khib proteome in S. cerevisiae, logical aging assay (36), we confirmed that CLS in the H4K8A an enrichment-based method was applied (Fig. 4A), using Khib mutant was significantly shorter than WT (Fig. 3D), suggesting pan-antibody–conjugated beads to collect the modified peptides, that the modification of the H4K8 site might have an important which were subsequently analyzed by HPLC-MS/MS. Using this function in chronological aging. method, we identified 1,458 Khib sites on 369 proteins (Dataset To better understand the change in cellular metabolism and S2). Further bioinformatics analysis of the modified proteins re- function for the H4K8A mutant, we performed a transcriptome veals a strong enrichment in the ribosome and glycolysis/glyco- analysis using RNA-seq. A total of 236 genes were identified as genesis pathways, suggesting a possible function of Khib in differentially expressed between H4K8A and WT (P < 0.05, Dataset regulating cellular glucose metabolism (Fig. 4 B and C). In addi- S1). Of these genes, 26 were up-regulated over twofold in H4K8A tion, the modified proteins were also enriched in the aminoacyl- versus WT, whereas 23 were down-regulated. Interestingly, we found tRNA biosynthesis pathway and in some amino acid metabolism that genes involved in the carbohydrate metabolic process (P = pathways, which indicates a possible function of this modification 0.00019) and carbohydrate transport process (P = 0.00098) were in coordinating carbon metabolism with nitrogen metabolism. highly enriched. Particularly, genes in glucose transportation, treha- Lysine acetylome and succinylome have been studied exten- lose metabolism, and gluconeogenesis were repressed, whereas sively in S. cerevisiae recently. These two types of acylation and genes in fatty acid β-oxidation and amino acid deamination were up- 2-hydroxyisobutyrylation have many features in common. For regulated. These data are consistent with cellular responses during example, they are evolutionarily conserved and can use their glucose starvation, inferring that alternative pathways are used, corresponding acyl-CoAs as cofactors for modifying lysine presumably, to provide more intermediates for glycolysis and the (16, 37). By comparing the published data with ours, we found trichloroacetic acid (TCA) cycle to ensure energy supply (Fig. 3E). that 206 proteins were modified by all three acylations, sug- Putting all data together, we proposed a regulatory and func- gesting a comprehensive cross-talk among them (Fig. 4D and tional linkage among glucose metabolism, H4K8hib,andCLSas Dataset S3). The Kyoto Encyclopedia of Genes and Genomes shown in Fig. 3F. When glucose is present, the 2-hydroxyisobutyryl- (KEGG) pathway enrichment analysis of the 206 proteins re- CoA is abundant and a high H4K8hib level is maintained; when veals a close relationship among the three acylations with ri- glucose is absent, the Rpd3p and Hos3p act together to reduce the bosome and glycolysis/gluconeogenesis pathways, which further H4K8hib level to orchestrate the glucose level, which signals the cells enhances the correlation of these three acylations with glucose to change their transcriptome and expedite chronological aging. metabolism (Fig. 4E) (38, 39). Although many proteins are modified by these three modifications, there are some proteins The Lysine 2-Hydroxyisobutyrylation Proteome Is Intricately Interlinked that are modified by only one or two types of acylations (Fig. to Glucose Metabolism in S. cerevisiae. The identification of H4K8hib 4D). Interestingly, we found that each part of the electron as a glucose-responsive epigenetic element prompts us to ask transfer chain complex I–V contains at least one protein that is

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1700796114 Huang et al. Downloaded by guest on September 26, 2021 AB D BY4741 cells at log phase in YPD medium

Total protein extracon

C E Trypc digeson

Khib pepde enrichment

HPLC-MS/MS

Idenfying Khib pepdes

Fig. 4. Landscape of the lysine 2-hydroxyisobutyrylation proteome. (A) Schematic representation of the workflow used for HPLC-MS/MS–based Khib 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 Khib pan-antibody–conjugated beads. The purified peptides were analyzed by HPLC-MS/MS. (B) Gene ontology (GO) enrichment analysis of Khib 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 Khib 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 Kac,Ksucci, and Khib proteins. The Kac and Ksucci proteome data are from two previously published papers (19, 37). (E) KEGG pathway enrichment analysis of the common proteins with Kac,Ksucci, and Khib using DAVID software. The top three enriched pathways are shown. Detailed information can be found in Dataset S3.

2-hydroxyisobutyrylated specifically (Fig. S4 ). Site-directed also be influenced in a similar manner. However, other possi- BIOCHEMISTRY mutagenesis analysis showed that these sites are critical for bilities, such as regulation of the activities of acyltransferase or normal yeast growth (Fig. S4B). deacylase, cannot be ruled out, and more efforts are needed to Given that histone H4K8hib correlates with the availability of detail the regulation mechanism. Overall, the stress response glucose, we tested if the entire 2-hydroxyisobutyrylation pro- of H4K8hib gives us a good model to explore the regulation and teome was affected similarly using Western blot and stable iso- function details of these new histone acylations. tope labeling by amino acid in cell culture (SILAC). We found HPLC-MS/MS–based proteome analysis is a widely used that nearly all enzymes involved in the glycolysis pathway were 2- method for dissecting the possible functions of protein modifica- hydroxyisobutyrylated (Fig. S5A), and the Khib level was differ- tion. The proteome study of lysine acetylation and succinylation entially regulated under different glucose concentrations (Fig. S5 (Ksucc) suggests a broad function in cellular metabolism and sig- B and C and Dataset S4). Therefore, by analyzing the lysine 2- naling (7–9, 11). Since all types of acylation require the corre- hydroxyisobutyrylation proteome, we revealed an intricate link sponding acyl-CoAs as donors, it is not surprising that a strong between Khib and glucose metabolism. linkage existed between these new acylations and metabolism (3, 12, 40, 41). However, many of the metabolic enzymes are heavily Discussion acylated, making it difficult to study the exact functions of indi- Protein posttranslational modification is a key regulatory mecha- vidual modification. Given that the H4K8hib is also regulated by nism used by the cell to fine-tune protein functions. Recently, many glucose availability and the glycolysis pathway, we speculate that new acylation forms have been identified on histones (6, 8, 10, 21). the link between Khib and glucose metabolism exists at both epi- Compared with the functions of histone N-tail acetylation, very genetic and proteome levels. A similar link may also exist for other little is known about the function and regulation of these new types of lysine acylations, such as succinylation and crotonylation. S. cerevisiae modifications. Using as a model organism, we dem- Our study shows that the removal of H4K8hib during glucose onstrated that the newly identified histone H4K8hib is a histone starvation requires both Rpd3p (class I HDACs) and Hos3p (class mark responsive to carbon starvation. We further showed that only II HDACs) in budding yeast. Consistently, the mammalian the preferred carbon sources (glucose and fructose) could restore HDAC3 (class I HDACs) was capable of carrying out the de-2– the modification rapidly through a pathway depending on glycol- hydroxyisobutyrylation reaction in vitro (10). In addition, several ysis. And interestingly, the fully active glycolysis pathway is not studies have demonstrated that Sirt5 (class III) is a de-succinylase, required for its maintenance in YPD medium. The selectivity of de-malonylase, and de-glutarylase both in vitro and in vivo (7, 11, carbon source and glycolysis-dependent rapid restoration of this 26). Furthermore, at least one HAT, p300, in human cells has modification further strengthen the previously proposed link be- been reported as a propionylase, butyrylase, and crotonylase (6, tween protein acylations and metabolism (3, 12, 40, 41), suggesting 24). Together, these results suggest that many HATs and HDACs a delicate active regulation of these new acylation forms by cells. might function promiscuously and catalyze different acylations. Previous findings have shown that glucose availability affects How cells decide to add or remove a particular type of acyl histone acetylation (42, 43), which very likely affects the pro- group onto or from the same residue and differentiate them will duction and cellular concentration of acetyl-CoA and, therefore, be of great interest. A possible explanation is that this will help a influences histone acetylation and cell proliferation or differen- delicate regulation of gene expression in response to the complex tiation (44–46). Similarly, it is possible that the decreased metabolic state of the cell and environmental nutrient change. H4K8hib level as glucose deprivation may result from the re- Since the different acyl groups have distinct structures and charge duced production of 2-hydroxyisobutyl-CoA, the potential donor states, a differential transcription response is expected based on for Khib. Given that glucose metabolism is the center of carbon the known epigenetic models (23). As reported previously, the and energy metabolism in a cell and the closed relationship be- H4K8hib may be a better indicator of high transcriptional activity, tween acy-CoA and energy metabolism (12), other acylation may and an additive effect is seen between H4K8hib and H4K8ac.The

Huang et al. PNAS Early Edition | 5of6 Downloaded by guest on September 26, 2021 cellular crotonyl-CoA regulates histone crotonylation through H4K8hib antibody, catalog no. PTM-804 for the pan-Khib antibody). The p300, and the histone crotonylation up-regulates transcription to a H4K8Ac antibody was purchased from Abcam (catalog no. ab15823). All greater extent than histone acetylation (10, 24). It will also be antibodies were used according to the product manuals. important to evaluate whether some specific “readers” exist to distinguish different acylation forms. Stress Resistance Assay and Chronological Life Span Assay. Both assays were Our data clearly show a correlation between H4K8A and re- conducted by following the protocols in previously reports (36). A detailed duced CLS. However, we cannot attribute the effect to a particular description of the assays is included in SI Materials and Methods. modification since H4K8 is known to be acetylated as well. Stable Isotope Labeling and Mass Spectrometry Analysis. The yeast strain Acetylation on H4K8 (H4K8ac) recruits the SWI/SNF complex, a ZLY018 (arg4::kanMX4 leu2Δ0lys2Δ0ura3Δ0his3Δ1) was inoculated in synthetic chromatin-remodeling complex involved in gene transcription, 13 complete medium containing 2% glucose plus arginine (arg) ( C6) and lysine (lys) and correlates with opening the chromatin domain (47, 48). 13 12 ( C6) or in synthetic complete medium containing 0.2% glucose plus arg ( C6) Similarly, H4K8hib is also reported as an active marker (10), 12 suggesting a general role of H4K8 modification in transcription and lys ( C6). After 24 h, the cells were reinoculated into the same medium and activation. In addition, both H4K8 and H4K8 decreased after grown until log phase before harvesting and subsequently disrupted with glass ac hib beads. The total proteins were isolated by 20% TCA and digested by trypsin. The entering the stationary phase (42) and are closely related to glu- – cose metabolism, which implies that the calorie restriction af- 2-Hydroxyisobutyrylated peptides were enriched with pan-anti-Khib conjugated resin (PTM BioLabs, catalog no. PTM-804) and subjected to HPLC-MS/MS analysis. fecting CLS may be partly through modifying the modifications on “ ” H4K8. Identifying the readers that distinguish these acylation ACKNOWLEDGMENTS. We thank the following agencies for financial support: forms will help to dissect the functions of these histone acylations. the National Key Research and Development Program of China (Grant 2017YFA0505103); the Research Fund for the Doctoral Program of Higher Materials and Methods Education of China (Grant 20120002110022); the National Key Program for Strains and Antibodies Used in This Study. Strains used in this study are listed in Basic Research of China (Grants 2012CB910603 and 2014CBA02001); and the Table S1, except those from the yeast YKO library (49). Standard methods for National Natural Science Foundation of China (Grants 31471254, 81530021, gene disruption and transformation were applied. Both H4K8hib and pan- 31100591, and 21235001). Y.Z. was supported by National Institutes of Health Khib antibodies were purchased from PTM Biolabs (catalog no. PTM-805 for Grants GM105933, DK107868, and GM115961.

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