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Characterization of histone acylations links chromatin modifications with metabolism

Characterization of histone acylations links chromatin modifications with metabolism ARTICLE DOI: 10.1038/s41467-017-01384-9 OPEN Characterization of histone acylations links chromatin modifications with metabolism 1 1 1 1 1 2 Johayra Simithy , Simone Sidoli , Zuo-Fei Yuan , Mariel Coradin , Natarajan V. Bhanu , Dylan M. Marchione , 3 4 5 6 3 Brianna J. Klein , Gleb A. Bazilevsky , Cheryl E. McCullough , Robert S. Magin , Tatiana G. Kutateladze , 7 8 1 Nathaniel W. Snyder , Ronen Marmorstein & Benjamin A. Garcia Over the last decade, numerous histone acyl post-translational modifications (acyl-PTMs) have been discovered, of which the functional significance is still under intense study. Here, we use high-resolution mass spectrometry to accurately quantify eight acyl-PTMs in vivo and after in vitro enzymatic assays. We assess the ability of seven histone acetyltransferases (HATs) to catalyze acylations on histones in vitro using short-chain acyl-CoA donors, proving that they are less efficient towards larger acyl-CoAs. We also observe that acyl-CoAs can acylate histones through non-enzymatic mechanisms. Using integrated metabolomic and proteomic approaches, we achieve high correlation (R > 0.99) between the abundance of acyl-CoAs and their corresponding acyl-PTMs. Moreover, we observe a dose-dependent increase in histone acyl-PTM abundances in response to acyl-CoA supplementation in in nucleo reactions. This study represents a comprehensive profiling of scarcely investigated low-abundance histone marks, revealing that concentrations of acyl-CoAs affect histone acyl-PTM abundances by both enzymatic and non-enzymatic mechanisms. 1 2 Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA. Graduate Group in Cell and Molecular Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA. Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. 7 8 AJ Drexel Autism Institute, Drexel University, 3020 Market Street Suite 560, Philadelphia, PA 19104, USA. Department of Biochemistry and Biophysics, Abramson Family Cancer Research Institute, and the Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA. Correspondence and requests for materials should be addressed to B.A.G. (email: [email protected]) NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications 1 | | | 1234567890 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 ysine acetylation is the most extensively studied histone addition of acetyl groups using acetyl-CoA as a cofactor, and post-translational modification (PTM). Discovered more histone deacetylases (HDACs), which remove these groups . Lthan 50 years ago, it has been recognized to play a funda- The activities of both HATs and HDACs are regulated by mental role in transcriptional activation, metabolic regulation and the metabolic state of the cell . Thus, endogenous metabolite other central cellular processes . Mechanistically, lysine acetyla- concentrations are proposed to provide signaling that can directly tion neutralizes the positive charge of histone tails, reducing the influence acetylation dynamics in chromatin . physical interaction between histones and DNA, thereby allowing Over the last decade, a growing number of lysine modifications 2,3 the access of gene-activating transcription factors . Acetylation chemically related to acetylation (propionylation, malonylation, can also influence chromatin function by serving as a binding site crotonylation, butyrylation, succinylation, glutarylation, for bromodomain-containing remodeling complexes that can 2-hydroxyisobutyrylation and β-hydroxybutyrylation) have been directly stimulate trasnscription by recruiting transciptional identified on histones using mass spectrometry (MS)-based 4 8–14 co-activators . Acetylation dynamics in the nucleosome are the proteomic approaches . These findings have raised numerous result of the net activities between two different families of questions regarding their functional significance, possible enzymes: histone acetyltransferases (HATs), which catalyze the implications in metabolic pathways and the existence of a b O O O O Recombinant H3 or H4 HN HN HN HN 10 μg Incubation 1h at 30°C HN R Short-chain acyl-CoA COOH COOH COOH 0.5mM H N COOH H N 2 H N H N 2 2 Kbu Kcr Kac Kpr O O O OH O OH HAT COOH H N 2 HN HN HN HN OH 0.5 μg 2× propionylation Kacyl O Trypsin digestion 2× propionylation Stage-tip COOH COOH COOH H N COOH 2 H N H N H N 2 2 2 Kglu Kbhb Kmal Ksuc Time LC-ESI-MS Automated data analysis analysis EpiProfile software z-score Histone H3 Kglu, 2% Kbhb, 2% –1 0 2 Ksuc, 4% Mass shift (Da) Kmal, 4% 42.0105 56.0262 68.0262 70.0418 86.0003 86.0368 100.0160 114.0281 Kcr, 1% Kac Kpr Kcr Kbu Kmal Kbhb Ksuc Kglu HAT CBP (H3) GCN5 (H3) p300 (H3) Kac PCAF (H3) 43% Kpr 29% NatA (H4) Tip60 (H4) Kbu MOF (H4) 15% Histone H3 Histone H4 Histone H4 60% 30% Kbhb 50% 25% 5% Kglu Kac 8% Kbu 40% 20% Kpr Kac Ksuc 32% 11% Kcr 30% 15% Kmal 20% 10% Kmal Ksuc 12% Kglu 10% 5% Kbu Kpr Kbhb 12% 0% 0% 20% Kcr 0% Modification site Modification site Fig. 1 Overview of histone acetyltransferases (HATs) in vitro acylation activity and specificity. a Schematic representation of in vitro acylation assay. b Chemical structures of histone acyl modifications evaluated in this study. Lysine modifications and abbreviations are: acetyl (Kac), propionyl (Kpr), butyryl (Kbu), crotonyl (Kcr), malonyl (Kmal), succinyl (Ksuc), β-hydroxybutyryl (Kbhb), and glutaryl (Kglu). c Heat map displaying the in vitro acylation activity profiles of different HATs in the presence of acetyl-, propionyl-, crotonyl-, butyryl-, malonyl-, β-hydroxybutyryl-, succinyl- and glutaryl-CoA. Molecular mass shift of the various acylated lysines residues are shown in the table headers. Different HATs were assayed against histones H3 or H4 as specified in the first column. To generate the heat map, we averaged the relative abundance of acyl-PTMs on the quantified peptides and then normalized (z-scores) those values across the different HATs, i.e. row normalization. d Pie chart showing the average relative frequency of in vitro acylated peptides, divided in results for histone H3 (top) and histone H4 (bottom). e Bar plots depict the specificity for all HATs on the histone sequence. The x axis represents the modification site at histones H3 (left) and histone H4 (right), and the y axis represents the relative abundance shown as the average contribution of HATs to all acylated peptides. For histone H4 N-terminal peptide (G4-R17), the number of acylations on the sequence are displayed using the code 2, 3 or 4 mods. This is because it was not always possible to discriminate modification sites on the multiply modified H4 peptide. All values shown were corrected by the contribution of non-enzymatic acylation. All results are shown as the average of 3 independent experiments 2 NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications | | | K4 K9 K14 K9K14 K18 K23 K18K23 K27 K36 K27K36 K56 K64 K79 K122 K5 K8 K12 K16 2MODS 3MODS 4MODS K20 K31 K44 K59 K77 K79 K91 Relative abundance Relative abundance Relative abundance NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 ARTICLE regulatory enzymes beyond the well-established acetylation subset of acylations on histones. Because these marks are mechanisms that could govern these marks. While most of these dependent on their corresponding short-chain acyl-CoA questions remain to be answered, many studies have provided metabolic intermediates, we employ a targeted metabolomics new insights into the roles that acyl marks can play in genome approach to measure the concentrations of acyl-CoA metabolites function. For example, it has been reported that lysine crotony- in HeLa cells and in proliferative and differentiated human lation mediated by the HAT p300 can stimulate gene transcrip- myogenic cells. We find that the cellular concentrations of tion in vitro and in vivo seemingly to a greater degree than lysine different acyl-CoA metabolites span orders of magnitude and are acetylation, and that this mechanism is highly regulated by tightly correlated with the relative abundances of the acyl marks the metabolic concentrations of the crotonyl-CoA co-factor . identified in vivo. These findings support the notion of a direct Histone crotonylation has also been found to be enriched at link between cellular metabolism and epigenetic regulation, where transcriptionally active X/Y sex-linked genes during post-meiotic the relative abundances of different acyl marks in histones are 8,16 sex inactivation in mouse , and the YEATS domains Taf14, driven by the cellular concentrations of their respective metabolic 29,30 AF9 and YEATS2 have been reported to preferentially bind intermediates . 17–20 crotonylated over acetylated lysines residues in vitro . More recently, lysine butyrylation has been reported to directly sti- mulate gene transcription and compete with acetylation for the Results binding of the testis specific gene expression-driver Brdt in In vitro acylation of histones H3 and H4. Previous studies have spermatogenic cells . In addition, β-hydroxybutyrylation, was reported that the HATs CBP, p300 and PCAF can mediate 12 12,31 15 found to be induced during starvation or streptozotocin-induced propionylation , butyrylation , and crotonylation of lysine diabetic ketoacidosis, and to activate transcription of specific residues in vitro. These observations prompted us to investigate genes associated with starvation-responsive metabolic path- whether these and other known HATs can catalyze the acylation ways . These studies suggest that newly identified histone acyl- of human recombinant histones H3 and H4 using a broader PTMs may have unique or similar roles to acetylation in tran- range of acyl-CoA donors. We performed in vitro HAT activity scriptional activation. Such observations are supported by several assays with the HAT domains of PCAF, Gcn5, and the full-length reports showing that SIRT5, a member of the class III HDACs CBP and p300 enzymes against histone H3, and with the HAT can preferentially remove acidic acyl modifications, including domains of MOF, Tip60 and NatA against histone H4. Each 22 22,23 13 malonyl , succinyl and glutaryl , whereas propionyl, reaction was carried out individually in the presence of eight crotonyl and butyryl marks can be removed by various other different short-chain acyl-CoA donors, followed by bottom-up sirtuins . However, it remains unclear whether the same group nano-LC-MS/MS analysis (Fig. 1a, b). Figure 1c summarizes the of enzymes involved in the establishment of acetylation could also in vitro activity profiles of all HATs evaluated in this study. The mediate the establishment of these histone modifications in vivo. heat map shows that most HATs could utilize acetyl- Another aspect that has been underexplored is the relative propionyl- and butyryl-CoA with relatively high efficiency, 32,33 abundance of acyl marks, which is an important step towards supporting recent findings . However, acidic acyl-CoA donors understanding their biological relevance. This gap in knowledge is including malonyl-, succinyl- and glutaryl-CoA, and branched- mainly due to the biases inherent in the use of antibody-based chain acyl donors like β-hydroxybutyryl CoA are utilized by enrichment methods commonly employed prior to MS detec- HATs less efficiently. Interestingly, enzymes did not seem to tion . Although stoichiometry at individual sites has been utilize crotonyl-CoA for the catalysis of acyl marks as effectively reported for propionylation (7%) at H3K23 in a leukemia cell as propionyl- and butyryl-CoA despite the structural similarity 26 27 line , butyrylation (31%) at H3K115 in mouse brain , and within these cofactors. These data are in agreement with previous crotonylation (1–3%) at H2AK36, H2BK5, H3K23 and H4K12 in observations suggesting that HATs activity is weaker with brain histones , a global overview of the abundances relative to crotonyl-CoA due to the planarity and rigidity imparted by the 8,20,32 histone acetylation is lacking. The dearth of quantitative data for C-C double bond in the crotonyl moeity . these non-canonical acyl-PTMs has led to the hypothesis that When taking a closer look at the individual acylation activities they might arise due to the chemical reactivity of acyl-CoAs. of all HATs (Supplementary Table 1), we observed that enzymes Indeed, this has been observed in the context of acetylation and have different trends in their substrate preference. For instance, succinylation in mitochondrial proteins . Thus, it is still open to histone H3 is known to be selectively acylated at the lysine residue 33 34 debate whether these modifications are strategically positioned on 14 (H3K14) by Gcn5 and PCAF . Therefore, if we look at the the chromatin by enzymes or whether their presence is instead relative abundances for all acylations at H3K14, under our assay the result of non-enzymatic chemical reactivity. conditions, Gcn5 was able to butyrylate ~ 78% of the H3 peptide In this study, we sought to characterize the acylation of at position 14, whereas acetylation and propionylation were histones, including their overall abundance and their likelihood to found at ~ 32% and ~ 11%, respectively (Supplementary Table 1). be products of HAT catalysis rather than chemical reactivity of Likewise, PCAF displayed ~ 88% butyrylation, followed by ~ 5% acyl-CoAs. First, we investigate the ability of several recombinant crotonylation and ~ 2% acetylation at position H3K14. However, HATs to catalyze the acylation of histones H3 and H4 using when looking at the average sum of the relative abundances of all different acyl-CoA donors employing an MS-based in vitro acylated peptides by HATs on H3, there seems to be a trend bioassay. Our data show that most HATs can catalyze histone in the order of substrate preference: acetyl > propionyl > butyryl acylation using different acyl-CoA substrates to variable extents > malonyl > succinyl > β-hydroxybutyryl > glutaryl > crotonyl when tested individually. However, in competition assays (Fig. 1d; Supplementary Table 1). This trend inversely correlates performed in the presence of equimolar concentrations of acyl- with the increasing size of the side chain of the acyl donor (except CoA and acetyl-CoA, almost all HATs strongly prefer to utilize for crotonyl-CoA and β-hydroxybutyryl), supporting the notion acetyl-CoA to modify histones. Our data also confirm that that the activity of HATs gets weaker with increasing acyl-chain histones can be modified non-enzymatically through the chemical length . Interestingly, p300 and PCAF were the enzymes with reactivity of the different acyl-CoA donors alone. We also employ the highest crotonylation activities on H3 (Supplementary a proteomics approach to characterize several acyl-PTMs in Table 1). nucleo and in vivo without the use of enrichment strategies, Moreover, the average activities of HATs on histone H4 which allow us to determine the relative abundance of a diverse showed patterns that were consistent with the trend mentioned NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications 3 | | | ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 a c GCN5 vs. non-enzymatic acylations Non-enzymatic acylations H3 3.0 50% 2.5 40% Kbhb 2.0 Kglu 30% GCN5 1.5 Ksuc Non-enzymatic Kmal 20% 1.0 Kcr Kpr 10% 0.5 Kbu 0.0 0% Kac Acyl-PTM Modification site Histone H3 Histone H4 Non-enzymatic acylations H4 40% 1 1 0.9 0.9 0.8 0.8 30% Kbhb 0.7 0.7 Kglu 0.6 0.6 Ksuc 0.5 0.5 20% Kmal 0.4 0.4 Kcr 0.3 0.3 10% 0.2 0.2 Kpr 0.1 0.1 Kbu 0 0 0% Kac Acyl-PTM Acyl-PTM Modification site Non-enzymatic Enzymatic Enzymatic Non-enzymatic Fig. 2 Non-enzymatic versus enzymatic acylation of histones in vitro. a Comparison of non-enzymatic versus GCN5-catalyzed acylations on histone H3. The y axis (arbitrary units) represents the sum of the relative abundances of all enzymatically and non-enzymatically acylated peptides from histones H3. We can observe that the contributions for Ksuc, Kmal, Kbhb, Kglu and Kcr in the experiments with GCN5 were mostly the result of non-enzymatic acylations. b Stacked column representation of non-enzymatic reactivity divided by enzymatic reactivity of the eight acyl-CoA donors on histone H3 (left) and histone H4 (right). The fractional reactivity represents the ratio of PTM intensity in presence of all seven enzymes tested versus PTM intensity in absence of enzymes, i.e., 0.5 corresponds to identical intensities with and without enzyme. For instance, crotonylation is an overall low abundance PTM, although the majority detected on histone peptides is the result of an enzymatic catalysis. c, d Bar plot showing the relative quantitation of non- enzymatically acylated sites on c histones H3 and d histone H4. All results are shown as the average of three independent experiments and error bars represent the S.D. before in terms of substrate preference (Fig. 1d). However, non-enzymatically modified in vitro, we incubated histones H3 individual acylation activities suggest that, while MOF seems to and H4 with 0.5 mM acyl-CoAs in the absence of acetyl- follow the same trend when looking at the sum of all acylated transferases. We found that all acyl-CoAs can chemically acylate peptides, Tip60 prefers to utilize butyryl-CoA as a cofactor, histones, as seen in Fig. 2a, showing a comparison between the followed by succinyl-CoA and acetyl-CoA (Supplementary non-enzymatic acylation profiles of various acyl-CoA donors on Table 1). Nonetheless, all results reported in Fig. 1 are based on histone H3 with acylations mediated by GCN5. We showed that the average contribution of both groups of HATs rather than most non-enzymatically catalyzed acylations have site specificities individual acylation activities. Detailed acylation site specificities that were different from those enzymatically modified sites for all HATs can be found in Supplementary Table 1. Our data (Figs. 1e, 2c, d). The most prevalent sites observed for also showed that the N-terminal acetyltransferase NatA can non-enzymatic acylations on histone H3 were K36, K56, K64, catalyze N-terminal propionylation and butyrylation of histone K79 and K122 (Supplementary Fig. 1; Fig. 2c). On H4, the most H4 in vitro (Supplementary Fig. 2). prevalent sites were K31, K59, K79 and K91 (Supplementary Fig. 1; Fig. 2d). Interestingly, these sites are closer to the C terminus of histones, whereas enzymes showed higher specificity Histones are non-enzymatically acylated in vitro. for sites at the N-terminal tails (Fig. 1e). These observations suggest that non-enzymatic acylations may be enhanced by some Non-enzymatic acylation of proteins has been reported to occur level of structural and conformational dynamics on histones. In through the nucleophilic attack of the unprotonated ɛ-amino 28,35 agreement, most succinylated and malonylated sites identified by group of lysine residues to the acyl group of acyl-CoAs . Xie et al. in vivo have been reported to occur also at the globular This mechanism is facilitated by an alkaline pH and high levels domain and C terminus of histones H3 and H4 rather than at the of acyl-CoA. In mitochondria, where both conditions are met N-terminal tails. and evidence of the existence of acetyltransferases is lacking, a large body of evidence suggests that high levels of protein We then estimated which acylations are more likely to occur through enzymatic or non-enzymatic reactivity in vitro. As acylation observed in this organelle are the result of 36,37 non-enzymatic mechanisms . To test whether histones can be seen in Fig. 2b, the average contribution for acetylation, 4 NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications | | | Kac Kpr Kbu Ksuc Kmal Kbhb Kglu Kcr Kac Kpr Kbu Kcr Kbhb Kmal K4 Ksuc K9 Kglu K14 K9K14 K18 Kac Kbu K23 Kpr K18K23 Kcr Kmal K27 Ksuc K36 Kglu K27K36 Kbhb K56 K64 K79 K122 K5 K8 K12 K16 2mods 3mods 4mods K20 K31 K44 K59 K77 K79 K91 Fractional reactivity Relative abundance (AU) Fractional reactivity Relative abundance Relative abundance NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 ARTICLE a b HAT CBP (H3) GCN5 (H3) p300 (H3) Pcaf (H3) NatA (H4) Tip60 (H4) MOF (H4) Neg (H3) Neg (H4) CoAs Ace/Pro Recombinant H3 or H4 Ace/Cro 10 μg Ace/But Ace/Mal Ace/Bhb Acetyl-CoA10 μM Ace/Suc Acyl-CoA 10 μM Ace/Glu Log 2 HAT –1 0 11 0.5 μg c d 6 12 GCN5 p300 Glutaryl β-hydroxybutyryl Glutaryl 114.02 86.04 Da R = 0.94 (without crotonyl) 114.02 β-hydroxybutyryl 4 8 R = 0.94 (without crotonyl) Succinyl 86.04 Da Succinyl 100.02 Da 100.02 Da 3 6 Butyryl Malonyl 70.04 Da 2 Propionyl 86.00 Da Propionyl Malonyl 56.03 Da Butyryl 56.03 Da 86.00 Da 70.04 Da 50 60 70 80 90 100 110 120 50 60 70 80 90 100 110 120 Modification M.W (Da) Modification M.W (Da) MOF β-hydroxybutyryl Glutaryl 86.04 Da 114.02 Da 5 R = 0.91 (without crotonyl) Succinyl 100.02 Da Butyryl 70.04 Da Malonyl 86.00 Da Propionyl 56.03 Da 50 60 70 80 90 100 110 120 Modification M.W (Da) Fig. 3 In vitro acetylation competition assay. a Schematic representation of the in vitro competition acetylation assay. b Heat map displaying in vitro acylation specificities of HATs during acyl-CoA competition assays. Different HATs were assayed against histones H3 or H4 as specified in the table headers in the presence of equimolar concentrations of acetyl-CoA and a competing acyl-CoA donor. Negative controls with no enzyme are also shown. c Correlation between the molecular weight and the acylation preference for different acyl donors displayed for the HATs GCN5 on histone H3, d p300 on histone H3 and e MOF on histone H4. For each modification, molecular weight is indicated. Results show that the preference for acetyl-CoA over the other acyl donor tightly correlates with the molecular weight of the acyl donor. Crotonylation was not included in the correlations, as its molecular weight did not correlate well with the preference of the enzymes over acetyl-CoA. All graphs are shown as the average of log2 ratios between the relative abundances of all acetylated peptides and the relative abundances of the corresponding competing acylated peptide propionylation and butyrylation marks in the presence of all it is important to mention that concentrations of acyl-CoAs used HATs was more abundant in in vitro experiments, whereas acidic in this experiment were far above the known physiological acyl modifications (malonylation, succinylation and glutaryla- concentrations of CoA derivatives in whole cells , so the extent tion) and β-hydroxybutyrylation occurred to a greater extent of chemical acylation observed in this study is likely an through non-enzymatic mechanisms in both histones H3 and H4. overestimation. This higher concentration was required to ensure Again, we observed a trend in which the ratio of enzymatic/ proper sensitivity to the in vitro assay. Even though it has been chemical reactivity of acyl groups is inversely correlated with the previously demostrated that protein lysine acylation can occur at size of the side chain, supporting the idea that most known HATs physiological acyl-CoA concentrations in vitro , our study does 32,33 catalyze larger acylations less efficiently . When looking not represent a suitable extrapolation for the reactivity of these individually at non-enzymatic acylations in histones, crotonyl- intermediates in cells. As such, our data cannot rule out the CoA showed the lowest acylation levels through chemical possibility of the existence of enzymes that play a major role in catalysis (Fig. 2c, d). As expected, crotonyl-CoA presents a lower catalyzing these marks in vivo as compared to non-enzymatic chemical reactivity towards lysine nucleophiles due to the reactions. resonance properties of the beta unsaturated C-C bond . The correlation between sites prone to chemical acylation (this study) and sites identified in other in vivo studies suggests that HATs prefer acetyl-CoA for acylation of histones. Previously, it histone acyl modifications in cells could be the result of both has been demonstrated that some HATs are able to catalyze the enzymatic and non-enzymatic mechanisms after direct exposure transfer of propionyl and butyryl groups in vitro with similar 12,31 to intrinsically reactive acyl-CoA metabolites. This is supported specificites but different efficiencies than acetyl groups . These by the fact that enzymes with distinctive acyltransferase activities observations lead to the following question: what determines the have not been identified in any cellular compartment . However, acyl group to be transferered if HATs are indeed mediating NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications 5 | | | Log 2 Kac/Kacyl Log 2 Kac/Kacyl Log 2 Kac/Kacyl ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 various acylations in vivo? It has been shown that fibroblasts of decarboxylase (MCD) and short-chain acyl-CoA dehydrogenase patients with inherited metabolic disorders have high levels of (SCAD), respectively. However, the mechanism by which this propionyl-, malonyl- and butyryl-CoA and high levels of the increase in protein acylation is mediated has not been explored. 43,44 corresponding lysine acylations . These disorders include We hypothesized that under such conditions, other acyl-CoAs deficiencies in propionyl-CoA carboxylase (PCC), malonyl-CoA could rival the levels of acetyl-CoA and induce HATs to use non- ab HeLa cells Myogenic cells 50 40 *** Myoblasts *** Myotubes 25 20 10 ** ** 0 0 PTMs PTMs c d HeLa cells Myogenic cells –5 4×10 –5 3×10 –7 –7 6×10 2.5×10 –7 Myoblasts –5 2×10 –5 *** 3×10 Myotubes 3×10 –7 –7 4×10 1.5×10 –7 1×10 –5 –5 3×10 –7 2×10 –8 2×10 5×10 –5 –5 2×10 2×10 –6 –5 3×10 1×10 ** –6 2×10 –6 –6 5×10 1×10 CoA metabolites CoA metabolites 2.0 HeLa cells 1.5 1.0 0.5 R = 0.99 0.0 –7 –6 –6 –6 –6 0 5×10 1×10 1.5×10 2×10 2.5×10 R = 0.99 –5 –5 –5 0 1×10 2×10 3×10 CoA metabolites pmol/cell Myoblasts Myotubes 30 15 1.5 1.4 1.0 1.2 0.5 R = 0.99 1.0 R = 0.95 0.0 –7 –6 –6 –6 0.8 0 5×10 1×10 1.5×10 2×10 –8 –7 –7 –7 5×10 1×10 1.5×10 2×10 R = 0.99 R = 0.92 R = 0.99 (without Kcr,crotonyl-CoA) –5 –5 –5 –6 –5 –5 –5 0 2×10 4×10 6×10 0 5×10 1×10 1.5×10 2×10 CoA metabolites pmol/cell CoA metabolites pmol/cell 6 NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications | | | Acetyl-CoA Acetyl-CoA Succinyl-CoA Succinyl-CoA Propionyl-CoA Propionyl-CoA Kme1 Kme2 Kac Kme3 Kpr Ksu Kbhb Kbu Kmal Kcr Kglu Kme1 Butyryl-CoA Malonyl-CoA Kac Crotonyl-CoA Glutaryl-CoA Kme2 Kme3 Ksu Kpr Kbu Kcr Kbhb Kmal Kglu Butyryl-CoA Malonyl-CoA Glutaryl-CoA Crotonyl-CoA Acyl-PTMs normalized abundance % Normalized relative abundance % pmol/cell Acyl-PTMs normalized abundance % pmol/cell Acyl-PTMs normalized abundance % Normalized relative abundance % NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 ARTICLE native cofactors. To test this, we performed in vitro HAT com- between 15 and –30% (Fig. 4a,b; Supplementary Fig. 5). Using our petition assays in the presence of equimolar concentrations of workflow, we measured the relative abundances at individual acetyl-CoA and other acyl-CoAs (Fig. 3a). As shown in Fig. 3b, sites. Acetylation was found at high relative abundances at most HATs preferred to utilize acetyl-CoA than any other acyl- positions H3K18 and H3K23 ranging from 17 to –35%, and CoA donor. It is important to mention that at 10 µM of acyl- between 15–30% at H4K12 and H4K16 (Supplementary Table 2), CoAs, we observed non-enzymatic acylation of histones H3 and which is in accordance with previous findings . Interestingly, H4, as shown in Fig. 3b. Consistent with previous in vitro similar relative abundances were observed at H3K14 (3–8%) for experiments, for most HATs, the preference for the competing acetylation and propionylation marks (Supplementary Table 2). cofactor, if any, largely depended on the size of the acyl donor Overall, most non-acetyl acyl marks were found at levels below side chain. As shown in Fig. 3c–e, for the enzymes GCN5, p300 2%, mainly at the N-terminal domains of histones H3 and H4. and MOF we observed an inverse correlation between the HAT Surprisingly, some acidic acyl marks were found at the globular preference for the competing cofactor and the increasing mole- domains and C terminus, showing abundances as high as 10% cular weight of the acyl donor side chain, with the exception of (Supplementary Table 2), which coincide with the sites that were crotonyl-CoA. Relative abundances for all peptides in competi- more susceptible to non-enzymatic acylation in our in vitro tion assays are shown in Supplementary Table 3. Altogether, our experiments (Fig. 2c, d). Detailed site specificity for all acyl marks data suggest that even in the highly unlikely chance that any other is shown in Supplementary Table 2. Importantly, we did detect acyl-CoA accumulated to the extent that its concentration rivaled low levels of hydroxybutyrylation; however, our MS acquisition that of acetyl-CoA, HATs would still mostly utilize acetyl-CoA. method cannot discriminate between possible isoforms of this We thus shifted our focus to investigate how abundant these acyl mark, including bhb (β-hydroxybutyryl or 3hb), 2- marks are in vivo, and whether their abundance can be justified hydroxybutyryl (2hb), 3-hydroxyisobutyryl (bhib), 2 hydroxyiso- 10,14 by the abundance of acyl-CoA intermediates. butyryl (2hib) or 4-hydroxybutyryl (4hb) . Likewise, peptides bearing Kbu (butyryl) marks may also represent isobutyryl marks. Our analysis of myoblasts showed dynamic changes in global Relative abundances of histone acyl-PTMs in mammalian cells. histone acylations upon their fusion to form multinucleated myotube cells. We observed that the global levels of lysine Owing to the low abundance of lysine acyl marks, current MS- based approaches involve the use of antibody-based enrichments acetylation, propionylation, butyrylation, malonylation, succiny- lation and glutarylation were significantly decreased upon to increase the sensitivity of the MS analysis for identification and quantification. Although these approaches are helpful for esti- myogenic differentiation, whereas the levels of lysine crotonyla- tion were increased (Fig. 4b). The role of differential histone mating the relative changes of modifications across multiple conditions, they cannot provide direct information on relative acylation in cellular differentiation is poorly understood; how- abundances, as the peptide with the modification and the ever, various lines of evidence suggest that nutrition and 47,48 unmodified peptide end up in different sample pools. In addition, metabolism play a key role in the differentiation of cells . evaluation of the specificity of several commercially available pan For example, a previous study investigating the role of carbon anti-acyl-PTM antibodies by dot blot analysis revealed significant metabolism in the differentiation of myogenic cells demonstrated cross-reactivity among differentially acylated peptides, compli- that siRNA knock down of ATP citrate lyase (ACLY) induces cating their further application for immunoenrichment of histone differentiation of mouse myoblasts . The same study also acyl-PTMs (Supplementary Fig. 3). showed that the levels of histone acetylation in shACLY-treated cells were reduced, hypothesizing that the deposition of acetyl- Thus, to accurately detect and quantify histone acyl-PTMs in vivo using label-free approaches, we used the retention time CoA, and in turn histone acetylation levels, play an important role in the differentiation of myoblasts. Because there is little and mass shift information from the in vitro experiments to optimize the MS acquisition and in-house quantification soft- evidence of how newly identified histones acylations may be implicated in the differentiation of cells and/or epigenetic ware . Here, we refer to our quantitative values as “relative abundances”, as we are aware that differences in the ionization regulations, we next sought to investigate whether the cellular efficiencies of modified peptides or biases in trypsin digestion in concentrations of other acyl-CoA metabolites have a direct the presence of certain modifications can affect the assessment of relationship with histone acylation levels. accurate PTM stoichiometry, which was observed for the differentially acylated peptide H3 aa 18–26 (KQLATKAAR) (Supplementary Fig. 4). Acyl-CoA donors dictate the levels of histone acylation. Various Analysis of acid-extracted histones from wild-type HeLa and studies in mammalian cells have shown that chromatin mod- myogenic cells showed that all acyl-PTMs combined, excluding ifications are sensitive to changes in intracellular concentrations acetylation, were found at relative abundances between 6 and of metabolic intermediates, linking cell metabolism to epigenetic 6,50 –15% of all detectable modified peptides of canonical histones H3 changes . However, the mechanisms and enzymes mediating and H4 (Supplementary Fig. 5). Individually, most acyl-PTMs these processes have not been fully explored. So far, it has been showed low relative abundances ranging from 1 to –5% (Figs. 4a, demonstrated that changes in the levels of acetyl-CoA can b), as opposed to acetylation with global relative abundances influence global histone acetylation levels . These findings led us Fig. 4 Overview of the relative abundances of endogenous acyl-PTMs and intracellular metabolite concentrations. Bar plots showing the relative abundances of several lysine PTMs in a HeLa cells, b proliferative myogenic cells (myoblasts) and differentiated myogenic cells (myotubes). PTMs are shown as percentages representing the normalized relative abundances of all detectable peptides of canonical histones H3 and H4. c, d Bar plots showing the concentrations of acyl-CoA metabolites in c HeLa cells and d myogenic cells normalized to cell number. Metabolic concentrations of butyryl-CoA may also represent concentrations for isobutyryl-CoA. e–g Global histone acylation levels correlation with intracellular concentrations of acyl-CoA metabolic intermediates in e HeLa cells, f myoblasts and g myotubes. Correlations are calculated between the normalized net abundances of all detectable acylated peptides in H3 and H4 and the global concentrations CoA metabolites in pmol per cell. Insets show zoom in graphs with the linear correlation of the data without the values for acetyl-CoA and Kac. All results are shown as the average of three biological replicates and error bars represent the S.D. Summary of p-values is as follows; *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. p-values were generated by unpaired Student’s t-test NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications 7 | | | ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 a b In nucleo acylation 100% Histone extraction Propionylation Trypsin digestion 80% Propionylation Cell 0 μM Acyl-CoA LC/MS Nucleus 1 μM Acyl-CoA 60% 5 μM Acyl-CoA Incubation at 30 °C for 40% 2-4 h 20% Nucleus 0% Nucleus Acyl CoAs (0 –5 μM) 45% H3K56 PTMs H3K9-K14 PTMs 40% In nucleo propionylation In nucleo malonylation 35% 80% 30% 70% 25% 60% 20% 50% 15% 40% 10% 30% 5% 0% 20% 0 μM_Mal-CoA 1 μM_Mal-CoA 5 μM_Mal-CoA H3_54_63 K56pr 0.03% 0.03% 0.00% 10% H3_54_63 K56bu 0.01% 0.02% 0.05% 0% H3_54_63 K56cr 0.00% 0.03% 0.00% 0 μM_Pro-CoA 1 μM_Pro-CoA 5 μM_Pro-CoA Others 0.07% 0.50% 0.50% H3_54_63 K56su 0.02% 0.08% 0.01% H3_54_63 K56ma 1.11% 14.66% 26.65% H3_9_17 K14pr 3.45% 7.94% 10.21% H3_54_63 K56hi 0.12% 0.11% 0.08% H3_9_17 K9pr 1.34% 1.68% 1.72% H3_9_17 K14ac 3.94% 2.80% 1.69% H3_54_63 K56gl 0.36% 0.15% 0.33% H3_54_63 K56ac 0.33% 0.67% 0.52% H3_9_17 K9ac 0.24% 0.37% 0.66% H3_54_63 K56me3 0.08% 0.19% 0.07% H3_9_17 K9me3 10.04% 11.42% 12.91% H3_9_17 K9me2 16.48% 19.00% 10.72% H3_54_63 K56me2 12.23% 8.20% 10.30% H3_54_63 K56me1 1.92% 1.00% 0.72% H3_9_17 K9me1 30.51% 29.15% 28.72% Fig. 5 Analysis of in nucleo acylation. a Schematic representation of in nucleo acylation assay. b Bar plots showing the dose-dependent acylation of histones upon treatment with 0, 1 and 5 μM of acetyl-, butyryl-, malonyl-, glutaryl-, propionyl-, succinyl-, β-hydroxybutyryl and crotonyl-CoA, respectively. Values represent the sum of the relative abundances of all acylated peptides from histones H3 and H4. c Example of in nucleo acylation resulting in an increase in malonylation of H3K56 after treatment with increasing concentrations of malonyl-CoA. d Example of in nucleo acylation showing that induced propionylation by increasing the concentration of propionyl-CoA resulted into a reduced relative abundance of acetylation on the site H3K14 to consider whether the levels of other histone acylations may also measurable increase in the levels of crotonyl-CoA, this did not be influenced by the intracellular concentrations of their respec- correlate well with the increase in lysine crotonylation observed in tive acyl-CoA donors. Using a stable isotope dilution MS myotubes (Fig. 4g). Acyl-CoA intermediates are derived from approach , we accurately measured the concentrations of seven various metabolic pathways including the TCA cycle, fatty acid acyl-CoA metabolic intermediates in HeLa (Fig. 4c) and myo- synthesis, β-oxidation and amino acid metabolism. Although it genic cells (Fig. 4d). Our metabolomics analysis revealed that remains poorly understood which metabolic pathway leading to acetyl-, propionyl- and succinyl-CoA were the most abundant the production of different acyl-CoAs might serve as a substrate CoA thioesters in our cell models, with concentrations around 12, for the acylation of nuclear histones, the global concentrations of 1 and 0.5 μM, respectively, when normalized to cell volume of metabolites determined in this study are consistent with the HeLa cells (Supplementary Fig. 6). In myogenic cells, when abundance of lysine acylations. Collectively, our data demonstrate normalizing the data to cell number, we observed that the a clear quantitative link between metabolism and differential intracellular concentrations of metabolites also appear to undergo histone acylations. regulation from myoblast to myotube differentiation. Our data showed that the levels of acetyl-, succinyl-, propionyl-, butyryl and malonyl-CoA were significantly decreased upon differentia- Acylation of histones in nucleo. Our in vivo and metabolomics tion (Fig. 4d), whereas the levels of glutaryl- and crotonyl-CoA studies strongly implicated metabolism in histone acylations. As did not show significant changes. the concentrations of intracellular metabolites are known to When looking at the levels of acyl lysine PTMs in HeLa cells, change in response to diet or physiological conditions, we next our data showed a strong positive correlation with the metabolic turned to the question of whether alterations in the levels levels of acyl donors (Fig. 4e). A similar observation could be of metabolites could affect the corresponding levels of histone made for myoblasts (Fig. 4f). Even though we observed a acylations. To further explore this idea, we turned to an in nucleo 8 NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications | | | Kac Kbu Kmal Kglu Kpr Ksu Kbhb Kcr Relative abundance (sum) Relative abundance (sum) Relative abundance (sum) NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 ARTICLE system, where purified nuclei can be treated with artificial levels To further explore the possible mechanisms underlying the of metabolites. We isolated nuclei from HeLa cells under hypo- establishment of acyl marks, we performed in vitro acylation tonic conditions, treated with varying concentrations of eight assays in the absence of HATs (Fig. 2). We found that histones different acyl-CoA donors and performed histone extraction, can be acylated by the chemical reactivity of all acyl-CoA meta- digestion and derivatization following standard procedures bolites evaluated in this study. While most HATs showed strong (Fig. 5a). preference for the acylation of residues at the N-terminal MS analysis revealed that histone acylations can be induced in a domains, i.e., residues K9–K36 in H3 and K5–K16 in H4, concentration-dependent manner. Specifically, by adding 1 or 5 µM non-enzymatic acylation sites were more prevalent closer to the C of acyl-CoAs, we induced an increase of the respective acylation on terminus of histones, i.e., residues K56–K122 in histone H3 and histone peptides (Fig. 5b). Since the in nucleo experiment preserves K59–K91 in histone H4. Acidic acyl-PTMs including malonyla- the natural state of nuclear processes, it can be used to observe tion, succinylation and glutarylation were among those most histone acylations in native chromatin. Our in vitro experiment easily catalyzed in the absence of enzymes (Fig. 2b). These marks demonstrated that acylations can occur by both enzymatic and are different from acetyl marks as they add bulkier groups to non-enzymatic mechanisms, but such simplified assay cannot lysine residues and they carry a negative charge under physio- accurately represent the balances of a nuclear environment. We logical conditions. As such, it has been suggested that these acidic compared the two assays by performing an in nucleo–in vitro acyl marks could disrupt the interactions between histones and Spearman’s rank-order correlation analysis by using corrected DNA, resulting in a more profound effect in chromatin unfolding in vitro enzymatic data (subtracting the non-enzymatic contribu- than lysine acetylation . Emerging hypotheses have suggested a tion). We observed a good correlation for some residues, including model where non-enzymatic chemical reactions are a significant H3K9acyl and H3K18acyl sites (Supplementary Fig. 7) that were contributor to the landscape of lysine acylations in nuclear highly acylated only in presence of enzymes in vitro. This suggests histones . They also suggest that sirtuin enzymes showing that specific sites are likely more accessible to enzymatic activity specificity for the removal of acyl marks may represent a than others, and that this reactivity is also a function of the acyl- constitutive programming to suppress potential damaging effects 56,57 CoA utilized. However, a generalized conclusion cannot be drawn, caused by the presence of these PTMs . Interestingly, our as the in nucleo assay cannot discriminate enzymatic catalysis from study showed that those succinylated and malonylated sites chemical reactions, and physiological acylation turnover (equili- highly susceptible to non-enzymatic acylation in vitro were brium deposition/removal). among the sites reported previously in in vivo studies . While Additionally, our in nucleo results showed that histones more studies are required, our data suggest that histone lysine accommodate acylation in two ways; by simply increasing residues are prone to be modified by several free acyl-CoAs with the modified state or by removing pre-existing modifications to and without enzymatic assistance. maintain the same level of total modified form. For example, The accurate quantification and elucidation of potential func- upon treatment with malonyl-CoA, levels of H3K56mal increased tional roles of acyl marks have been hampered by their low with almost no changes in the other modifications on that peptide abundance. To provide an accurate estimate of the levels of acyl (Fig. 5c). On the other hand, after treatment with increasing marks, we employed a label-free approach using DIA-MS. By concentrations of propionyl-CoA, the levels of H3K14pr analyzing in the same mixture modified and unmodified forms of increased, whereas the levels of H3K14ac showed a measurable histone peptides, we could report the relative levels of acyl-PTMs decrease (Fig. 5d). Detailed in nucleo acylation relative abun- expressed as a percentage of the total histone. Analysis of human dances can be found in Supplementary Table 4. Taken together, cervical cancer cells (HeLa) and human myogenic cells revealed our in nucleo studies demonstrated that modifications in that acyl marks together represent around 6–15% of all detected chromatin are sensitive to changes in the concentrations of modifications on histones H3 and H4 (Fig. 4a,b; Supplementary cellular metabolites, consistent with previous observations con- Fig. 5). Our myoblast/myotube comparison strongly indicates necting the metabolic state of the cell with chromatin that the differentiation of pluripotent cells is marked by a 7,53 regulation . decrease in global levels of histone acetylation. This was not surprising, as this mechanism has been shown to be driven by a decrease in acetyl-CoA production mediated through the inhi- 51,58 Discussion bition of glycolysis . In agreenment with these findings, our A comprehensive screen of the major families of histone acetyl- data showed a significant decrease of the bulk levels of histone transferases (HATs) confirmed that most enzymes can catalyze acetylation once myoblasts fused to form multinucleated myo- the acylation of histones utilizing acetyl, propionyl and butyryl- tubes. Intriguingly, while the levels of most histone acylations CoA cofactors with similar efficiencies (Fig. 1), as previously decreased upon differentiation, myotube cells showed an increase reported . However, they were less efficient catalyzing the acy- in the global levels of lysine crotonylation (Fig. 4b). The under- lation of histones with charged, branched or planar acyl-CoA standing of how these chromatin modifications could be involved cofactors. Even though these acyl donors are structurally similar in driving myoblast differentiation is beyond the scope of this to acetyl-CoA, the universal cofactor of HATs, our data showed study and the subject of studies to come. This is a comprehensive that the ability of enzymes to utilize other cofactors largely report providing quantitative information on the levels of a broad depended on the size of the acyl group, which is in close agree- number of histone acyl marks in HeLa and human myogenic ment with recent data demonstrating the structural incompat- cells, thus representing an important resource for future work ibility of the active sites of p300 and GCN5 with long-chain acyl aiming to understand cellular function and the dynamics of acyl- 32,33 donors . This observation was further confirmed by in vitro PTMs in the complex mammalian epigenetic mechanisms. HAT assays performed at equal concentrations of acetyl-CoA and In general, we osberved a strong correlation between a competing cofactor (Fig. 3). The data showed a similar trend in histone acylations and their corresponding metabolic substrates. which the preference for a cofactor different from acetyl-CoA was One exception was crotonylation, as the observed increase in inversely proportional to the molecular weight of the competing lysine crotonylation in myotubes was not accompanied by a donor, except for crotonyl-CoA, which, unlike the other acyl statistically significant increase in the levels of crotonyl-CoA groups, possesses an unsaturated moiety that seems to render its (Fig. 4b,d,g). This specific experiment cannot prove whether use by most HATs unfavorable. crotonylation in myotubes is regulated by enzymatic mechanisms NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications 9 | | | ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 induction with 0.5 mM IPTG and grown at 17 °C overnight. The cells were that are not strictly regulated by crotonyl-CoA levels. However, collected by centrifugation at 4000 r.p.m. at 4 °C and lysed in 25 mM HEPES pH we performed an intermediate experiment between in vivo and 7.5, 0.150 M NaCl, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 100 ug per in vitro, namely in nucleo, to test whether there are corre- mL DNase I, and 100 ug per mL lysozyme. PCAF was purified from the lysate sponding changes in the levels of histone acylations upon through Ni-NTA affinity as described above with 5 mM imidazole in 25 mM HEPES pH 7.5, 0.150 M NaCl, 1 mM DTT and eluted with 200 mM imidazole in manipulation of the concentrations of metabolites. This experi- the same. The protein was then transferred to 6–8 kDa MWCO dialysis tubing ment showed that the bulk levels of lysine acylations can be (Spectrum Labs) and dialyzed overnight into 20 mM sodium citrate pH 6.0, induced in a dose-dependent manner, resulting in either a net 0.150 M NaCl, and 1 mM DTT buffer. This was then followed by additional increase or a dynamic exchange of modifications in response to purification through SP Sepharose ion exchange and Superdex 75 (GE) gel filtration. The protein was concentrated to ~ 30 mg per mL, flash frozen, and stored increasing concentrations of acyl-CoA metabolites (Fig. 5). at −70 °C in a buffer containing 20 mM Na-citrate pH 6.0, 150 mM NaCl, 1 mM Despite the evidence that the concentrations of metabolites can DTT. regulate the global levels of lysine acylations, it is premature to Recombinant histones H3 and H4 were expressed in Rosetta BL21 [DE3] pLysS 64,65 pinpoint how this metabolic regulation is involved in complex cells and purified as monomers using standard procedures . processes including gene expression, cell differentiation and apoptosis, or in diseases such as cancer that are characterized by altered metabolic states. Such a scenario is further complicated In vitro histone acylation assay. Based on substrate specificity, histone H3 was considering that metabolite precursors of histone modifications assayed with HATs p300, CBP, PCAF and Gcn5, and histone H4 with HATs MOF, NatA and Tip60. In vitro enzymatic assays were carried by incubating 0.5 µgof exist in different pools derived from various biological pathways each HAT with 10 µg of recombinant histones H3 or H4 in the presence of 0.5 mM that are regulated in response to cellular physiological conditions. of short-chain acyl-CoAs (acetyl-, crotonyl-, malonyl-, succinyl-, propionyl-, Continued work in this area will help elucidate remaining ques- butyryl-, glutaryl- and β-hydroxybutyryl CoA;—Sigma-Aldrich) in 1X HAT buffer tions surrounding the role of acyl-PTMs, such as the following: (i) (25 mM Tris-HCl pH = 8, 25 mM KCl, 1 mM DTT, 0.1 mM AEBSF and 5 mM sodium butyrate) for 60 min at 30 °C; the final volume was 50 µL. For competition do these PTMs compete with lysine acetylation, or (ii) do they assays, reactions were carried out in the presence of 10 µM of acetyl-CoA and 10 work in concert to regulate epigenetic mechanisms? Various µM of other acyl-CoAs. Background control reactions were performed in the studies have shown that the availability of acetyl-CoA can reg- absence of HATs. Reactions were stopped by freezing and samples were dried in a ulate the abundance of lysine acetylation. Our conclusion is that SpeedVac and resuspended in 20 µL of 100 mM ammonium bicarbonate (pH 8.0). Histones were derivatized with propionic anhydride (or d -propionic anhydride this study provides compelling evidence that this metabolic reg- 10 when analyzing lysine propionylation). For this procedure, fresh propionylation ulation is not restricted to acetylation, but also extends to the reagent was prepared by mixing propionic anhydride with acetonitrile in the ratio regulation of newly identified acyl marks in chromatin. 1:3 (v/v). Propionylation reagent was added to each sample in 1:4 (v/v). Ammo- nium bicarbonate was quickly added to the solution to re-establish pH 8.0. Samples were dried down to 10–20 µL in a SpeedVac, reconstituted with 20 µL of ammo- Methods nium bicarbonate and the propionylation procedure was repeated one more time. Protein expression and purification. Human p300 and human CBP sequences Samples were then digested with trypsin at a 1:10 ratio (wt/wt) for 6 h at 37 °C. containing an N-terminal His tag, and C-terminal Strep2 and FLAG tags, were Samples were desalted by C18 stage-tip. For this procedure, a small piece of a synthesized and cloned by Genewiz (Cambridge, MA) into the pVL1393 vector for C18 solid phase extraction disk was deposited into a pipette tip to create a stage-tip. baculovirus expression. The plasmid was transfected into Sf9 cells utilizing the BD The C18 resin was flushed with 100% acetonitrile by slow centrifugation using a (Franklin Lake, NJ) BaculoGold transfection system. Then, p300 was expressed in centrifuge adaptor to hold the stage-tips in place in a 1.5 ml Eppendorf tube. Sf9 cells and purified using a GE Healthcare (Piscataway, NJ) HiTrap column. The resin was then equilibrated by flushing 80 µl of 0.1% TFA. Samples were The protein identity and purity were confirmed through protein staining with acidified to pH 4.0 or lower with acetic acid and loaded onto the disk by slow Coomassie dye . centrifugation. Samples were then washed by flushing 70–80 µL of 0.1% TFA The NatA (Naa10p/Naa15p) protein complex from Schizosaccharomyces pombe and eluted into a clean Eppendorf tube by flushing 70 µL of 75% acetonitrile and was prepared essentially as previously described . Briefly, protein expression 0.5% acetic acid by slow centrifugation. Samples were dried in a SpeedVac and vectors encoding 6XHis-tagged Naa15p and residues 1–156 of Naa10p were resuspended at 0.5 μg per μL in 0.1 M acetic acid for nano LC-MS/MS analysis . overexpressed in Rosetta (DE3) pLysS E. coli cells, and purified to homogeneity using a combination of Ni-resin affinity, 6XHis-TEV protease treatment to remove the 6XHis-tag, Ni-resin affinity to remove the 6XHis-TEV protease, HiTrap SP ion Cell culture and histone extraction. HeLa S3 mammalian cells were cultured at exchange and Superdex 200 gel filtration. The protein complex was concentrated to 37 °C and 5% CO in spinner flasks in Joklik’s modified Eagle’s medium supple- ~ 10 mg per mL in a buffer containing 25 mM HEPES (pH 7.0), 200 mM NaCl and 2 mented with 10% (v/v) newborn calf serum (HyClone), penicillin-streptomycin 1 mM TCEP and stored frozen at −70 °C until use. (1:100), and 1% (v/v) GlutaMAX (Invitrogen). Cells were harvested, washed with The HAT domain of human hMOF was prepared previously essentially as PBS, and stored at −80 °C. Human myogenic cell line, LHCN-M2 (a kind gift from described . Briefly, a protein expression vector encoding 6xHis-tagged hMOF Dr. Woodring Wright, UT Southwestern Medical Center at Dallas, Dallas, TX, HAT domain (residues 174–449) was overexpressed in Escherichia coli cells and the USA) was cultured in proliferation medium described elsewhere . In brief, protein purified to homogeneity using a combination of Ni-resin affinity and confluent cells were differentiated in 2% horse serum. Matched cultures for HiLoad Superdex 75 gel filtration. The protein was concentrated to ~ 20 mg per mL proliferation and differentiation were set up, the myoblasts harvested at 80% in a buffer containing 20 mM HEPES (pH 7.5), 0.5 M NaCl), and stored as confluence and differentiation cultures harvested on day 5–7 when the myotubes described for the NatA complex. are formed fully. Briefly, 0.05% trypsin-EDTA was used to detach cells after The HAT domain of S. cerevisiae Gcn5 (residues 99–262) was overexpressed in washing with sterile PBS. The cells were collected by centrifugation, washed again bacteria from a PRSETA/yGCN5 vector and purified similarly as previously in PBS, snap-frozen in liquid nitrogen, and stored at −80 °C until further analysis. described . The plasmid was transformed into E. coli strain BL21 (DE3) and Three biological replicates were used. Histone extraction and digestion was carried overexpressed by induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside out according to standard procedures . Briefly, nuclei were isolated by suspension (IPTG) and grown at 17 °C overnight. The cells were collected by centrifugation at of cells in 10X volume of nuclear isolation buffer (15 mM Tris-HCl pH = 7.5, 4000 r.p.m. at 4 °C and lysed in 50 mM potassium phosphate pH 7.5, 0.500 M 60 mM KCl, 15 mM NaCl, 5 mM MgCl , 1 mM CaCl and 250 mM sucrose, 0.2% NaCl, 5% glycerol, 1 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl 2 2 NP-40) with 1 mM DTT, 5 nM microcystin, 0.5 mM ASBSF and 10 mM sodium fluoride (PMSF), 100 ug per mL DNase I, and 100 μg per mL lysozyme. The butyrate at 4 °C. Nuclei were pelleted by centrifugation at 1000× g for 5 min at 4 °C supernatant liquid was passed over Ni-NTA resin, washed with 10 column volumes and washed twice with nuclear isolation buffer in the absence of NP-40. To the of lysis buffer with 5 mM imidazole but without PMSF, DNase, or lysozyme. The pelleted nuclei, 0.4 N H SO was added to a final ratio of 5:1 and incubated for 2 h 2 4 protein was then transferred to 6–8 kDa MWCO dialysis tubing (Spectrum Labs) with shaking at 4 °C. Samples were centrifuged at 3400× g for 10 min at 4 °C and and dialyzed overnight into 50 mM potassium phosphate pH 7.5, 20 mM NaCl, 5% the supernatants were collected and incubated on ice with ¼ volume of 100% TCA glycerol, and 1 mM DTT buffer. This was then followed by additional purification for 1 h. Precipitated histones were collected by centrifugation at 3400× g for 10 min through SP Sepharose ion exchange and Superdex 75 gel filtration. Purified protein at 4 °C and pellets were rinsed once with ice-cold acetone containing 0.1% HCl was concentrated to ~ 20 mg per mL in buffer containing 50 mM potassium and once with ice-cold acetone. Protein concentration was determined using a phosphate, pH 7.5, 500 mM NaCl, 5% glycerol and 1 mM DTT, and flash frozen Bradford assay. For in vivo analyses of histone acyl marks, 50 μg of histones and stored at −70 °C. were resuspended in 20 μL of 50 mM ammonium bicarbonate and subjected to The DNA sequence encoding residues 443–658 (including an N-terminal Met derivatization with propionic anhydride (or d-10 propionic anhydride when residue) of human PCAF was amplified by PCR and subcloned into the pET28-A identifying propionyl and butyryl histone marks), digested with trypsin for vector (Invitrogen) for overexpression similarly as previously described .The 6 h at 37 °C and desalted with C18 stage-tips as described above. plasmid was transformed into E. coli strain BL21 (DE3) and overexpressed by 10 NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications | | | NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 ARTICLE In nucleo acylation assay. HeLa S3 mammalian cells were harvested and washed common acyl-CoAs to reflect cellular levels. Yeast SILEC growth media was twice in ice-cold PBS. Cells were incubated on ice with hypotonic lysis buffer prepared with 200 μg biotin, 200 μg folic acid, 200 mg inositol, 40 mg niacin, 20 mg containing 10 mM Hepes-NaOH pH = 7.9, 10 mM KCl, 1.5 mM MgCl , 0.5 mM p-aminobenzoic acid, 40 mg pyridoxine-HCl, 20 mg riboflavin, 40 mg thiamine 13 15 DTT, 0.1% (v/v) NP-40, and 1X Halt protease and phosphate inhibitors for 10 min HCl, 20 g dextrose, 400 μg[ C N ]-pantothenate, 2.0 g Drop-out Mix Complete 3 1 with intermittent agitation. Nuclei were collected by centrifugation at 600 g for w/o Yeast Nitrogen Base, 1.7 g Yeast Nitrogen Base w/o AA & w/o AS, w/o 10 min at 4 °C, and washed once with a buffer containing 20 mM Hepes-NaOH Vitamins, and 5.0 g ammonium sulfate dissolved in 1 L of distilled H O. The 13 15 pH = 7.9, 50 mM KCl, 1.5 mM MgCl , 0.5 mM DTT, 0.2 mM EDTA, 20% (v/v) vitamins and [ C N ]-pantothenate were filter sterilized and the dextrose, drop- 2 3 1 glycerol and 1 X Halt protease and phosphate inhibitors. Nuclei were aliquoted at out mix, yeast nitrogen base mix, and ammonium sulfate were autoclaved. To ≈ 5× 10 cells per mL and used immediately by resuspending in 50 μL acylation confirm pantothenate auxotrophy, agar plates were prepared, with one batch reaction buffer (50 mM Tris-HCl pH = 8.0, 50 mM NaCl, 1 mM EDTA, 0.5 mM omitting pantothenate from the growth media. The plates were inoculated with DTT, 10% (w/v) sucrose, 1 X Halt protease and phosphate inhibitors), containing pan6Δ yeast, and then incubated at 37 °C for 24 h. After confirming auxotrophy, 0, 1 or 5 µM of short-chain acyl-CoAs (acetyl-, crotonyl-, malonyl-, succinyl-, 1 L of media was inoculated with pan6Δ S. cerevisiae and incubated at 30 °C while propionyl-, butyryl-, glutaryl- and β-hydroxybutyryl CoA). Reactions were agitating overnight with 500 mL in two 2 L Erlenmeyer flasks covered loosely with incubated at 30 °C for 3 h with gentle agitation. Nuclei were washed with acylation aluminum foil. After approximately 31 h from the onset of the culture, the yeast reaction buffer and the reactions were terminated by resuspending nuclei in 0.4 N cells were removed from the incubator, divided into 50 mL aliquots, and pelleted at H SO for histone extraction. Extracted histones were derivatized with d10- 500×g. The cells were resuspended in ice-cold 10% TCA extraction, respectively. 2 4 propionic anhydride and digested as described above. Samples were dried in The cells were pulse sonicated for 30 half-second pulses, on ice. Samples were spun a SpeedVac and resuspended at 0.5 μg per μL in 0.1 M acetic acid for nano at 16,000×g for 10 min at 4 °C to remove unbroken cells and debris. The final LC-MS/MS analysis. supernatant was transferred to a separate tube and stored at −80 °C until use as the SILEC internal standard. Dot blot analysis.2 μg of full-length acylated histones H3 or H4 from in vitro Acyl-CoAs metabolites extraction and quantitation. HeLa and myogenic cells reactions were spotted onto a nitrocellulose membrane. The membrane was collected from two confluent 15-cm plates (roughly 10–30 × 10 cells) were washed blocked with either 5% BSA or 5% nonfat milk and incubated with the twice with PBS, scraped into a 15 mL conical tube and pelleted by centrifugation at primary antibodies at 1:1,000 dilution (pan anti-crotonyl-lysine: PTM-501, 800× g for 3 min at 4 °C. Acyl-CoA metabolites were extracted following proce- pan anti-propionyl-lysine: PTM-201, pan anti-malonyl-lysine: PTM-901, pan anti- dures already described . In brief, pelleted cells were metabolically quenched and succinyl-lysine: PTM-401, pan anti-butyryl-lysine: PTM-301, pan anti-glytaryl- resuspended in 0.9 mL of ice-cold 10% TCA solution along with 0.1 mL of SILEC lysine: PTM-1151, all purchased from PTM-Biolabs, Hangzhou, China) according CoA internal standards, and sonicated with a probe tip sonicator for 30 s on to the manufacturers’ instructions overnight at 4 °C. The membrane was washed ice with a 1 pulse per 2 s rate. Cell lysates were centrifuged at 14,000×g for 10 min with TBS-T three times for 10 min each, incubated with secondary antibody (Goat anti-Rabbit IgG Fc, Pierce 31463) at a 1:10,000 dilution for 60 min at room at 4 °C to precipitate cellular debris and the supernatant containing the metabolites were kept on ice. Standards for calibration curves were prepared from commer- temperature and then probed with ECL Western Blot Substrate (Pierce). cially available acyl-CoAs (acetyl-, crotonyl-, malonyl-, propionyl-, butyryl-, glutaryl- and β-hydroxybutyryl CoA; —Sigma-Aldrich) as a master mix of 1 mM Nano LC-MS/MS analysis. A total of 2.5 µg of peptides were injected into a 75 μm and diluted from 1 µM to 10 nM in 10% TCA. Succinyl-CoA was run as a separate i.d × 17 cm Reprosil-Pur C -AQ (3 μm; Dr. Maisch GmbH, Germany) nano- 18 standard curve due to lower purity of the standard used, but otherwise prepared column (packed in-house) using an EASY-nLC nano HPLC (Thermo Scientific, identically. 50 μL of each standard solution was mixed with 0.85 mL of 10% TCA Odense, Denmark). The mobile phases consisted of water with 0.1% (v/v) formic and spiked with 0.1 mL of SILEC CoA internal standard. Standard calibration acid (A) and acetonitrile with 0.1% (v/v) formic acid (B). For analysis of in vitro curve samples were also subjected to sonication and solid phase extraction (SPE) in samples, peptides were eluted using a gradient of 0–30% B for 20 min followed by the same manner as the experimental samples to account for matrix effects, sample 30–98% B for 5 min and maintained over 10 minutes at 300 nL per min. For in losses and analyte stability. Samples and standards were purified using SPE car- nucleo and in vivo samples, the gradient consisted of 0–26% B over 45 min tridges (Oasis HLB 10 mg) that were conditioned with 1 mL of methanol and followed by 26–98% B over 5 min and maintained for 10 min at 300 nL per min. equilibrated with 1 mL of water utilizing a vacuum manifold. Acid-extracted The nano HPLC was coupled to a LTQ Orbitrap Elite or an Orbitrap Fusion mass supernatants were loaded onto the cartridges and washed with 1 mL of water. Acyl- spectrometer (Thermo Scientific, San Jose, California). Spray voltage was set at 2. CoA metabolites were eluted with 1 mL of 25 mM ammonium acetate in methanol 4 kV and capillary temperature was set at 275 °C. For DDA, the mass spectrometer and dried under nitrogen. Samples were resuspended in 50 µL of 5% (w/v) 5- was set to perform a full MS scan (290–1,400 m/z) in the Orbitrap with a resolution sulfosalicyilic acid and 10 µL injections were analyzed for acyl-CoAs by LC-HRMS of 60,000 (at 400 m/z), followed by a series of targeted MS/MS scans of each and LC-MS/HRMS. modified H3 and H4 peptide, followed by MS/MS scans of the top four most intense abundant ions from the first scan. All MS/MS scans were performed in the Data availability. The authors declare that all data supporting the findings of this ion trap mass analyzer (normal scan rate) using collision induced dissociation study are available within the article and its supplementary information files or (CID) with a normalized collision energy of 35 and an isolation window of 2.0 m/z. from the corresponding author upon reasonable request. All mass spectrometry Maximum injection times of 50 ms were defined for both MS and MS/MS scans. 6 4 raw files have been deposited in Chorus (https://chorusproject.org) under the AGC values were set to 1 × 10 for MS and 3 × 10 for MS/MS. MS data were project number 1376. collected in profile mode and MS/MS data were collected in centroid mode. For DIA, a full scan MS spectrum (m/z 300−1,100) was acquired in the Orbitrap with a resolution of 120,000 (at 200 m/z) and an AGC target of 5 × 10 or in the ion Received: 17 November 2016 Accepted: 14 September 2017 trap with an AGC target of 3 × 10 . MS/MS was performed with an AGC target 4 68 of 3 × 10 using an injection time limit of 30 or 60 ms . All acquisitions were performed in positive mode polarity. Data analysis was performed using our in-house software EpiProfile with a 10-ppm tolerance for extracting peak areas from raw files . The relative abundances of acyl-PTMs were calculated by dividing the intensity the modified peptide by the sum of all modified and unmodified peptides sharing the same References sequence, across all detectable charge states. For isobaric peptides (e.g., 1. Verdone, L., Caserta, M. & Di Mauro, E. Role of histone acetylation in the K STGGKAPR and KSTGGK APR), the relative abundances were estimated PTM PTM control of gene expression. Biochem. Cell Biol. Biochim. Biol. Cell 83, 344–353 by extracting the area under the curve of unique fragment ions. DDA was used to (2005). confirm peptide elution time by performing Mascot database searching (via 2. Mersfelder, E. L. & Parthun, M. R. 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Low resolution Proteomics 11, 100–107 (2012). data-independent acquisition in an ltq-orbitrap allows for simplified and fully 12 NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications | | | NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 ARTICLE supervised the study. All authors discussed the results and commented on the untargeted analysis of histone modifications. Anal. Chem. 87, 11448–11454 manuscript. (2015). 69. Basu, S. S. & Blair, I. A. SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards. Nat. Protoc. 7, Additional information 1–12 (2011). Supplementary Information accompanies this paper at doi:10.1038/s41467-017-01384-9. Competing interests: The authors declare no competing financial interests. Acknowledgements Reprints and permission information is available online at http://npg.nature.com/ We gratefully acknowledge Dr. Ben Black from the University of Pennsylvania for reprintsandpermissions/ supplying the recombinant histones and Dr. Andrew Andrews from Fox Chase Cancer Center for supplying the recombinant enzymes CBP and p300. This work was supported by NIH grants AI118891, GM110174, CA196539 and AG031862 to B.A.G.; GM101664 Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. to T.G.K.; K22ES26235, R21HD087866, R03CA211820 and a Pennsylvania Department of Health CURE grant to N.W.S.; R01 GM060293, R35 GM118090 and P01 AG031862 to R.M.; the NIH training grant fellowship T32GM008275 to D.M.M., and the NIH grant GM110174-S1 to M.C. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, Author contributions adaptation, distribution and reproduction in any medium or format, as long as you give J.S. performed most of the experiments, analyzed the data and wrote the manuscript; appropriate credit to the original author(s) and the source, provide a link to the Creative S.S. contributed to data analysis, made technical and intellectual contributions and Commons license, and indicate if changes were made. The images or other third party helped writing the manuscript. Z.-F.Y. developed the software for analysis of histone material in this article are included in the article’s Creative Commons license, unless marks and aided in the analysis of the histone data. M.C. performed the competition indicated otherwise in a credit line to the material. If material is not included in the assays and the analysis of the ionization efficiencies of the differentially acylated peptide article’s Creative Commons license and your intended use is not permitted by statutory of histone H3. N.V.B. provided the myogenic cells; D.M.M. designed and performed regulation or exceeds the permitted use, you will need to obtain permission directly from initial experiments for CoA analysis and contributed to manuscript writing. B.J.K. and the copyright holder. To view a copy of this license, visit http://creativecommons.org/ T.G.K. prepared the nucleosomal figures; G.A.B., C.E.M., and R.S.M. expressed and licenses/by/4.0/. purified the recombinant HATs; N.W.S. performed the CoA sample preparation and data analysis and contributed to editing the manuscript; R.M. provided the recombinant enzymes and was available for helpful discussions and B.A.G. conceived, designed and © The Author(s) 2017 NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications 13 | | | http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Communications Springer Journals

Characterization of histone acylations links chromatin modifications with metabolism

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Abstract

ARTICLE DOI: 10.1038/s41467-017-01384-9 OPEN Characterization of histone acylations links chromatin modifications with metabolism 1 1 1 1 1 2 Johayra Simithy , Simone Sidoli , Zuo-Fei Yuan , Mariel Coradin , Natarajan V. Bhanu , Dylan M. Marchione , 3 4 5 6 3 Brianna J. Klein , Gleb A. Bazilevsky , Cheryl E. McCullough , Robert S. Magin , Tatiana G. Kutateladze , 7 8 1 Nathaniel W. Snyder , Ronen Marmorstein & Benjamin A. Garcia Over the last decade, numerous histone acyl post-translational modifications (acyl-PTMs) have been discovered, of which the functional significance is still under intense study. Here, we use high-resolution mass spectrometry to accurately quantify eight acyl-PTMs in vivo and after in vitro enzymatic assays. We assess the ability of seven histone acetyltransferases (HATs) to catalyze acylations on histones in vitro using short-chain acyl-CoA donors, proving that they are less efficient towards larger acyl-CoAs. We also observe that acyl-CoAs can acylate histones through non-enzymatic mechanisms. Using integrated metabolomic and proteomic approaches, we achieve high correlation (R > 0.99) between the abundance of acyl-CoAs and their corresponding acyl-PTMs. Moreover, we observe a dose-dependent increase in histone acyl-PTM abundances in response to acyl-CoA supplementation in in nucleo reactions. This study represents a comprehensive profiling of scarcely investigated low-abundance histone marks, revealing that concentrations of acyl-CoAs affect histone acyl-PTM abundances by both enzymatic and non-enzymatic mechanisms. 1 2 Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA. Graduate Group in Cell and Molecular Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA. Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. 7 8 AJ Drexel Autism Institute, Drexel University, 3020 Market Street Suite 560, Philadelphia, PA 19104, USA. Department of Biochemistry and Biophysics, Abramson Family Cancer Research Institute, and the Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA. Correspondence and requests for materials should be addressed to B.A.G. (email: [email protected]) NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications 1 | | | 1234567890 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 ysine acetylation is the most extensively studied histone addition of acetyl groups using acetyl-CoA as a cofactor, and post-translational modification (PTM). Discovered more histone deacetylases (HDACs), which remove these groups . Lthan 50 years ago, it has been recognized to play a funda- The activities of both HATs and HDACs are regulated by mental role in transcriptional activation, metabolic regulation and the metabolic state of the cell . Thus, endogenous metabolite other central cellular processes . Mechanistically, lysine acetyla- concentrations are proposed to provide signaling that can directly tion neutralizes the positive charge of histone tails, reducing the influence acetylation dynamics in chromatin . physical interaction between histones and DNA, thereby allowing Over the last decade, a growing number of lysine modifications 2,3 the access of gene-activating transcription factors . Acetylation chemically related to acetylation (propionylation, malonylation, can also influence chromatin function by serving as a binding site crotonylation, butyrylation, succinylation, glutarylation, for bromodomain-containing remodeling complexes that can 2-hydroxyisobutyrylation and β-hydroxybutyrylation) have been directly stimulate trasnscription by recruiting transciptional identified on histones using mass spectrometry (MS)-based 4 8–14 co-activators . Acetylation dynamics in the nucleosome are the proteomic approaches . These findings have raised numerous result of the net activities between two different families of questions regarding their functional significance, possible enzymes: histone acetyltransferases (HATs), which catalyze the implications in metabolic pathways and the existence of a b O O O O Recombinant H3 or H4 HN HN HN HN 10 μg Incubation 1h at 30°C HN R Short-chain acyl-CoA COOH COOH COOH 0.5mM H N COOH H N 2 H N H N 2 2 Kbu Kcr Kac Kpr O O O OH O OH HAT COOH H N 2 HN HN HN HN OH 0.5 μg 2× propionylation Kacyl O Trypsin digestion 2× propionylation Stage-tip COOH COOH COOH H N COOH 2 H N H N H N 2 2 2 Kglu Kbhb Kmal Ksuc Time LC-ESI-MS Automated data analysis analysis EpiProfile software z-score Histone H3 Kglu, 2% Kbhb, 2% –1 0 2 Ksuc, 4% Mass shift (Da) Kmal, 4% 42.0105 56.0262 68.0262 70.0418 86.0003 86.0368 100.0160 114.0281 Kcr, 1% Kac Kpr Kcr Kbu Kmal Kbhb Ksuc Kglu HAT CBP (H3) GCN5 (H3) p300 (H3) Kac PCAF (H3) 43% Kpr 29% NatA (H4) Tip60 (H4) Kbu MOF (H4) 15% Histone H3 Histone H4 Histone H4 60% 30% Kbhb 50% 25% 5% Kglu Kac 8% Kbu 40% 20% Kpr Kac Ksuc 32% 11% Kcr 30% 15% Kmal 20% 10% Kmal Ksuc 12% Kglu 10% 5% Kbu Kpr Kbhb 12% 0% 0% 20% Kcr 0% Modification site Modification site Fig. 1 Overview of histone acetyltransferases (HATs) in vitro acylation activity and specificity. a Schematic representation of in vitro acylation assay. b Chemical structures of histone acyl modifications evaluated in this study. Lysine modifications and abbreviations are: acetyl (Kac), propionyl (Kpr), butyryl (Kbu), crotonyl (Kcr), malonyl (Kmal), succinyl (Ksuc), β-hydroxybutyryl (Kbhb), and glutaryl (Kglu). c Heat map displaying the in vitro acylation activity profiles of different HATs in the presence of acetyl-, propionyl-, crotonyl-, butyryl-, malonyl-, β-hydroxybutyryl-, succinyl- and glutaryl-CoA. Molecular mass shift of the various acylated lysines residues are shown in the table headers. Different HATs were assayed against histones H3 or H4 as specified in the first column. To generate the heat map, we averaged the relative abundance of acyl-PTMs on the quantified peptides and then normalized (z-scores) those values across the different HATs, i.e. row normalization. d Pie chart showing the average relative frequency of in vitro acylated peptides, divided in results for histone H3 (top) and histone H4 (bottom). e Bar plots depict the specificity for all HATs on the histone sequence. The x axis represents the modification site at histones H3 (left) and histone H4 (right), and the y axis represents the relative abundance shown as the average contribution of HATs to all acylated peptides. For histone H4 N-terminal peptide (G4-R17), the number of acylations on the sequence are displayed using the code 2, 3 or 4 mods. This is because it was not always possible to discriminate modification sites on the multiply modified H4 peptide. All values shown were corrected by the contribution of non-enzymatic acylation. All results are shown as the average of 3 independent experiments 2 NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications | | | K4 K9 K14 K9K14 K18 K23 K18K23 K27 K36 K27K36 K56 K64 K79 K122 K5 K8 K12 K16 2MODS 3MODS 4MODS K20 K31 K44 K59 K77 K79 K91 Relative abundance Relative abundance Relative abundance NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 ARTICLE regulatory enzymes beyond the well-established acetylation subset of acylations on histones. Because these marks are mechanisms that could govern these marks. While most of these dependent on their corresponding short-chain acyl-CoA questions remain to be answered, many studies have provided metabolic intermediates, we employ a targeted metabolomics new insights into the roles that acyl marks can play in genome approach to measure the concentrations of acyl-CoA metabolites function. For example, it has been reported that lysine crotony- in HeLa cells and in proliferative and differentiated human lation mediated by the HAT p300 can stimulate gene transcrip- myogenic cells. We find that the cellular concentrations of tion in vitro and in vivo seemingly to a greater degree than lysine different acyl-CoA metabolites span orders of magnitude and are acetylation, and that this mechanism is highly regulated by tightly correlated with the relative abundances of the acyl marks the metabolic concentrations of the crotonyl-CoA co-factor . identified in vivo. These findings support the notion of a direct Histone crotonylation has also been found to be enriched at link between cellular metabolism and epigenetic regulation, where transcriptionally active X/Y sex-linked genes during post-meiotic the relative abundances of different acyl marks in histones are 8,16 sex inactivation in mouse , and the YEATS domains Taf14, driven by the cellular concentrations of their respective metabolic 29,30 AF9 and YEATS2 have been reported to preferentially bind intermediates . 17–20 crotonylated over acetylated lysines residues in vitro . More recently, lysine butyrylation has been reported to directly sti- mulate gene transcription and compete with acetylation for the Results binding of the testis specific gene expression-driver Brdt in In vitro acylation of histones H3 and H4. Previous studies have spermatogenic cells . In addition, β-hydroxybutyrylation, was reported that the HATs CBP, p300 and PCAF can mediate 12 12,31 15 found to be induced during starvation or streptozotocin-induced propionylation , butyrylation , and crotonylation of lysine diabetic ketoacidosis, and to activate transcription of specific residues in vitro. These observations prompted us to investigate genes associated with starvation-responsive metabolic path- whether these and other known HATs can catalyze the acylation ways . These studies suggest that newly identified histone acyl- of human recombinant histones H3 and H4 using a broader PTMs may have unique or similar roles to acetylation in tran- range of acyl-CoA donors. We performed in vitro HAT activity scriptional activation. Such observations are supported by several assays with the HAT domains of PCAF, Gcn5, and the full-length reports showing that SIRT5, a member of the class III HDACs CBP and p300 enzymes against histone H3, and with the HAT can preferentially remove acidic acyl modifications, including domains of MOF, Tip60 and NatA against histone H4. Each 22 22,23 13 malonyl , succinyl and glutaryl , whereas propionyl, reaction was carried out individually in the presence of eight crotonyl and butyryl marks can be removed by various other different short-chain acyl-CoA donors, followed by bottom-up sirtuins . However, it remains unclear whether the same group nano-LC-MS/MS analysis (Fig. 1a, b). Figure 1c summarizes the of enzymes involved in the establishment of acetylation could also in vitro activity profiles of all HATs evaluated in this study. The mediate the establishment of these histone modifications in vivo. heat map shows that most HATs could utilize acetyl- Another aspect that has been underexplored is the relative propionyl- and butyryl-CoA with relatively high efficiency, 32,33 abundance of acyl marks, which is an important step towards supporting recent findings . However, acidic acyl-CoA donors understanding their biological relevance. This gap in knowledge is including malonyl-, succinyl- and glutaryl-CoA, and branched- mainly due to the biases inherent in the use of antibody-based chain acyl donors like β-hydroxybutyryl CoA are utilized by enrichment methods commonly employed prior to MS detec- HATs less efficiently. Interestingly, enzymes did not seem to tion . Although stoichiometry at individual sites has been utilize crotonyl-CoA for the catalysis of acyl marks as effectively reported for propionylation (7%) at H3K23 in a leukemia cell as propionyl- and butyryl-CoA despite the structural similarity 26 27 line , butyrylation (31%) at H3K115 in mouse brain , and within these cofactors. These data are in agreement with previous crotonylation (1–3%) at H2AK36, H2BK5, H3K23 and H4K12 in observations suggesting that HATs activity is weaker with brain histones , a global overview of the abundances relative to crotonyl-CoA due to the planarity and rigidity imparted by the 8,20,32 histone acetylation is lacking. The dearth of quantitative data for C-C double bond in the crotonyl moeity . these non-canonical acyl-PTMs has led to the hypothesis that When taking a closer look at the individual acylation activities they might arise due to the chemical reactivity of acyl-CoAs. of all HATs (Supplementary Table 1), we observed that enzymes Indeed, this has been observed in the context of acetylation and have different trends in their substrate preference. For instance, succinylation in mitochondrial proteins . Thus, it is still open to histone H3 is known to be selectively acylated at the lysine residue 33 34 debate whether these modifications are strategically positioned on 14 (H3K14) by Gcn5 and PCAF . Therefore, if we look at the the chromatin by enzymes or whether their presence is instead relative abundances for all acylations at H3K14, under our assay the result of non-enzymatic chemical reactivity. conditions, Gcn5 was able to butyrylate ~ 78% of the H3 peptide In this study, we sought to characterize the acylation of at position 14, whereas acetylation and propionylation were histones, including their overall abundance and their likelihood to found at ~ 32% and ~ 11%, respectively (Supplementary Table 1). be products of HAT catalysis rather than chemical reactivity of Likewise, PCAF displayed ~ 88% butyrylation, followed by ~ 5% acyl-CoAs. First, we investigate the ability of several recombinant crotonylation and ~ 2% acetylation at position H3K14. However, HATs to catalyze the acylation of histones H3 and H4 using when looking at the average sum of the relative abundances of all different acyl-CoA donors employing an MS-based in vitro acylated peptides by HATs on H3, there seems to be a trend bioassay. Our data show that most HATs can catalyze histone in the order of substrate preference: acetyl > propionyl > butyryl acylation using different acyl-CoA substrates to variable extents > malonyl > succinyl > β-hydroxybutyryl > glutaryl > crotonyl when tested individually. However, in competition assays (Fig. 1d; Supplementary Table 1). This trend inversely correlates performed in the presence of equimolar concentrations of acyl- with the increasing size of the side chain of the acyl donor (except CoA and acetyl-CoA, almost all HATs strongly prefer to utilize for crotonyl-CoA and β-hydroxybutyryl), supporting the notion acetyl-CoA to modify histones. Our data also confirm that that the activity of HATs gets weaker with increasing acyl-chain histones can be modified non-enzymatically through the chemical length . Interestingly, p300 and PCAF were the enzymes with reactivity of the different acyl-CoA donors alone. We also employ the highest crotonylation activities on H3 (Supplementary a proteomics approach to characterize several acyl-PTMs in Table 1). nucleo and in vivo without the use of enrichment strategies, Moreover, the average activities of HATs on histone H4 which allow us to determine the relative abundance of a diverse showed patterns that were consistent with the trend mentioned NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications 3 | | | ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 a c GCN5 vs. non-enzymatic acylations Non-enzymatic acylations H3 3.0 50% 2.5 40% Kbhb 2.0 Kglu 30% GCN5 1.5 Ksuc Non-enzymatic Kmal 20% 1.0 Kcr Kpr 10% 0.5 Kbu 0.0 0% Kac Acyl-PTM Modification site Histone H3 Histone H4 Non-enzymatic acylations H4 40% 1 1 0.9 0.9 0.8 0.8 30% Kbhb 0.7 0.7 Kglu 0.6 0.6 Ksuc 0.5 0.5 20% Kmal 0.4 0.4 Kcr 0.3 0.3 10% 0.2 0.2 Kpr 0.1 0.1 Kbu 0 0 0% Kac Acyl-PTM Acyl-PTM Modification site Non-enzymatic Enzymatic Enzymatic Non-enzymatic Fig. 2 Non-enzymatic versus enzymatic acylation of histones in vitro. a Comparison of non-enzymatic versus GCN5-catalyzed acylations on histone H3. The y axis (arbitrary units) represents the sum of the relative abundances of all enzymatically and non-enzymatically acylated peptides from histones H3. We can observe that the contributions for Ksuc, Kmal, Kbhb, Kglu and Kcr in the experiments with GCN5 were mostly the result of non-enzymatic acylations. b Stacked column representation of non-enzymatic reactivity divided by enzymatic reactivity of the eight acyl-CoA donors on histone H3 (left) and histone H4 (right). The fractional reactivity represents the ratio of PTM intensity in presence of all seven enzymes tested versus PTM intensity in absence of enzymes, i.e., 0.5 corresponds to identical intensities with and without enzyme. For instance, crotonylation is an overall low abundance PTM, although the majority detected on histone peptides is the result of an enzymatic catalysis. c, d Bar plot showing the relative quantitation of non- enzymatically acylated sites on c histones H3 and d histone H4. All results are shown as the average of three independent experiments and error bars represent the S.D. before in terms of substrate preference (Fig. 1d). However, non-enzymatically modified in vitro, we incubated histones H3 individual acylation activities suggest that, while MOF seems to and H4 with 0.5 mM acyl-CoAs in the absence of acetyl- follow the same trend when looking at the sum of all acylated transferases. We found that all acyl-CoAs can chemically acylate peptides, Tip60 prefers to utilize butyryl-CoA as a cofactor, histones, as seen in Fig. 2a, showing a comparison between the followed by succinyl-CoA and acetyl-CoA (Supplementary non-enzymatic acylation profiles of various acyl-CoA donors on Table 1). Nonetheless, all results reported in Fig. 1 are based on histone H3 with acylations mediated by GCN5. We showed that the average contribution of both groups of HATs rather than most non-enzymatically catalyzed acylations have site specificities individual acylation activities. Detailed acylation site specificities that were different from those enzymatically modified sites for all HATs can be found in Supplementary Table 1. Our data (Figs. 1e, 2c, d). The most prevalent sites observed for also showed that the N-terminal acetyltransferase NatA can non-enzymatic acylations on histone H3 were K36, K56, K64, catalyze N-terminal propionylation and butyrylation of histone K79 and K122 (Supplementary Fig. 1; Fig. 2c). On H4, the most H4 in vitro (Supplementary Fig. 2). prevalent sites were K31, K59, K79 and K91 (Supplementary Fig. 1; Fig. 2d). Interestingly, these sites are closer to the C terminus of histones, whereas enzymes showed higher specificity Histones are non-enzymatically acylated in vitro. for sites at the N-terminal tails (Fig. 1e). These observations suggest that non-enzymatic acylations may be enhanced by some Non-enzymatic acylation of proteins has been reported to occur level of structural and conformational dynamics on histones. In through the nucleophilic attack of the unprotonated ɛ-amino 28,35 agreement, most succinylated and malonylated sites identified by group of lysine residues to the acyl group of acyl-CoAs . Xie et al. in vivo have been reported to occur also at the globular This mechanism is facilitated by an alkaline pH and high levels domain and C terminus of histones H3 and H4 rather than at the of acyl-CoA. In mitochondria, where both conditions are met N-terminal tails. and evidence of the existence of acetyltransferases is lacking, a large body of evidence suggests that high levels of protein We then estimated which acylations are more likely to occur through enzymatic or non-enzymatic reactivity in vitro. As acylation observed in this organelle are the result of 36,37 non-enzymatic mechanisms . To test whether histones can be seen in Fig. 2b, the average contribution for acetylation, 4 NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications | | | Kac Kpr Kbu Ksuc Kmal Kbhb Kglu Kcr Kac Kpr Kbu Kcr Kbhb Kmal K4 Ksuc K9 Kglu K14 K9K14 K18 Kac Kbu K23 Kpr K18K23 Kcr Kmal K27 Ksuc K36 Kglu K27K36 Kbhb K56 K64 K79 K122 K5 K8 K12 K16 2mods 3mods 4mods K20 K31 K44 K59 K77 K79 K91 Fractional reactivity Relative abundance (AU) Fractional reactivity Relative abundance Relative abundance NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 ARTICLE a b HAT CBP (H3) GCN5 (H3) p300 (H3) Pcaf (H3) NatA (H4) Tip60 (H4) MOF (H4) Neg (H3) Neg (H4) CoAs Ace/Pro Recombinant H3 or H4 Ace/Cro 10 μg Ace/But Ace/Mal Ace/Bhb Acetyl-CoA10 μM Ace/Suc Acyl-CoA 10 μM Ace/Glu Log 2 HAT –1 0 11 0.5 μg c d 6 12 GCN5 p300 Glutaryl β-hydroxybutyryl Glutaryl 114.02 86.04 Da R = 0.94 (without crotonyl) 114.02 β-hydroxybutyryl 4 8 R = 0.94 (without crotonyl) Succinyl 86.04 Da Succinyl 100.02 Da 100.02 Da 3 6 Butyryl Malonyl 70.04 Da 2 Propionyl 86.00 Da Propionyl Malonyl 56.03 Da Butyryl 56.03 Da 86.00 Da 70.04 Da 50 60 70 80 90 100 110 120 50 60 70 80 90 100 110 120 Modification M.W (Da) Modification M.W (Da) MOF β-hydroxybutyryl Glutaryl 86.04 Da 114.02 Da 5 R = 0.91 (without crotonyl) Succinyl 100.02 Da Butyryl 70.04 Da Malonyl 86.00 Da Propionyl 56.03 Da 50 60 70 80 90 100 110 120 Modification M.W (Da) Fig. 3 In vitro acetylation competition assay. a Schematic representation of the in vitro competition acetylation assay. b Heat map displaying in vitro acylation specificities of HATs during acyl-CoA competition assays. Different HATs were assayed against histones H3 or H4 as specified in the table headers in the presence of equimolar concentrations of acetyl-CoA and a competing acyl-CoA donor. Negative controls with no enzyme are also shown. c Correlation between the molecular weight and the acylation preference for different acyl donors displayed for the HATs GCN5 on histone H3, d p300 on histone H3 and e MOF on histone H4. For each modification, molecular weight is indicated. Results show that the preference for acetyl-CoA over the other acyl donor tightly correlates with the molecular weight of the acyl donor. Crotonylation was not included in the correlations, as its molecular weight did not correlate well with the preference of the enzymes over acetyl-CoA. All graphs are shown as the average of log2 ratios between the relative abundances of all acetylated peptides and the relative abundances of the corresponding competing acylated peptide propionylation and butyrylation marks in the presence of all it is important to mention that concentrations of acyl-CoAs used HATs was more abundant in in vitro experiments, whereas acidic in this experiment were far above the known physiological acyl modifications (malonylation, succinylation and glutaryla- concentrations of CoA derivatives in whole cells , so the extent tion) and β-hydroxybutyrylation occurred to a greater extent of chemical acylation observed in this study is likely an through non-enzymatic mechanisms in both histones H3 and H4. overestimation. This higher concentration was required to ensure Again, we observed a trend in which the ratio of enzymatic/ proper sensitivity to the in vitro assay. Even though it has been chemical reactivity of acyl groups is inversely correlated with the previously demostrated that protein lysine acylation can occur at size of the side chain, supporting the idea that most known HATs physiological acyl-CoA concentrations in vitro , our study does 32,33 catalyze larger acylations less efficiently . When looking not represent a suitable extrapolation for the reactivity of these individually at non-enzymatic acylations in histones, crotonyl- intermediates in cells. As such, our data cannot rule out the CoA showed the lowest acylation levels through chemical possibility of the existence of enzymes that play a major role in catalysis (Fig. 2c, d). As expected, crotonyl-CoA presents a lower catalyzing these marks in vivo as compared to non-enzymatic chemical reactivity towards lysine nucleophiles due to the reactions. resonance properties of the beta unsaturated C-C bond . The correlation between sites prone to chemical acylation (this study) and sites identified in other in vivo studies suggests that HATs prefer acetyl-CoA for acylation of histones. Previously, it histone acyl modifications in cells could be the result of both has been demonstrated that some HATs are able to catalyze the enzymatic and non-enzymatic mechanisms after direct exposure transfer of propionyl and butyryl groups in vitro with similar 12,31 to intrinsically reactive acyl-CoA metabolites. This is supported specificites but different efficiencies than acetyl groups . These by the fact that enzymes with distinctive acyltransferase activities observations lead to the following question: what determines the have not been identified in any cellular compartment . However, acyl group to be transferered if HATs are indeed mediating NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications 5 | | | Log 2 Kac/Kacyl Log 2 Kac/Kacyl Log 2 Kac/Kacyl ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 various acylations in vivo? It has been shown that fibroblasts of decarboxylase (MCD) and short-chain acyl-CoA dehydrogenase patients with inherited metabolic disorders have high levels of (SCAD), respectively. However, the mechanism by which this propionyl-, malonyl- and butyryl-CoA and high levels of the increase in protein acylation is mediated has not been explored. 43,44 corresponding lysine acylations . These disorders include We hypothesized that under such conditions, other acyl-CoAs deficiencies in propionyl-CoA carboxylase (PCC), malonyl-CoA could rival the levels of acetyl-CoA and induce HATs to use non- ab HeLa cells Myogenic cells 50 40 *** Myoblasts *** Myotubes 25 20 10 ** ** 0 0 PTMs PTMs c d HeLa cells Myogenic cells –5 4×10 –5 3×10 –7 –7 6×10 2.5×10 –7 Myoblasts –5 2×10 –5 *** 3×10 Myotubes 3×10 –7 –7 4×10 1.5×10 –7 1×10 –5 –5 3×10 –7 2×10 –8 2×10 5×10 –5 –5 2×10 2×10 –6 –5 3×10 1×10 ** –6 2×10 –6 –6 5×10 1×10 CoA metabolites CoA metabolites 2.0 HeLa cells 1.5 1.0 0.5 R = 0.99 0.0 –7 –6 –6 –6 –6 0 5×10 1×10 1.5×10 2×10 2.5×10 R = 0.99 –5 –5 –5 0 1×10 2×10 3×10 CoA metabolites pmol/cell Myoblasts Myotubes 30 15 1.5 1.4 1.0 1.2 0.5 R = 0.99 1.0 R = 0.95 0.0 –7 –6 –6 –6 0.8 0 5×10 1×10 1.5×10 2×10 –8 –7 –7 –7 5×10 1×10 1.5×10 2×10 R = 0.99 R = 0.92 R = 0.99 (without Kcr,crotonyl-CoA) –5 –5 –5 –6 –5 –5 –5 0 2×10 4×10 6×10 0 5×10 1×10 1.5×10 2×10 CoA metabolites pmol/cell CoA metabolites pmol/cell 6 NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications | | | Acetyl-CoA Acetyl-CoA Succinyl-CoA Succinyl-CoA Propionyl-CoA Propionyl-CoA Kme1 Kme2 Kac Kme3 Kpr Ksu Kbhb Kbu Kmal Kcr Kglu Kme1 Butyryl-CoA Malonyl-CoA Kac Crotonyl-CoA Glutaryl-CoA Kme2 Kme3 Ksu Kpr Kbu Kcr Kbhb Kmal Kglu Butyryl-CoA Malonyl-CoA Glutaryl-CoA Crotonyl-CoA Acyl-PTMs normalized abundance % Normalized relative abundance % pmol/cell Acyl-PTMs normalized abundance % pmol/cell Acyl-PTMs normalized abundance % Normalized relative abundance % NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 ARTICLE native cofactors. To test this, we performed in vitro HAT com- between 15 and –30% (Fig. 4a,b; Supplementary Fig. 5). Using our petition assays in the presence of equimolar concentrations of workflow, we measured the relative abundances at individual acetyl-CoA and other acyl-CoAs (Fig. 3a). As shown in Fig. 3b, sites. Acetylation was found at high relative abundances at most HATs preferred to utilize acetyl-CoA than any other acyl- positions H3K18 and H3K23 ranging from 17 to –35%, and CoA donor. It is important to mention that at 10 µM of acyl- between 15–30% at H4K12 and H4K16 (Supplementary Table 2), CoAs, we observed non-enzymatic acylation of histones H3 and which is in accordance with previous findings . Interestingly, H4, as shown in Fig. 3b. Consistent with previous in vitro similar relative abundances were observed at H3K14 (3–8%) for experiments, for most HATs, the preference for the competing acetylation and propionylation marks (Supplementary Table 2). cofactor, if any, largely depended on the size of the acyl donor Overall, most non-acetyl acyl marks were found at levels below side chain. As shown in Fig. 3c–e, for the enzymes GCN5, p300 2%, mainly at the N-terminal domains of histones H3 and H4. and MOF we observed an inverse correlation between the HAT Surprisingly, some acidic acyl marks were found at the globular preference for the competing cofactor and the increasing mole- domains and C terminus, showing abundances as high as 10% cular weight of the acyl donor side chain, with the exception of (Supplementary Table 2), which coincide with the sites that were crotonyl-CoA. Relative abundances for all peptides in competi- more susceptible to non-enzymatic acylation in our in vitro tion assays are shown in Supplementary Table 3. Altogether, our experiments (Fig. 2c, d). Detailed site specificity for all acyl marks data suggest that even in the highly unlikely chance that any other is shown in Supplementary Table 2. Importantly, we did detect acyl-CoA accumulated to the extent that its concentration rivaled low levels of hydroxybutyrylation; however, our MS acquisition that of acetyl-CoA, HATs would still mostly utilize acetyl-CoA. method cannot discriminate between possible isoforms of this We thus shifted our focus to investigate how abundant these acyl mark, including bhb (β-hydroxybutyryl or 3hb), 2- marks are in vivo, and whether their abundance can be justified hydroxybutyryl (2hb), 3-hydroxyisobutyryl (bhib), 2 hydroxyiso- 10,14 by the abundance of acyl-CoA intermediates. butyryl (2hib) or 4-hydroxybutyryl (4hb) . Likewise, peptides bearing Kbu (butyryl) marks may also represent isobutyryl marks. Our analysis of myoblasts showed dynamic changes in global Relative abundances of histone acyl-PTMs in mammalian cells. histone acylations upon their fusion to form multinucleated myotube cells. We observed that the global levels of lysine Owing to the low abundance of lysine acyl marks, current MS- based approaches involve the use of antibody-based enrichments acetylation, propionylation, butyrylation, malonylation, succiny- lation and glutarylation were significantly decreased upon to increase the sensitivity of the MS analysis for identification and quantification. Although these approaches are helpful for esti- myogenic differentiation, whereas the levels of lysine crotonyla- tion were increased (Fig. 4b). The role of differential histone mating the relative changes of modifications across multiple conditions, they cannot provide direct information on relative acylation in cellular differentiation is poorly understood; how- abundances, as the peptide with the modification and the ever, various lines of evidence suggest that nutrition and 47,48 unmodified peptide end up in different sample pools. In addition, metabolism play a key role in the differentiation of cells . evaluation of the specificity of several commercially available pan For example, a previous study investigating the role of carbon anti-acyl-PTM antibodies by dot blot analysis revealed significant metabolism in the differentiation of myogenic cells demonstrated cross-reactivity among differentially acylated peptides, compli- that siRNA knock down of ATP citrate lyase (ACLY) induces cating their further application for immunoenrichment of histone differentiation of mouse myoblasts . The same study also acyl-PTMs (Supplementary Fig. 3). showed that the levels of histone acetylation in shACLY-treated cells were reduced, hypothesizing that the deposition of acetyl- Thus, to accurately detect and quantify histone acyl-PTMs in vivo using label-free approaches, we used the retention time CoA, and in turn histone acetylation levels, play an important role in the differentiation of myoblasts. Because there is little and mass shift information from the in vitro experiments to optimize the MS acquisition and in-house quantification soft- evidence of how newly identified histones acylations may be implicated in the differentiation of cells and/or epigenetic ware . Here, we refer to our quantitative values as “relative abundances”, as we are aware that differences in the ionization regulations, we next sought to investigate whether the cellular efficiencies of modified peptides or biases in trypsin digestion in concentrations of other acyl-CoA metabolites have a direct the presence of certain modifications can affect the assessment of relationship with histone acylation levels. accurate PTM stoichiometry, which was observed for the differentially acylated peptide H3 aa 18–26 (KQLATKAAR) (Supplementary Fig. 4). Acyl-CoA donors dictate the levels of histone acylation. Various Analysis of acid-extracted histones from wild-type HeLa and studies in mammalian cells have shown that chromatin mod- myogenic cells showed that all acyl-PTMs combined, excluding ifications are sensitive to changes in intracellular concentrations acetylation, were found at relative abundances between 6 and of metabolic intermediates, linking cell metabolism to epigenetic 6,50 –15% of all detectable modified peptides of canonical histones H3 changes . However, the mechanisms and enzymes mediating and H4 (Supplementary Fig. 5). Individually, most acyl-PTMs these processes have not been fully explored. So far, it has been showed low relative abundances ranging from 1 to –5% (Figs. 4a, demonstrated that changes in the levels of acetyl-CoA can b), as opposed to acetylation with global relative abundances influence global histone acetylation levels . These findings led us Fig. 4 Overview of the relative abundances of endogenous acyl-PTMs and intracellular metabolite concentrations. Bar plots showing the relative abundances of several lysine PTMs in a HeLa cells, b proliferative myogenic cells (myoblasts) and differentiated myogenic cells (myotubes). PTMs are shown as percentages representing the normalized relative abundances of all detectable peptides of canonical histones H3 and H4. c, d Bar plots showing the concentrations of acyl-CoA metabolites in c HeLa cells and d myogenic cells normalized to cell number. Metabolic concentrations of butyryl-CoA may also represent concentrations for isobutyryl-CoA. e–g Global histone acylation levels correlation with intracellular concentrations of acyl-CoA metabolic intermediates in e HeLa cells, f myoblasts and g myotubes. Correlations are calculated between the normalized net abundances of all detectable acylated peptides in H3 and H4 and the global concentrations CoA metabolites in pmol per cell. Insets show zoom in graphs with the linear correlation of the data without the values for acetyl-CoA and Kac. All results are shown as the average of three biological replicates and error bars represent the S.D. Summary of p-values is as follows; *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. p-values were generated by unpaired Student’s t-test NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications 7 | | | ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 a b In nucleo acylation 100% Histone extraction Propionylation Trypsin digestion 80% Propionylation Cell 0 μM Acyl-CoA LC/MS Nucleus 1 μM Acyl-CoA 60% 5 μM Acyl-CoA Incubation at 30 °C for 40% 2-4 h 20% Nucleus 0% Nucleus Acyl CoAs (0 –5 μM) 45% H3K56 PTMs H3K9-K14 PTMs 40% In nucleo propionylation In nucleo malonylation 35% 80% 30% 70% 25% 60% 20% 50% 15% 40% 10% 30% 5% 0% 20% 0 μM_Mal-CoA 1 μM_Mal-CoA 5 μM_Mal-CoA H3_54_63 K56pr 0.03% 0.03% 0.00% 10% H3_54_63 K56bu 0.01% 0.02% 0.05% 0% H3_54_63 K56cr 0.00% 0.03% 0.00% 0 μM_Pro-CoA 1 μM_Pro-CoA 5 μM_Pro-CoA Others 0.07% 0.50% 0.50% H3_54_63 K56su 0.02% 0.08% 0.01% H3_54_63 K56ma 1.11% 14.66% 26.65% H3_9_17 K14pr 3.45% 7.94% 10.21% H3_54_63 K56hi 0.12% 0.11% 0.08% H3_9_17 K9pr 1.34% 1.68% 1.72% H3_9_17 K14ac 3.94% 2.80% 1.69% H3_54_63 K56gl 0.36% 0.15% 0.33% H3_54_63 K56ac 0.33% 0.67% 0.52% H3_9_17 K9ac 0.24% 0.37% 0.66% H3_54_63 K56me3 0.08% 0.19% 0.07% H3_9_17 K9me3 10.04% 11.42% 12.91% H3_9_17 K9me2 16.48% 19.00% 10.72% H3_54_63 K56me2 12.23% 8.20% 10.30% H3_54_63 K56me1 1.92% 1.00% 0.72% H3_9_17 K9me1 30.51% 29.15% 28.72% Fig. 5 Analysis of in nucleo acylation. a Schematic representation of in nucleo acylation assay. b Bar plots showing the dose-dependent acylation of histones upon treatment with 0, 1 and 5 μM of acetyl-, butyryl-, malonyl-, glutaryl-, propionyl-, succinyl-, β-hydroxybutyryl and crotonyl-CoA, respectively. Values represent the sum of the relative abundances of all acylated peptides from histones H3 and H4. c Example of in nucleo acylation resulting in an increase in malonylation of H3K56 after treatment with increasing concentrations of malonyl-CoA. d Example of in nucleo acylation showing that induced propionylation by increasing the concentration of propionyl-CoA resulted into a reduced relative abundance of acetylation on the site H3K14 to consider whether the levels of other histone acylations may also measurable increase in the levels of crotonyl-CoA, this did not be influenced by the intracellular concentrations of their respec- correlate well with the increase in lysine crotonylation observed in tive acyl-CoA donors. Using a stable isotope dilution MS myotubes (Fig. 4g). Acyl-CoA intermediates are derived from approach , we accurately measured the concentrations of seven various metabolic pathways including the TCA cycle, fatty acid acyl-CoA metabolic intermediates in HeLa (Fig. 4c) and myo- synthesis, β-oxidation and amino acid metabolism. Although it genic cells (Fig. 4d). Our metabolomics analysis revealed that remains poorly understood which metabolic pathway leading to acetyl-, propionyl- and succinyl-CoA were the most abundant the production of different acyl-CoAs might serve as a substrate CoA thioesters in our cell models, with concentrations around 12, for the acylation of nuclear histones, the global concentrations of 1 and 0.5 μM, respectively, when normalized to cell volume of metabolites determined in this study are consistent with the HeLa cells (Supplementary Fig. 6). In myogenic cells, when abundance of lysine acylations. Collectively, our data demonstrate normalizing the data to cell number, we observed that the a clear quantitative link between metabolism and differential intracellular concentrations of metabolites also appear to undergo histone acylations. regulation from myoblast to myotube differentiation. Our data showed that the levels of acetyl-, succinyl-, propionyl-, butyryl and malonyl-CoA were significantly decreased upon differentia- Acylation of histones in nucleo. Our in vivo and metabolomics tion (Fig. 4d), whereas the levels of glutaryl- and crotonyl-CoA studies strongly implicated metabolism in histone acylations. As did not show significant changes. the concentrations of intracellular metabolites are known to When looking at the levels of acyl lysine PTMs in HeLa cells, change in response to diet or physiological conditions, we next our data showed a strong positive correlation with the metabolic turned to the question of whether alterations in the levels levels of acyl donors (Fig. 4e). A similar observation could be of metabolites could affect the corresponding levels of histone made for myoblasts (Fig. 4f). Even though we observed a acylations. To further explore this idea, we turned to an in nucleo 8 NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications | | | Kac Kbu Kmal Kglu Kpr Ksu Kbhb Kcr Relative abundance (sum) Relative abundance (sum) Relative abundance (sum) NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 ARTICLE system, where purified nuclei can be treated with artificial levels To further explore the possible mechanisms underlying the of metabolites. We isolated nuclei from HeLa cells under hypo- establishment of acyl marks, we performed in vitro acylation tonic conditions, treated with varying concentrations of eight assays in the absence of HATs (Fig. 2). We found that histones different acyl-CoA donors and performed histone extraction, can be acylated by the chemical reactivity of all acyl-CoA meta- digestion and derivatization following standard procedures bolites evaluated in this study. While most HATs showed strong (Fig. 5a). preference for the acylation of residues at the N-terminal MS analysis revealed that histone acylations can be induced in a domains, i.e., residues K9–K36 in H3 and K5–K16 in H4, concentration-dependent manner. Specifically, by adding 1 or 5 µM non-enzymatic acylation sites were more prevalent closer to the C of acyl-CoAs, we induced an increase of the respective acylation on terminus of histones, i.e., residues K56–K122 in histone H3 and histone peptides (Fig. 5b). Since the in nucleo experiment preserves K59–K91 in histone H4. Acidic acyl-PTMs including malonyla- the natural state of nuclear processes, it can be used to observe tion, succinylation and glutarylation were among those most histone acylations in native chromatin. Our in vitro experiment easily catalyzed in the absence of enzymes (Fig. 2b). These marks demonstrated that acylations can occur by both enzymatic and are different from acetyl marks as they add bulkier groups to non-enzymatic mechanisms, but such simplified assay cannot lysine residues and they carry a negative charge under physio- accurately represent the balances of a nuclear environment. We logical conditions. As such, it has been suggested that these acidic compared the two assays by performing an in nucleo–in vitro acyl marks could disrupt the interactions between histones and Spearman’s rank-order correlation analysis by using corrected DNA, resulting in a more profound effect in chromatin unfolding in vitro enzymatic data (subtracting the non-enzymatic contribu- than lysine acetylation . Emerging hypotheses have suggested a tion). We observed a good correlation for some residues, including model where non-enzymatic chemical reactions are a significant H3K9acyl and H3K18acyl sites (Supplementary Fig. 7) that were contributor to the landscape of lysine acylations in nuclear highly acylated only in presence of enzymes in vitro. This suggests histones . They also suggest that sirtuin enzymes showing that specific sites are likely more accessible to enzymatic activity specificity for the removal of acyl marks may represent a than others, and that this reactivity is also a function of the acyl- constitutive programming to suppress potential damaging effects 56,57 CoA utilized. However, a generalized conclusion cannot be drawn, caused by the presence of these PTMs . Interestingly, our as the in nucleo assay cannot discriminate enzymatic catalysis from study showed that those succinylated and malonylated sites chemical reactions, and physiological acylation turnover (equili- highly susceptible to non-enzymatic acylation in vitro were brium deposition/removal). among the sites reported previously in in vivo studies . While Additionally, our in nucleo results showed that histones more studies are required, our data suggest that histone lysine accommodate acylation in two ways; by simply increasing residues are prone to be modified by several free acyl-CoAs with the modified state or by removing pre-existing modifications to and without enzymatic assistance. maintain the same level of total modified form. For example, The accurate quantification and elucidation of potential func- upon treatment with malonyl-CoA, levels of H3K56mal increased tional roles of acyl marks have been hampered by their low with almost no changes in the other modifications on that peptide abundance. To provide an accurate estimate of the levels of acyl (Fig. 5c). On the other hand, after treatment with increasing marks, we employed a label-free approach using DIA-MS. By concentrations of propionyl-CoA, the levels of H3K14pr analyzing in the same mixture modified and unmodified forms of increased, whereas the levels of H3K14ac showed a measurable histone peptides, we could report the relative levels of acyl-PTMs decrease (Fig. 5d). Detailed in nucleo acylation relative abun- expressed as a percentage of the total histone. Analysis of human dances can be found in Supplementary Table 4. Taken together, cervical cancer cells (HeLa) and human myogenic cells revealed our in nucleo studies demonstrated that modifications in that acyl marks together represent around 6–15% of all detected chromatin are sensitive to changes in the concentrations of modifications on histones H3 and H4 (Fig. 4a,b; Supplementary cellular metabolites, consistent with previous observations con- Fig. 5). Our myoblast/myotube comparison strongly indicates necting the metabolic state of the cell with chromatin that the differentiation of pluripotent cells is marked by a 7,53 regulation . decrease in global levels of histone acetylation. This was not surprising, as this mechanism has been shown to be driven by a decrease in acetyl-CoA production mediated through the inhi- 51,58 Discussion bition of glycolysis . In agreenment with these findings, our A comprehensive screen of the major families of histone acetyl- data showed a significant decrease of the bulk levels of histone transferases (HATs) confirmed that most enzymes can catalyze acetylation once myoblasts fused to form multinucleated myo- the acylation of histones utilizing acetyl, propionyl and butyryl- tubes. Intriguingly, while the levels of most histone acylations CoA cofactors with similar efficiencies (Fig. 1), as previously decreased upon differentiation, myotube cells showed an increase reported . However, they were less efficient catalyzing the acy- in the global levels of lysine crotonylation (Fig. 4b). The under- lation of histones with charged, branched or planar acyl-CoA standing of how these chromatin modifications could be involved cofactors. Even though these acyl donors are structurally similar in driving myoblast differentiation is beyond the scope of this to acetyl-CoA, the universal cofactor of HATs, our data showed study and the subject of studies to come. This is a comprehensive that the ability of enzymes to utilize other cofactors largely report providing quantitative information on the levels of a broad depended on the size of the acyl group, which is in close agree- number of histone acyl marks in HeLa and human myogenic ment with recent data demonstrating the structural incompat- cells, thus representing an important resource for future work ibility of the active sites of p300 and GCN5 with long-chain acyl aiming to understand cellular function and the dynamics of acyl- 32,33 donors . This observation was further confirmed by in vitro PTMs in the complex mammalian epigenetic mechanisms. HAT assays performed at equal concentrations of acetyl-CoA and In general, we osberved a strong correlation between a competing cofactor (Fig. 3). The data showed a similar trend in histone acylations and their corresponding metabolic substrates. which the preference for a cofactor different from acetyl-CoA was One exception was crotonylation, as the observed increase in inversely proportional to the molecular weight of the competing lysine crotonylation in myotubes was not accompanied by a donor, except for crotonyl-CoA, which, unlike the other acyl statistically significant increase in the levels of crotonyl-CoA groups, possesses an unsaturated moiety that seems to render its (Fig. 4b,d,g). This specific experiment cannot prove whether use by most HATs unfavorable. crotonylation in myotubes is regulated by enzymatic mechanisms NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications 9 | | | ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 induction with 0.5 mM IPTG and grown at 17 °C overnight. The cells were that are not strictly regulated by crotonyl-CoA levels. However, collected by centrifugation at 4000 r.p.m. at 4 °C and lysed in 25 mM HEPES pH we performed an intermediate experiment between in vivo and 7.5, 0.150 M NaCl, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 100 ug per in vitro, namely in nucleo, to test whether there are corre- mL DNase I, and 100 ug per mL lysozyme. PCAF was purified from the lysate sponding changes in the levels of histone acylations upon through Ni-NTA affinity as described above with 5 mM imidazole in 25 mM HEPES pH 7.5, 0.150 M NaCl, 1 mM DTT and eluted with 200 mM imidazole in manipulation of the concentrations of metabolites. This experi- the same. The protein was then transferred to 6–8 kDa MWCO dialysis tubing ment showed that the bulk levels of lysine acylations can be (Spectrum Labs) and dialyzed overnight into 20 mM sodium citrate pH 6.0, induced in a dose-dependent manner, resulting in either a net 0.150 M NaCl, and 1 mM DTT buffer. This was then followed by additional increase or a dynamic exchange of modifications in response to purification through SP Sepharose ion exchange and Superdex 75 (GE) gel filtration. The protein was concentrated to ~ 30 mg per mL, flash frozen, and stored increasing concentrations of acyl-CoA metabolites (Fig. 5). at −70 °C in a buffer containing 20 mM Na-citrate pH 6.0, 150 mM NaCl, 1 mM Despite the evidence that the concentrations of metabolites can DTT. regulate the global levels of lysine acylations, it is premature to Recombinant histones H3 and H4 were expressed in Rosetta BL21 [DE3] pLysS 64,65 pinpoint how this metabolic regulation is involved in complex cells and purified as monomers using standard procedures . processes including gene expression, cell differentiation and apoptosis, or in diseases such as cancer that are characterized by altered metabolic states. Such a scenario is further complicated In vitro histone acylation assay. Based on substrate specificity, histone H3 was considering that metabolite precursors of histone modifications assayed with HATs p300, CBP, PCAF and Gcn5, and histone H4 with HATs MOF, NatA and Tip60. In vitro enzymatic assays were carried by incubating 0.5 µgof exist in different pools derived from various biological pathways each HAT with 10 µg of recombinant histones H3 or H4 in the presence of 0.5 mM that are regulated in response to cellular physiological conditions. of short-chain acyl-CoAs (acetyl-, crotonyl-, malonyl-, succinyl-, propionyl-, Continued work in this area will help elucidate remaining ques- butyryl-, glutaryl- and β-hydroxybutyryl CoA;—Sigma-Aldrich) in 1X HAT buffer tions surrounding the role of acyl-PTMs, such as the following: (i) (25 mM Tris-HCl pH = 8, 25 mM KCl, 1 mM DTT, 0.1 mM AEBSF and 5 mM sodium butyrate) for 60 min at 30 °C; the final volume was 50 µL. For competition do these PTMs compete with lysine acetylation, or (ii) do they assays, reactions were carried out in the presence of 10 µM of acetyl-CoA and 10 work in concert to regulate epigenetic mechanisms? Various µM of other acyl-CoAs. Background control reactions were performed in the studies have shown that the availability of acetyl-CoA can reg- absence of HATs. Reactions were stopped by freezing and samples were dried in a ulate the abundance of lysine acetylation. Our conclusion is that SpeedVac and resuspended in 20 µL of 100 mM ammonium bicarbonate (pH 8.0). Histones were derivatized with propionic anhydride (or d -propionic anhydride this study provides compelling evidence that this metabolic reg- 10 when analyzing lysine propionylation). For this procedure, fresh propionylation ulation is not restricted to acetylation, but also extends to the reagent was prepared by mixing propionic anhydride with acetonitrile in the ratio regulation of newly identified acyl marks in chromatin. 1:3 (v/v). Propionylation reagent was added to each sample in 1:4 (v/v). Ammo- nium bicarbonate was quickly added to the solution to re-establish pH 8.0. Samples were dried down to 10–20 µL in a SpeedVac, reconstituted with 20 µL of ammo- Methods nium bicarbonate and the propionylation procedure was repeated one more time. Protein expression and purification. Human p300 and human CBP sequences Samples were then digested with trypsin at a 1:10 ratio (wt/wt) for 6 h at 37 °C. containing an N-terminal His tag, and C-terminal Strep2 and FLAG tags, were Samples were desalted by C18 stage-tip. For this procedure, a small piece of a synthesized and cloned by Genewiz (Cambridge, MA) into the pVL1393 vector for C18 solid phase extraction disk was deposited into a pipette tip to create a stage-tip. baculovirus expression. The plasmid was transfected into Sf9 cells utilizing the BD The C18 resin was flushed with 100% acetonitrile by slow centrifugation using a (Franklin Lake, NJ) BaculoGold transfection system. Then, p300 was expressed in centrifuge adaptor to hold the stage-tips in place in a 1.5 ml Eppendorf tube. Sf9 cells and purified using a GE Healthcare (Piscataway, NJ) HiTrap column. The resin was then equilibrated by flushing 80 µl of 0.1% TFA. Samples were The protein identity and purity were confirmed through protein staining with acidified to pH 4.0 or lower with acetic acid and loaded onto the disk by slow Coomassie dye . centrifugation. Samples were then washed by flushing 70–80 µL of 0.1% TFA The NatA (Naa10p/Naa15p) protein complex from Schizosaccharomyces pombe and eluted into a clean Eppendorf tube by flushing 70 µL of 75% acetonitrile and was prepared essentially as previously described . Briefly, protein expression 0.5% acetic acid by slow centrifugation. Samples were dried in a SpeedVac and vectors encoding 6XHis-tagged Naa15p and residues 1–156 of Naa10p were resuspended at 0.5 μg per μL in 0.1 M acetic acid for nano LC-MS/MS analysis . overexpressed in Rosetta (DE3) pLysS E. coli cells, and purified to homogeneity using a combination of Ni-resin affinity, 6XHis-TEV protease treatment to remove the 6XHis-tag, Ni-resin affinity to remove the 6XHis-TEV protease, HiTrap SP ion Cell culture and histone extraction. HeLa S3 mammalian cells were cultured at exchange and Superdex 200 gel filtration. The protein complex was concentrated to 37 °C and 5% CO in spinner flasks in Joklik’s modified Eagle’s medium supple- ~ 10 mg per mL in a buffer containing 25 mM HEPES (pH 7.0), 200 mM NaCl and 2 mented with 10% (v/v) newborn calf serum (HyClone), penicillin-streptomycin 1 mM TCEP and stored frozen at −70 °C until use. (1:100), and 1% (v/v) GlutaMAX (Invitrogen). Cells were harvested, washed with The HAT domain of human hMOF was prepared previously essentially as PBS, and stored at −80 °C. Human myogenic cell line, LHCN-M2 (a kind gift from described . Briefly, a protein expression vector encoding 6xHis-tagged hMOF Dr. Woodring Wright, UT Southwestern Medical Center at Dallas, Dallas, TX, HAT domain (residues 174–449) was overexpressed in Escherichia coli cells and the USA) was cultured in proliferation medium described elsewhere . In brief, protein purified to homogeneity using a combination of Ni-resin affinity and confluent cells were differentiated in 2% horse serum. Matched cultures for HiLoad Superdex 75 gel filtration. The protein was concentrated to ~ 20 mg per mL proliferation and differentiation were set up, the myoblasts harvested at 80% in a buffer containing 20 mM HEPES (pH 7.5), 0.5 M NaCl), and stored as confluence and differentiation cultures harvested on day 5–7 when the myotubes described for the NatA complex. are formed fully. Briefly, 0.05% trypsin-EDTA was used to detach cells after The HAT domain of S. cerevisiae Gcn5 (residues 99–262) was overexpressed in washing with sterile PBS. The cells were collected by centrifugation, washed again bacteria from a PRSETA/yGCN5 vector and purified similarly as previously in PBS, snap-frozen in liquid nitrogen, and stored at −80 °C until further analysis. described . The plasmid was transformed into E. coli strain BL21 (DE3) and Three biological replicates were used. Histone extraction and digestion was carried overexpressed by induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside out according to standard procedures . Briefly, nuclei were isolated by suspension (IPTG) and grown at 17 °C overnight. The cells were collected by centrifugation at of cells in 10X volume of nuclear isolation buffer (15 mM Tris-HCl pH = 7.5, 4000 r.p.m. at 4 °C and lysed in 50 mM potassium phosphate pH 7.5, 0.500 M 60 mM KCl, 15 mM NaCl, 5 mM MgCl , 1 mM CaCl and 250 mM sucrose, 0.2% NaCl, 5% glycerol, 1 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl 2 2 NP-40) with 1 mM DTT, 5 nM microcystin, 0.5 mM ASBSF and 10 mM sodium fluoride (PMSF), 100 ug per mL DNase I, and 100 μg per mL lysozyme. The butyrate at 4 °C. Nuclei were pelleted by centrifugation at 1000× g for 5 min at 4 °C supernatant liquid was passed over Ni-NTA resin, washed with 10 column volumes and washed twice with nuclear isolation buffer in the absence of NP-40. To the of lysis buffer with 5 mM imidazole but without PMSF, DNase, or lysozyme. The pelleted nuclei, 0.4 N H SO was added to a final ratio of 5:1 and incubated for 2 h 2 4 protein was then transferred to 6–8 kDa MWCO dialysis tubing (Spectrum Labs) with shaking at 4 °C. Samples were centrifuged at 3400× g for 10 min at 4 °C and and dialyzed overnight into 50 mM potassium phosphate pH 7.5, 20 mM NaCl, 5% the supernatants were collected and incubated on ice with ¼ volume of 100% TCA glycerol, and 1 mM DTT buffer. This was then followed by additional purification for 1 h. Precipitated histones were collected by centrifugation at 3400× g for 10 min through SP Sepharose ion exchange and Superdex 75 gel filtration. Purified protein at 4 °C and pellets were rinsed once with ice-cold acetone containing 0.1% HCl was concentrated to ~ 20 mg per mL in buffer containing 50 mM potassium and once with ice-cold acetone. Protein concentration was determined using a phosphate, pH 7.5, 500 mM NaCl, 5% glycerol and 1 mM DTT, and flash frozen Bradford assay. For in vivo analyses of histone acyl marks, 50 μg of histones and stored at −70 °C. were resuspended in 20 μL of 50 mM ammonium bicarbonate and subjected to The DNA sequence encoding residues 443–658 (including an N-terminal Met derivatization with propionic anhydride (or d-10 propionic anhydride when residue) of human PCAF was amplified by PCR and subcloned into the pET28-A identifying propionyl and butyryl histone marks), digested with trypsin for vector (Invitrogen) for overexpression similarly as previously described .The 6 h at 37 °C and desalted with C18 stage-tips as described above. plasmid was transformed into E. coli strain BL21 (DE3) and overexpressed by 10 NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications | | | NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 ARTICLE In nucleo acylation assay. HeLa S3 mammalian cells were harvested and washed common acyl-CoAs to reflect cellular levels. Yeast SILEC growth media was twice in ice-cold PBS. Cells were incubated on ice with hypotonic lysis buffer prepared with 200 μg biotin, 200 μg folic acid, 200 mg inositol, 40 mg niacin, 20 mg containing 10 mM Hepes-NaOH pH = 7.9, 10 mM KCl, 1.5 mM MgCl , 0.5 mM p-aminobenzoic acid, 40 mg pyridoxine-HCl, 20 mg riboflavin, 40 mg thiamine 13 15 DTT, 0.1% (v/v) NP-40, and 1X Halt protease and phosphate inhibitors for 10 min HCl, 20 g dextrose, 400 μg[ C N ]-pantothenate, 2.0 g Drop-out Mix Complete 3 1 with intermittent agitation. Nuclei were collected by centrifugation at 600 g for w/o Yeast Nitrogen Base, 1.7 g Yeast Nitrogen Base w/o AA & w/o AS, w/o 10 min at 4 °C, and washed once with a buffer containing 20 mM Hepes-NaOH Vitamins, and 5.0 g ammonium sulfate dissolved in 1 L of distilled H O. The 13 15 pH = 7.9, 50 mM KCl, 1.5 mM MgCl , 0.5 mM DTT, 0.2 mM EDTA, 20% (v/v) vitamins and [ C N ]-pantothenate were filter sterilized and the dextrose, drop- 2 3 1 glycerol and 1 X Halt protease and phosphate inhibitors. Nuclei were aliquoted at out mix, yeast nitrogen base mix, and ammonium sulfate were autoclaved. To ≈ 5× 10 cells per mL and used immediately by resuspending in 50 μL acylation confirm pantothenate auxotrophy, agar plates were prepared, with one batch reaction buffer (50 mM Tris-HCl pH = 8.0, 50 mM NaCl, 1 mM EDTA, 0.5 mM omitting pantothenate from the growth media. The plates were inoculated with DTT, 10% (w/v) sucrose, 1 X Halt protease and phosphate inhibitors), containing pan6Δ yeast, and then incubated at 37 °C for 24 h. After confirming auxotrophy, 0, 1 or 5 µM of short-chain acyl-CoAs (acetyl-, crotonyl-, malonyl-, succinyl-, 1 L of media was inoculated with pan6Δ S. cerevisiae and incubated at 30 °C while propionyl-, butyryl-, glutaryl- and β-hydroxybutyryl CoA). Reactions were agitating overnight with 500 mL in two 2 L Erlenmeyer flasks covered loosely with incubated at 30 °C for 3 h with gentle agitation. Nuclei were washed with acylation aluminum foil. After approximately 31 h from the onset of the culture, the yeast reaction buffer and the reactions were terminated by resuspending nuclei in 0.4 N cells were removed from the incubator, divided into 50 mL aliquots, and pelleted at H SO for histone extraction. Extracted histones were derivatized with d10- 500×g. The cells were resuspended in ice-cold 10% TCA extraction, respectively. 2 4 propionic anhydride and digested as described above. Samples were dried in The cells were pulse sonicated for 30 half-second pulses, on ice. Samples were spun a SpeedVac and resuspended at 0.5 μg per μL in 0.1 M acetic acid for nano at 16,000×g for 10 min at 4 °C to remove unbroken cells and debris. The final LC-MS/MS analysis. supernatant was transferred to a separate tube and stored at −80 °C until use as the SILEC internal standard. Dot blot analysis.2 μg of full-length acylated histones H3 or H4 from in vitro Acyl-CoAs metabolites extraction and quantitation. HeLa and myogenic cells reactions were spotted onto a nitrocellulose membrane. The membrane was collected from two confluent 15-cm plates (roughly 10–30 × 10 cells) were washed blocked with either 5% BSA or 5% nonfat milk and incubated with the twice with PBS, scraped into a 15 mL conical tube and pelleted by centrifugation at primary antibodies at 1:1,000 dilution (pan anti-crotonyl-lysine: PTM-501, 800× g for 3 min at 4 °C. Acyl-CoA metabolites were extracted following proce- pan anti-propionyl-lysine: PTM-201, pan anti-malonyl-lysine: PTM-901, pan anti- dures already described . In brief, pelleted cells were metabolically quenched and succinyl-lysine: PTM-401, pan anti-butyryl-lysine: PTM-301, pan anti-glytaryl- resuspended in 0.9 mL of ice-cold 10% TCA solution along with 0.1 mL of SILEC lysine: PTM-1151, all purchased from PTM-Biolabs, Hangzhou, China) according CoA internal standards, and sonicated with a probe tip sonicator for 30 s on to the manufacturers’ instructions overnight at 4 °C. The membrane was washed ice with a 1 pulse per 2 s rate. Cell lysates were centrifuged at 14,000×g for 10 min with TBS-T three times for 10 min each, incubated with secondary antibody (Goat anti-Rabbit IgG Fc, Pierce 31463) at a 1:10,000 dilution for 60 min at room at 4 °C to precipitate cellular debris and the supernatant containing the metabolites were kept on ice. Standards for calibration curves were prepared from commer- temperature and then probed with ECL Western Blot Substrate (Pierce). cially available acyl-CoAs (acetyl-, crotonyl-, malonyl-, propionyl-, butyryl-, glutaryl- and β-hydroxybutyryl CoA; —Sigma-Aldrich) as a master mix of 1 mM Nano LC-MS/MS analysis. A total of 2.5 µg of peptides were injected into a 75 μm and diluted from 1 µM to 10 nM in 10% TCA. Succinyl-CoA was run as a separate i.d × 17 cm Reprosil-Pur C -AQ (3 μm; Dr. Maisch GmbH, Germany) nano- 18 standard curve due to lower purity of the standard used, but otherwise prepared column (packed in-house) using an EASY-nLC nano HPLC (Thermo Scientific, identically. 50 μL of each standard solution was mixed with 0.85 mL of 10% TCA Odense, Denmark). The mobile phases consisted of water with 0.1% (v/v) formic and spiked with 0.1 mL of SILEC CoA internal standard. Standard calibration acid (A) and acetonitrile with 0.1% (v/v) formic acid (B). For analysis of in vitro curve samples were also subjected to sonication and solid phase extraction (SPE) in samples, peptides were eluted using a gradient of 0–30% B for 20 min followed by the same manner as the experimental samples to account for matrix effects, sample 30–98% B for 5 min and maintained over 10 minutes at 300 nL per min. For in losses and analyte stability. Samples and standards were purified using SPE car- nucleo and in vivo samples, the gradient consisted of 0–26% B over 45 min tridges (Oasis HLB 10 mg) that were conditioned with 1 mL of methanol and followed by 26–98% B over 5 min and maintained for 10 min at 300 nL per min. equilibrated with 1 mL of water utilizing a vacuum manifold. Acid-extracted The nano HPLC was coupled to a LTQ Orbitrap Elite or an Orbitrap Fusion mass supernatants were loaded onto the cartridges and washed with 1 mL of water. Acyl- spectrometer (Thermo Scientific, San Jose, California). Spray voltage was set at 2. CoA metabolites were eluted with 1 mL of 25 mM ammonium acetate in methanol 4 kV and capillary temperature was set at 275 °C. For DDA, the mass spectrometer and dried under nitrogen. Samples were resuspended in 50 µL of 5% (w/v) 5- was set to perform a full MS scan (290–1,400 m/z) in the Orbitrap with a resolution sulfosalicyilic acid and 10 µL injections were analyzed for acyl-CoAs by LC-HRMS of 60,000 (at 400 m/z), followed by a series of targeted MS/MS scans of each and LC-MS/HRMS. modified H3 and H4 peptide, followed by MS/MS scans of the top four most intense abundant ions from the first scan. All MS/MS scans were performed in the Data availability. The authors declare that all data supporting the findings of this ion trap mass analyzer (normal scan rate) using collision induced dissociation study are available within the article and its supplementary information files or (CID) with a normalized collision energy of 35 and an isolation window of 2.0 m/z. from the corresponding author upon reasonable request. All mass spectrometry Maximum injection times of 50 ms were defined for both MS and MS/MS scans. 6 4 raw files have been deposited in Chorus (https://chorusproject.org) under the AGC values were set to 1 × 10 for MS and 3 × 10 for MS/MS. MS data were project number 1376. collected in profile mode and MS/MS data were collected in centroid mode. For DIA, a full scan MS spectrum (m/z 300−1,100) was acquired in the Orbitrap with a resolution of 120,000 (at 200 m/z) and an AGC target of 5 × 10 or in the ion Received: 17 November 2016 Accepted: 14 September 2017 trap with an AGC target of 3 × 10 . MS/MS was performed with an AGC target 4 68 of 3 × 10 using an injection time limit of 30 or 60 ms . All acquisitions were performed in positive mode polarity. Data analysis was performed using our in-house software EpiProfile with a 10-ppm tolerance for extracting peak areas from raw files . The relative abundances of acyl-PTMs were calculated by dividing the intensity the modified peptide by the sum of all modified and unmodified peptides sharing the same References sequence, across all detectable charge states. For isobaric peptides (e.g., 1. Verdone, L., Caserta, M. & Di Mauro, E. Role of histone acetylation in the K STGGKAPR and KSTGGK APR), the relative abundances were estimated PTM PTM control of gene expression. Biochem. Cell Biol. Biochim. Biol. Cell 83, 344–353 by extracting the area under the curve of unique fragment ions. DDA was used to (2005). confirm peptide elution time by performing Mascot database searching (via 2. Mersfelder, E. L. & Parthun, M. R. 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Low resolution Proteomics 11, 100–107 (2012). data-independent acquisition in an ltq-orbitrap allows for simplified and fully 12 NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications | | | NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01384-9 ARTICLE supervised the study. All authors discussed the results and commented on the untargeted analysis of histone modifications. Anal. Chem. 87, 11448–11454 manuscript. (2015). 69. Basu, S. S. & Blair, I. A. SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards. Nat. Protoc. 7, Additional information 1–12 (2011). Supplementary Information accompanies this paper at doi:10.1038/s41467-017-01384-9. Competing interests: The authors declare no competing financial interests. Acknowledgements Reprints and permission information is available online at http://npg.nature.com/ We gratefully acknowledge Dr. Ben Black from the University of Pennsylvania for reprintsandpermissions/ supplying the recombinant histones and Dr. Andrew Andrews from Fox Chase Cancer Center for supplying the recombinant enzymes CBP and p300. This work was supported by NIH grants AI118891, GM110174, CA196539 and AG031862 to B.A.G.; GM101664 Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. to T.G.K.; K22ES26235, R21HD087866, R03CA211820 and a Pennsylvania Department of Health CURE grant to N.W.S.; R01 GM060293, R35 GM118090 and P01 AG031862 to R.M.; the NIH training grant fellowship T32GM008275 to D.M.M., and the NIH grant GM110174-S1 to M.C. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, Author contributions adaptation, distribution and reproduction in any medium or format, as long as you give J.S. performed most of the experiments, analyzed the data and wrote the manuscript; appropriate credit to the original author(s) and the source, provide a link to the Creative S.S. contributed to data analysis, made technical and intellectual contributions and Commons license, and indicate if changes were made. The images or other third party helped writing the manuscript. Z.-F.Y. developed the software for analysis of histone material in this article are included in the article’s Creative Commons license, unless marks and aided in the analysis of the histone data. M.C. performed the competition indicated otherwise in a credit line to the material. If material is not included in the assays and the analysis of the ionization efficiencies of the differentially acylated peptide article’s Creative Commons license and your intended use is not permitted by statutory of histone H3. N.V.B. provided the myogenic cells; D.M.M. designed and performed regulation or exceeds the permitted use, you will need to obtain permission directly from initial experiments for CoA analysis and contributed to manuscript writing. B.J.K. and the copyright holder. To view a copy of this license, visit http://creativecommons.org/ T.G.K. prepared the nucleosomal figures; G.A.B., C.E.M., and R.S.M. expressed and licenses/by/4.0/. purified the recombinant HATs; N.W.S. performed the CoA sample preparation and data analysis and contributed to editing the manuscript; R.M. provided the recombinant enzymes and was available for helpful discussions and B.A.G. conceived, designed and © The Author(s) 2017 NATURE COMMUNICATIONS 8: 1141 DOI: 10.1038/s41467-017-01384-9 www.nature.com/naturecommunications 13 | | |

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