Large-Scale Identification of Protein Crotonylation Reveals Its Role in

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Large-scale identification of protein crotonylation reveals its role in multiple cellular functions Wei Wei, Anqi Mao, Bin Tang, Qiufang Zeng, Shennan Gao, Lu Lu, Wenpeng Li, James X. Du, Jiwen Li, Jiemin Wong, and Lujian Liao J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00012 • Publication Date (Web): 24 Feb 2017 Downloaded from http://pubs.acs.org on February 26, 2017

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Journal of Proteome Research

Large-scale identification of protein crotonylation reveals its role in multiple cellular functions

Wei Wei1, Anqi Mao1, Bin Tang1, Qiufang Zeng1, Shennan Gao1, Lu Lu1, Wenpeng Li1, James X. Du1, Jiwen Li1, Jiemin Wong1, 2*, Lujian Liao1,3*

1

Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China

Normal University, Shanghai 200241, China 2

Collaborative Innovation Center for Cancer Medicine, Sun Yat-Sen University Cancer

Center, Guangzhou 510060, China. 3

Lead contact

*Corresponding authors: [email protected] and [email protected]

1 ACS Paragon Plus Environment


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Abstract Lysine crotonylation on histones is a recently identified post-translational modification that has been demonstrated to associate with active promoters and to directly stimulate transcription. Given that crotonyl-CoA is essential for the acyl transfer reaction and it is a metabolic intermediate widely localized within the cell, we postulate that lysine crotonylation on non-histone proteins could also widely exist. Using specific antibody enrichment followed by high-resolution mass spectrometry analysis, we identified hundreds of crotonylated proteins and lysine residues. Bioinformatics analysis reveals that crotonylated proteins are particularly enriched for nuclear proteins involved in RNA processing, nucleic acid metabolism, chromosome organization and gene expression. Furthermore, we demonstrate that crotonylation regulates HDAC1 activity, expels HP1α from heterochromatin, and inhibits cell cycle progression through S-phase. Our data thus indicate that lysine crotonylation could occur in a large number of proteins and could have important regulatory roles in multiple nuclei-related cellular processes.

Key words: Lysine crotonylation, mass spectrometry, proteomics, DNA replication, cell cycle

Highlights • • •

First large-scale analysis of crotonylated proteome Crotonylated proteins are enriched in nuclear proteins Crotonylation regulates HDAC1 activity and changes HP1α localization

•

Crotonylation influences cell cycle progression through S-phase


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Introduction Post-translational modifications of lysine residues on histones by acyl groups are important epigenetic marks that exert a diverse array of biological functions. Among a number of acyl modifications, acetylation on histone tails has been studied extensively, leading to the notion that histone acetylation is associated with loose chromatin state and activation of gene transcription 1. Although initially identified on histones, our understanding of lysine acetylation has expanded to large number of non-histone proteins. Several landmark studies have revealed dramatically improved number of acetylated lysine residues and acetylated proteins, and these proteins play roles in nearly all cellular processes including chromatin remodeling, cell cycle, RNA splicing, cellular organization, metabolism etc.

23

, . As

acetyl-CoA is the most important hub molecule in the intermediate metabolism, it becomes non-surprise that many enzymes in energy metabolism are acetylated and their activities and/or stability are regulated by acetylation 3. Protein crotonylation on the other hand, is a recently discovered acyl modification. Lysine crotonylation on histones marks active promoters and is enriched in male germinal cells 4, and thus possibly plays a role driving male haploid cell gene expression 5. Histone crotonylation catalyzed by the histone acetyltransferase p300/CBP has been shown to directly stimulate transcription 6. Similar to acetylation, levels of histone crotonylation can be regulated by cellular concentration of crotonyl-CoA 6. In spite of these studies, crotonylation has only been described on histone lysine residues at current stage, whether crotonylation can be expanded to non-histone proteins and what are the functional implications of non-histone protein crotonylation remains unresolved. Traditionally, studies of low abundance posttranslational modifications entail affinity enrichment at the peptide level to facilitate mass spectrometry detection, as is the case in phosphorylation,

acetylation,

liquid-chromatography

ubiquitination,

fractionation,

affinity

etc.

78

,.

enrichment

Likewise, of

we

utilized

crotonylated

peptides,

high-resolution mass spectrometry to identify crotonylated peptides and proteins in a large scale. Here we report the identification of hundreds of crotonylated lysine residues and proteins and compared their biological functions with acetylated proteins. We show that



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protein crotonylation is particularly enriched in nuclear proteins, and crotonylation of a subset of proteins influence DNA replication and cell cycle.

Experimental Section Reagents The following antibodies were used in this study: anti-pan-lysine crotonylation (α-KCr, PTM-Biolabs 501, 1:3000), anti-pan-lysine acetylation (α-KAc, PTM-Biolabs 105, 1:3000), H3 (Abcam 1791, 1:5000), anti-HA (MBL, M180-3, 1:2,000), anti-Flag (Sigma, F3165, 1:10,000), anti-actin (Sigma, A1978, 1:10,000), anti-myc (Santa Cruz Biotechnology, sc-40, 1:2000), anti-GAPDH(Abmart, M20006, 1:10000), anti-HDAC1(ABclonal, A0238, 1:5000), anti-HP1α(abcam, ab77256, 1:3000), anti-H3S10P(Millipore, 17-685, 1:5000).The plasmids Flag-CUL4A, Flag-CUL4B, HA-HADAC1, MYC-P300, Flag-CBX3 (HP1γ), Flag-CBX5 (HP1α) and Flag-MTA2 were constructed and verified by DNA sequencing. C646, SGC-CBP30 and TSA was from Selleck, NAM was from Beyotime, Sodium crotonate (NaCr) was from Sigma. Cell Culture and protein extraction HeLa cells were cultured in DMEM (GIBCO) supplemented with 10% FBS (GEMINI) at 37ºC in 5% CO2. HeLa cells were treated with 20mM sodium crotonate (Sigma, MP207938) for 8 hours and then harvested. HeLa cells were harvested and re-suspended in 250µl urea lysis buffer (20 mM HEPES, pH 8.0, 6 M urea, 2 M thiourea, protease inhibitors, 1 mM sodium orthovanadate, 2.5 mM Sodium fluoride, 1 mM beta-glycerophosphate, 10 µM TSA, 10 mM nicotinamide and 50 mM Sodium butyrate), then homogenized using a bead-beating homogenizer (Xinzhi, Ningbo). The samples were sonicated at 20% power in a tip sonicator (XinZhi, Ningbo) and incubated at 37°C for 30 minutes. The supernatant were collected after 15-minute centrifugation at 20,000 xg at 4°C and the protein concentration were determined by Bradford assay (BIORAD). In-solution trypsin digestion was performed overnight, followed by centrifuging at 12,000 rpm for 15 minutes at room temperature. Enrichment of crotonylated peptides


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Basic reversed-phase (bRP) liquid chromatography separation of 5 mg HeLa cell digest was performed on a Zorbax 300 Extend-C18 column (9.4 x 250 mm, 300 Å, 5 µm, Agilent) using a 6460 HPLC system (Agilent). A 60-min gradient containing an initial increase from 100% solvent A (2% acetonitrile, 5 mM ammonium formate, pH 10) to 8% Solvent B (90% acetonitrile, 5 mM ammonium formate, pH 10) in 5 minutes, followed by a 38-minute linear phase with solvent B increased form 8% to 27% and ramp phases where the Solvent B amount was increased from 31% to 39% in 10 minutes and finally to 60% in 7 minutes. A total of 60 2ml fractions were collected every minute at a flow rate of 2 ml/min. Each fraction was combined into 6 fractions using a concatenated pooling strategy. Pooled samples were dried using a SpeedVac concentrator. For antibody enrichment of crotonylated peptides, either unfractionated cell lysates or the pooled peptide fractions after bRP separation were re-dissolved in binding buffer (50 mM MOPS, pH 7.2, 10 mM sodium phosphate, 50 mM NaCl). The samples were incubated at 4°C for 12 hours after adding α-KCr antibody and protein-A beads. Beads were collected after centrifugation at 500 xg and washed with 600 µl binding buffer three times, followed by three times washing with 600 µl LC-MS grade water. The peptides were eluted with 60 µl Elusion buffer (60% ACN, 0.15% TFA) twice and dried using SpeedVac. The dried peptides were desalted using C18 ZipTip. HPLC-MS/MS and data analysis The enriched crotonylated peptides were dissolved in 10 µl LC-MS buffer A (0.1% formic acid, 2% ACN in water). The peptide samples were analyzed using an in-house packed analytical column (15 cm length with 75 µm ID, packed with C18 resin, 3 µm particle size, 90 Å pore size, Phenomenex). Peptides were eluted with a 120-minute gradient from 12% to 32% buffer B (98% ACN, 0.1% formic acid) at a flow rate of 250 nl/min. One full scan MS from 400 to 1600 m/z followed by 12 MS/MS scan were continuously acquired in a Q-Exactive (Thermo) mass spectrometer using a nano-electrospray source. The resolution for MS was set to 70000 and for MS/MS was set to 12500. The nano-electrospray source conditions were: spray voltage, 1.8 KV; no sheath and auxiliary gas flow; heated capillary temperature, 275°C. For HCD, the isolation window was set to 2 m/z and the normalized collision energy of 27% was applied. 5 ACS Paragon Plus Environment


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The Proteome Discoverer 1.4 (Thermoscientific) and MaxQuant (version 1.4.1.2) software was used to analyze the MS/MS raw data. For the database searching parameters, the precursor mass tolerance was set to 15 ppm. Trypsin/P was set as the protease, accounting for in-source fragmentation of lysine or arginine residues followed by proline. Two missed cleavages were allowed. All data were searching against with the UniProt Human database (88817 sequences) including variable modification of crotonylation (lysine, C4H4O +68.0262) and fixed modification of cysteine residues by carbamidomethylation. A search for butyrylation was also performed using Proteome Discoverer with the same parameters except that a variable modification of butyrylation (lysine, C4H6O +70.0413) was applied. Bioinformatics analysis Functional annotation was performed with the David bioinformatics resource v6.7 (https://david.ncifcrf.gov/). The functional annotation term categories of GOTERM_BP_FAT, GOTERM_CC_FAT, and GOTERM_MF_FAT are analyzed. The protein counts for each term was sorted and top 10 terms were taken. Then, the percentage for these 10 terms was calculated for the pie chart. For

crotonylation

site

motif

analysis,

the

Motif

X

algorithm

(http://motif-x.med.harvard.edu) was applied to confidently localized crotonylated peptides. The localization score for each site was greater than or equal to 0.67 in order to be considered as confidently localized. The parameters used for the algorithm are for the most part default values, with the sequence width of 13 characters, occurrences of at least 20, and significance value of 0.00001. The IPI human proteome database was used as the background. Western blot analysis Western blotting was performed according to standard procedures. Cell pellets were washed with ice-cold PBS twice, lysed with a lysis buffer (10 mM HEPES-NaOH, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, and 0.5 mM beta-mercaptoethanol supplemented with protease inhibitor mixture and phosphatase inhibitors) by incubating on ice for 20 min. Then a final concentration of 1% NP-40 were added, vortexed and placed on ice for 2 min. The lysates were centrifuged at 16000 g for 15 min. Transfection and immunofluorescence staining


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Transient transfections of HeLa cells were carried out using Lipofectamine 2000 (Invitrogen) essentially according to the manufacturer’s instruction. For immunofluorescence staining, HeLa cells were washed with 1xPBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4) prior to fixation in 4% paraformaldehyde at room temperature for 30 min, incubated with 1% Triton X-100 on ice for 15 min, blocked with 5% BSA in 37 °C incubator for 60 min and incubated with mouse anti-Flag antibody for 2 hours. The coverslips were washed 3 times with PBST, followed by incubation with Texas-Green-conjugated secondary antibody against mouse. Images were acquired with an Olympus microscope system. Immunoprecipitation To harvest cells under non-denaturing conditions, the cells were washed with ice-cold PBS and lysed with 0.25 ml ice-cold lysis buffer (50 mM Tris-HCL, pH 8.0, 1% Triton X-100; 150 mM NaCl, 5 mM EDTA, protease inhibitor cocktail) by incubating on ice for 5 minutes. The lysates were scraped off and transfer to microcentrifuge tubes and further lysed on ice for 30 minutes. The lysates were centrifuged for 10 minutes at 14,000 g at 4°C, and the supernatant were transferred to a new tube. For each 150 ul of cell lysate, M2, MYC and HA antibody-conjugated beads were added and equilibrated with 200 ul binding buffer (50 mM Tris-HCl, pH 7.5, 0.1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 10% glycerol, protease inhibitor cocktail). The tubes were gently rocked overnight at 4°C. The beads were washed three times with 500 µl of Washing buffer (50 mM Tris-HCl,pH 7.5, 0.1% Triton X-100, 150 mM NaCl, 5 mM EDTA, protease inhibitor cocktail). The pellets were resuspended with 20 µl 3X SDS sample buffer, centrifuged for 30 seconds and then analyzed by western blotting. In vitro deacetylation For in vitro deacetylation assays, HA-tagged HDAC1 proteins were expressed and purified from HeLa S3 cells following a standard protocol. In vitro histone deacetylation assays were carried out at 37°C for 12 hours in reaction buffer (50 mM Tris pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM DTT,).

Crotonylated and acetylated core histones were

prepared from NaCr and TSA+NAM treated HeLa cells. The products were then subjected to western blot analysis with anti-pan-lysine acetylation antibody. Flow cytometry analysis 7 ACS Paragon Plus Environment


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Cells were washed twice with cold PBS and fixed with ice-cold 70% ethanol overnight at 4°C, Fixed cells were centrifuged at 200 g, 10 min at 4°C, washed three times again with cold PBS. Cells were then suspended in 500 µl PI/Triton X-100 staining solution（0. 1 % (v/v) Triton X-100，20mg/ml DNAse-free RNAse A and 500 µg/ml PI），incubated at 37°C for 15 minutes, and then transferred into tubes on ice for data acquisition on a flow cytometer (FACSAria， BD).

Results Protein crotonylation can be induced by sodium crotonate (NaCr) through enzymatic reaction. In order to detect crotonylated peptides and further enrich these peptides for mass spectrometry analysis, we made use of a previously described commercial α-KCr antibody raised against crotonylated peptides 4. A dot blot result using this antibody showed that it specifically recognized crotonylated peptides but not acetylated or propionylated peptides, while an α-KAc antibody only recognized acetylated peptides (Figure 1A). Using this antibody, we detected significantly increased histone crotonylation in cells treated with NaCr in six different cell types (Figure 1B), consistent with the previous report that treatment with NaCr enhances histone crotonylation most likely through conversion of NaCr to crotonyl-coA 6

. Importantly, using the same α-KCr antibody we observed that NaCr treatment also resulted

in significantly increased crotonylation of numerous non-histone proteins (Figure 1B). These results thereby demonstrated that lysine crotonylation also broadly occurs in non-histone proteins. To differentiate if NaCr-induced protein crotonylation is enzymatically catalyzed or simply chemical reaction due to increased concentration of crotonyl-coA, we compared HeLa cells over expressing control vector with myc-p300 vector. The acetyltransferase p300 is also known to catalyze lysine crotonylation 6, and as expected, over expression of p300 resulted in dramatically increased crotonylation as well as acetylation in both non-histone proteins and histones (Figure 1C). Furthermore, NaCr can specifically enhance protein crotonylation but not acetylation in untransfected HeLa cells, and this induction can be nearly completely eliminated by C646 and SGC-CBP30, two selective p300/CBP inhibitors (Figure 1D). Thus, we conclude that protein crotonylation induced by NaCr is enzymatically catalyzed.


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Large-scale analysis of lysine crotonylated proteins. We either analyzed HeLa cells without any treatment to identify the endogenously crotonylated proteins, or NaCr-treated HeLa cells with enhanced protein crotonylation. The protein extracts were prepared and digested with trypsin, and the resulting peptides were either directly enriched for crotonylated peptides

by

the

α-KCr

antibody

or

fractionated

through

basic

reversed-phase

high-performance liquid chromatography (HPLC) and then followed by antibody affinity enrichment from each fraction. The eluted peptides were analyzed by LC-MS/MS using high-resolution mass spectrometry (Figure 2A). From two biological replicate experiments we identified 74 endogenously crotonylated peptides representing 70 proteins with 5 overlapped peptides. And from the NaCr-treated biological replicates, we identified a total of 453 crotonylated proteins deriving from 1185 crotonylated peptides (Figure 2B, Table S1 and S2). Five of the 70 endogenously crotonylated proteins were also found in the NaCr-treated crotonylated proteome (Table S1). Remarkably, among the identified crotonylated proteins, nuclear proteins (about 62.3% of crotonylated proteins) are the most significantly enriched gene ontology terms covering a variety of molecular functions and biological processes intimately involved in DNA and RNA metabolism and cell cycle (Figure 1C), as well as covering a number of nuclear structures including chromatin, chromosome, pericentric heterochromatin, and nucleosome (Figure S1). Additional 9.4% of crotonylated proteins are categorized as having both nuclear and cytoplasmic localization. Other cellular components including plasma membrane, cytoplasm, golgi and unclassified proteins altogether cover only 28.3% of all crotonylated proteins. This is in sharp contrast with acetylated proteins, which covered a wide range of cellular components including nucleus, cytoplasm and mitochondrion 2. Because butyryl group is structurally very similar to crotonyl group 9, it is possible that the anti-KCr antibody would also recognize and capture butyrylated lysine (KBu) peptides. We therefore performed another round of database searches for captured KBu peptides. We identified 37 and 50 KBu peptideds from two replicates of the untreated HeLa cells, and 10 and 88 KBu peptides from two replicates of NaCr-treated HeLa cells (Figure S2 and Table S3). The low number of KBu peptides from replicate 1 of NaCr-treated cell samples was likely due to a lack of fractionation during the enrichment process. The KBu results from the 9 ACS Paragon Plus Environment


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four datasets nevertheless indicate that the anti-KCr antibody was able to capture KBu peptides to some degree, and that NaCr treatment selectively enhances lysine crotonylation. Crotonylated proteins are enriched in nuclear function. Using a binomial probability model to perform localization analysis of the modification sites 10, we confidently assigned 558 unique crotonylation sites. We compared our crotonylation dataset with datasets from two largest-scale protein acetylation studies

2, 11

. In total, 202 of the 558 crotonylation sites

overlap with the acetylation sites (Figure 3A), and 356 sites are not known for acetylation. At the protein level, 141 of the 453 crotonylated proteins are also known for acetylation, and 312 proteins are not known for acetylation (Figure 3B). Gene ontology analysis of these 312 crotonylated proteins reveals that RNA processing, nucleic acid metabolic process and chromosome organization are among the most enriched biological processes. The next level of highly enriched processes includes gene expression, DNA conformation change and packaging, chromatin organization as well as cell cycle (Figure 3C). These top-ranked cellular processes are exclusively nuclear events, among which there are large numbers of overlaps with a subset of the acetylated proteome that are also nuclear proteins 2. Since crotonylated proteins cover a wide range of nuclear functions, we constructed a protein-protein interaction map for proteins in eight of the most enriched gene ontology terms in biological processes (Figures 3D and S3). These proteins in each process are highly connected and form subgroups of interactions. For example, protein complexes regulating chromatin organization and cell cycle are closely interconnected. Proteins shared in these two processes include an ubiquitin ligase RNF2 and a conjugating enzyme UBE2E1, NCOR1 and RBBP4 that are components of histone deacetylase complexes 12, and SET that is known to inhibit histone acetylation

13

(Figure 3D). Among all crotonylated proteins in each cellular

process, known acetylated proteins are labeled green. Overall, these analyses indicate that acetylation and crotonylation may affect to large extent distinct sets of proteins. Analysis of crotonylation site motif. In order to gain a glimpse of sequence commonalities surrounding the crotonylation sites and to compare that with acetylation sites, we extracted sequence motifs from all 558 unambiguously localized crotonylated lysine residues (sites, Table S4, localization score >0.67), of which 356 sites are uniquely crotonylated and 202 sites share with acetylation. The analysis of all sites results in two motifs (KxxK) and (PxK) 10 ACS Paragon Plus Environment

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that show two distinct amino acid properties surrounding the modified lysine, where “x” represent any amino acid (Figure 4A). Crotonylation-only sites has a strong sequence preference for alanine at +3 or -6 position (Figure 4B) while common motifs shared by the two modifications are (KxxK) and (AxK) (Figure 4C). This is in contrast to acetylation site motifs in which bulky amino acids are enriched around acetylation sites 2, and in nuclear proteins acetylated lysine has a stronger preference for glycine and proline in its -1 and +1 positions

14

. Since it is currently known that the acetyl transferase p300/CBP is also the

primary enzyme for protein crotonylation, we downloaded currently reported 112 acetylated p300/CBP

substrates

and

their

associated

338

lysine

acetylation

sites

(http://bioinfo.bjmu.edu.cn/huac/). To our surprise, only 14 of the proteins and 17 of the sites are shared with our crotonylated proteins and sites. After extracting their sequence motifs, we found that these sites have a strong preference to basic residues surrounding the central lysine (Figure 4D), similar to one of the Cr-Ac shared sites shown in Figure 3c. These differences as well as similarities in sequence preference could in part explain why many of the crotonylated proteins are not known for acetylation, despite significant effort and technical advance in identifying acetylated proteins. Thus, although p300/CBP catalyze the acyl transfer reaction for both acetyl and crotonyl groups, sequence motifs in substrates may dictate whether they can be modified by acetylation or crotonylation or both. Biochemical validation of protein crotonylation. In order to validate our findings of protein crotonylation, we randomly selected five proteins from our list of crotonylated proteins, and expressed them exogenously in HeLa cells. The cells were treated with or without NaCr and the proteins of interest were immunoprecipitated and analyzed for crotonylation by western blotting analysis. We found that NaCr did not influence the expression of these proteins (Figure 4A). We effectively detected crotonylated CBX3, CBX5, MTA2, Cul4B and HDAC1, upon NaCr treatment (Figure 4B). As a negative control, we failed to detect crotonylation for Cul4A (Figure 4B), a protein that was not present in our list of crotonylated proteins. Note that crotonylation for HDAC1 could be detected even in the absence of NaCr treatment, suggesting crotonylation of some proteins occurs even under the regular cell culture condition. Confidently validated ms/ms spectra for these five crotonylated proteins are listed in Figures S5-S7. We thus conclude that most, if not all, of the crotonylated proteins identified in our 11 ACS Paragon Plus Environment


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study are likely truly crotonylated. Crotonylation of HDAC1 reduces its deacetylase activity. As acetylation of HDAC1 could reduce its deacetylation activity 15, we wondered whether crotonylation could also regulate HDAC1 deacetylase activity. We expressed HA-tagged HDAC1 in HeLa cells and found that the expression level was not affected by NaCr or a combined treatment with HDAC inhibitors trichostatin A (TSA) and nicotinamide (NAM) (Figure 6A, left panel). Immunoprecipitation of the exogenous HDAC1 followed by α-KCr or α-KAc detection showed that crotonylation of HDAC1 can be enhanced by NaCr, and that acetylation can be enhanced by TSA and NAM (Figure 6A, right panel). An in vitro deacetylation assay using immunoprecipitated HDAC1 showed that comparing to unmodified HDAC1, crotonylated HDAC1 reduced its deacetylase activity, to an extent similar to acetylated HDAC1, on its histone substrates (Figure 6B). Therefore, crotonylation of HDAC1 resulted in reduced deacylation activity. Crotonylation of HP1α alters its heterochromatin localization. HP1α (CBX5) is a member of the heterochromatin family and enriched in heterochromatin via binding with methylated histones

16

. Using antibody against endogenous HP1α, we found that in HeLa cells it

primarily localized at nucleus and accumulated at bright dot-like heterochromatin regions as demonstrated by co-staining with DAPI (Figure 6C). Treatment with either TSA or NaCr for 72 hours altered HP1α localization from the heterochromatin to the nucleoplasm (Figure 6C), while the morphology of the nucleus including the heterochromatin remained intact as demonstrated by DAPI staining. Co-immunoprecipitation experiment showed that crotonylated HP1α dramatically reduced its in vitro binding of tri-methylated H3 at K9 position, offering a possible explanation for the altered heterochromatin localization (Figure 6D). Protein crotonylation affects cell cycle. Since our protein-protein interaction network analysis of the crotonylated non-histone proteins identified enrichment in cell cycle, we further asked whether protein crotonylation could affect cell cycle. Treatment with NaCr not only strongly enhanced H3 crotonylation, but also increased H3 phosphorylation at Ser10 in a dose-dependent manner, which marks an increase of G2/M phase cells (Figure 7A). The increase in H3 phosphorylation at Ser10 was also confirmed by immunofluorescence staining with an antibody against phosphorylated H3 at Ser10 (Figure 7B). Flow cytometry analysis of 12 ACS Paragon Plus Environment

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cells treated with NaCr showed a reduction of cells in S phase and an increase of cells in G2 phase (Figures 7C and S4), and the increase in G2 cells was correlated with reduction in total cell numbers after NaCr treatment in a dose-dependent manner (Figure 7D). Thus, we have uncovered a novel function of nuclear protein crotonylation in regulation of cell cycle.

Discussion To our knowledge, our study is the first attempt to describe the crotonylated proteome. Even though it is well established that crotonylation of histones can happen in vivo 4 and in vitro and can be catalyzed by p300/CBP utilizing crotonyl-coA as donor 6, there has been no report demonstrating the crotonylation of non-histone proteins. In this study we treated HeLa cells without and with NaCr and identified crotonylated peptides by antibody affinity enrichment followed by high-resolution mass spectrometry analysis. This approach leads to the identification of 453 crotonylated proteins, 1185 crotonylated peptides and 558 high-confident crotonylation sites. Comparing to acetylation, our findings of crotonylated proteome fall short of scale. We note that the overlapped endogenous crotonylation events between the two biological replicate experiments were poor, and there were also poor overlaps between the endogenous and NaCr-induced crotonylation events, suggesting that we have only identified a subset of crotonylated proteins. One likely reason might be limited affinity of the anti-crotonylated antibody used, which makes enrichment of KCr peptides a somewhat stochastic process. In addition, the poor overlap may reflect a highly dynamic nature of KCr modification, dependent upon the metabolic states of the cell and the availability of crotonyl-CoA. Likewise, when we searched the datasets for lysine butyrylation, we also observed poor overlapped peptides between replicate experiments. Because anti-KCr antibody likely enriched KBu peptides as well, affinity of the antibody from batch to batch could contribute to the poor overlaps. More sensitive methods such as alternative antibodies or chemical biology approach targeting the double bond within the crotonyl group might help improve the depth of the coverage of crotonylated peptides, and potentially solve the overlap issue. Comparative analysis between crotonylation and acetylation revealed that these two acyl modifications share to some extent a common set of proteins and sites (Figures 3A and 3B).



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Given the almost exhaustive analysis and coverage of acetylated proteome and acetylation sites, it is rather surprising that the majority of crotonylated proteins and sites do not overlap with acetylation (Figure 3A and 3B, Figure 4). One reason could be that comparing to acetyl group, a bulkier crotonyl group requires a larger pocket at the vicinity of the lysine residues. Thus many lysine residues that can be acetylated are not accessible to crotonyl group. Extended analysis comparing other even bulkier acyl modifications such as succinylation or glutarylation could potentially clarify this. It is known that the acetyltransferase p300/CBP also catalyze protein crotonylation 6, then precisely how are the choices between acetylation and crotonylation of the substrates been made by essentially the same transferase? Deeper analyses of crontonylated proteome and mechanistic studies have the potential to elucidate these questions. Modifications of distinct acyl groups may share similar functional consequences but may also differentially affect protein structure and function and results in different functional regulations. We have shown that increasing protein crotonylation can have profound effect on cell cycle. Furthermore, acetylation at multiple lysine residues of the tumor suppressor p53 by p300/CBP promotes its transcription activity and expression of proapoptotic genes

17

;

while at the same time acetylation excludes p53 binding to its E3 ubiquitin ligase MDM2, thus promoting p53 stability 18. Conversely, MDM2 could recruit HDAC1 to deacetylate p53 and decrease its stability 19. We have evidence that p53 could also be crotonylated but this does not decrease p53 stability (data not shown). It will be interesting to reveal whether crotonylation of p53 share similar lysine residues with acetylation, whether crotonylation of p53 influence binding with MDM2. Our data reveal that crotonylation of different proteins could result in a number of different consequences, depending on the functional nature of the protein. Similarly to acetylation, crotonylation of HDAC1 clearly reduced its deacetylation activity to histone substrates. These data add another layer to the complexity that post-translational modification can have on key enzymes. On the other hand, crotonylation of the heterochromatin protein HP1α results in its redistribution in the nucleus. It is possible that crotonylation of HP1α reduced binding with methylated H3K9, which is highly enriched in the heterochromatin. It is known that acetylation of HP1α also reduce its binding with methylated H3K9 14 ACS Paragon Plus Environment

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, these

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results suggest that crotonylation of some proteins share similar functional role as acetylation. Protein-protein interaction network analyses of the crotonylated proteins identify an enrichment of protein network in cell cycle, including CDK7, MCM3 (Figure 3D). In metazoans, MCM family members 2-7 form a hexameric complex in DNA replication fork, dictating initiation of DNA replication 20,21. We have evidence showing that NaCr treatment results in a dramatic reduction of MCM proteins in the chromatin (data not shown), suggesting that DNA replication could be influenced by protein crotonylation. Together these could lead to an inhibition on DNA replication and thus influence cell cycle. Consistently, flow cytometry analysis shows that there are reduced cell populations in S phase and an accumulation of G2 cells. Although we could not completely exclude the possibility of cell toxicity caused by high concentration of NaCr, a plausible consequence for slowed cell cycle progression is the reduced cell division, as measured by reduced cell number (Figure 7D). Future studies are needed to investigate molecular details of how crotonylation of MCM3 and possible other MCM proteins results in its chromatin exclusion and the role of crotonylation in other biological processes such as RNA processing and nucleic acid metabolism. Nevertheless, our data show that multiple protein functions and cellular processes are affected by crotonylation. Data Availability. The mass spectrometry data has been deposited to ProteomeXchange with the accession number PXD004307. Author Contributions W.W., J.W. and L.L. conceived the project; W.W., A.M., W.L. and L.L. performed the experiments. W.W., Q.Z., B.T. and L.L. analyzed data; J.W. and L.L. wrote the paper. Acknowledgements The authors would like to acknowledge financial support from the Ministry of Science and Technology of China (2015CB910402), the National Natural Science Foundation of China (91419303) and The Science and Technology Commission of Shanghai Municipality

(14XD1401700,

11DZ2260300);

Shanghai

Pujiang

Talent

(14PJ1402900), and ECNU National “985” Project grant. SUPPORTING INFORMATION: The following files are available free of charge at ACS website http://pubs.acs.org:


Project


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201702_cover-sheet-for-SI. Introduction to supporting data. 201702_crotonylation-proteome-SFigures. Supporting figures S1-S7. TableS1_noNacr_2sets-cr-peptides.xlsx. Table S1, list of endogenous KCr peptides. TableS2_Nacr_2sets-cr-peptides.xlsx. Table S2, list of KCr peptides from NaCr treated HeLa cells. TableS3_KBu_peptides.xlsx. Table S3, list of KBu peptides from both untreated and NaCr treated HeLa cells. TableS4_Cr_Ac_site_motif.xlsx. Table S4, Peptide sequences frm confidently localized crotonylation sites and comparison with acetylation.

References 1.

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chromatin compaction by Polycomb repressive complexes. Cold Spring Harb Symp Quant Biol 2010, 75, 71-8. 2.

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Lysine propionylation and butyrylation are novel post-translational modifications in histones. Molecular & cellular proteomics : MCP 2007, 6 (5), 812-9. 10. Olsen, J. V.; Blagoev, B.; Gnad, F.; Macek, B.; Kumar, C.; Mortensen, P.; Mann, M., Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 2006, 127 (3), 635-48. 11. Schölz, C.; Weinert, B. T.; Wagner, S. A.; Beli, P.; Miyake, Y.; Qi, J.; Jensen, L. J.; Streicher, W.; McCarthy, A. R.; Westwood, N. J., Acetylation site specificities of lysine deacetylase inhibitors in human cells. Nature biotechnology 2015, 33 (4), 415-423. 12. (a) Jayne, S.; Zwartjes, C. G.; van Schaik, F. M.; Timmers, H. T., Involvement of the SMRT/NCoR-HDAC3 complex in transcriptional repression by the CNOT2 subunit of the human Ccr4-Not complex. The Biochemical journal 2006, 398 (3), 461-7; (b) Wang, Q.; Zhang, Y.; Yang, C.; Xiong, H.; Lin, Y.; Yao, J.; Li, H.; Xie, L.; Zhao, W.; Yao, Y., Acetylation of metabolic enzymes coordinates carbon source


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utilization and metabolic flux. Science 2010, 327 (5968), 1004-1007. 13. Seo, S. B.; McNamara, P.; Heo, S.; Turner, A.; Lane, W. S.; Chakravarti, D., Regulation of histone acetylation and transcription by INHAT, a human cellular complex containing the set oncoprotein. Cell 2001, 104 (1), 119-30. 14. Lundby, A.; Lage, K.; Weinert, B. T.; Bekker-Jensen, D. B.; Secher, A.; Skovgaard, T.; Kelstrup, C. D.; Dmytriyev, A.; Choudhary, C.; Lundby, C.; Olsen, J. V., Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns. Cell reports 2012, 2 (2), 419-31. 15. Qiu, Y.; Zhao, Y.; Becker, M.; John, S.; Parekh, B. S.; Huang, S.; Hendarwanto, A.; Martinez, E. D.; Chen, Y.; Lu, H.; Adkins, N. L.; Stavreva, D. A.; Wiench, M.; Georgel, P. T.; Schiltz, R. L.; Hager, G. L., HDAC1 acetylation is linked to progressive modulation of steroid receptor-induced gene transcription. Molecular cell 2006, 22 (5), 669-79. 16. Verschure, P. J.; van der Kraan, I.; de Leeuw, W.; van der Vlag, J.; Carpenter, A. E.; Belmont, A. S.; van Driel, R., In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation. Molecular and cellular biology 2005, 25 (11), 4552-64. 17. Brooks, C. L.; Gu, W., The impact of acetylation and deacetylation on the p53 pathway. Protein & cell 2011, 2 (6), 456-462. 18. Reed, S. M.; Hagen, J.; Tompkins, V. S.; Thies, K.; Quelle, F. W.; Quelle, D. E., Nuclear interactor of ARF and Mdm2 regulates multiple pathways to activate p53. Cell cycle 2014, 13 (8), 1288-98. 19. Ito, A.; Kawaguchi, Y.; Lai, C. H.; Kovacs, J. J.; Higashimoto, Y.; Appella, E.; Yao, T. P., MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation. The EMBO journal 2002, 21 (22), 6236-45. 20. Kinoshita, Y.; Johnson, E. M., Site-specific loading of an MCM protein complex in a DNA replication initiation zone upstream of the c-MYC gene in the HeLa cell cycle. The Journal of biological chemistry 2004, 279 (34), 35879-89. 21. Lei, M., The MCM complex: its role in DNA replication and implications for cancer therapy. Current cancer drug targets 2005, 5 (5), 365-80.



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Figure Legends Figure 1. Protein crotonylation can be induced by NaCr through enzymatic reaction. (A) Specificity of the pan-lysine crotonylation antibody used in this study as shown by peptide dot-blot assay. (B) Anti-crotonyl lysine antibody recognizes both crotonylated histone and crotonylated non-histone proteins induced by NaCr. (C) Immuno-blot analysis of protein crotonylation and acetylation in HeLa cells expressing myc-tagged p300. (D) Immuno-blot analysis of protein crotonylation after NaCr treatment in the absence or presence of p300/CBP inhibitors. Figure 2. Strategies for large-scale analysis of in vivo crotonylated proteins. (A) Strategies used in this study to enrich crotonylated peptides. (B) Overall identified crotonylated proteins, peptides and crotonylation sites. (C) Gene ontology analysis of the crotonylated proteins in the category of biological process (BP), molecular function (MF) and cellular component (CC). Figure 3. Gene ontology reveals that protein crotonylation appears dominantly in nuclear proteins. (A) Comparison between acetylation and crotonylation sites. (B) Comparison between acetylated and crotonylated proteins. (C) Enriched biological processes of uniquely crotonylated proteins that do not overlap with acetylation. (D) Crotonylated proteins form intricately interacted network that participate in chromatin organization and cell cycle. The brown dots indicate crotonylated proteins while the green dots are the proteins in the interaction database. Figure 4. Analysis of crotonylation site motif and comparison with acetylation sites. (A) All 558 confidently localized crotonylation sites display two sequence motifs. (B) Unique crotonylation sites display a distinct motif. (C) Shared modification sites between acetylation and crotonylation display a common motif. (D) Sequence motif of acetylation sites that are catalyzed by p300/CBP. Figure 5. Validation of protein crotonylation. (A) NaCr treatment had not effect on the level of exogenously expressed CBX3, CBX5, MAA2, Cul4A, Cul4B and HDAC1. (B) Except for Cul4A, all other exogenously expressed proteins show detectable and increased crotonylation after NaCr treatment. Figure 6. Functional effect of HDAC1 and HP1α crotonylation. (A) Expression of


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exogenous HDAC1 and its crotonylation and acetylation status in the presence of NaCr or HDAC inhibitors. (B) HDAC1 activity toward histone deacetylation after HDAC1 is crotonylated or acetylated. (C) Subcellular localization of HP1α after NaCr treatment. Scale bar: 5 µm. (D) Expression of HP1α in HeLa cells treated with or without NaCr and its binding affinity with H3K9me3 in vitro. Figure 7. Protein crotonylation alters cell cycle. (A) Phosphorylation of H3 at serine 10 is enhanced after NaCr treatment in a dose-dependent manner. (B) Immunofluorescence staining of phosphorylated H3 at serine 10 after NaCr treatment. Scale bar: 200 µm. (C) NaCr treatment alters cell cycle in a dose-dependent manner. (D) NaCr treatment inhibited cell proliferation in a dose-dependent manner.


Figure 1


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1 2 3 HeLa cell 4 5 treated with NaCr Replicate 1 6 Replicate 2 7 5 35 34 Digest with 8 K trypsin 9 Cr 10 11 Endogenous crotonylation bRP 12 13 14 15 Replicate 2 16 Ab enrich 874 Crotonylated 17 crotonylated 18 Proteins K K K K K K Cr Cr Cr Cr Cr Cr 19 peptides 303 453 20 21 Replicate 1 22 LC-MS/MS analysis 8 23 24 NaCr-induced crotonylation 25 26 27 28 nucleic acid metabolic process 29 RNA splicing DNA methylation on cytosine 30 chromatin silencing at rDNA 31 DNA methylation or demethylation 32 regulation of gene expression, epigenetic chromatin remodeling 33 BP DNA 34 replication−dependent nucleosome assembly cell cycle MF 35 macromolecule methylation 36 centromere complex assembly CC histone exchange 37 poly(A) RNA binding 38 heterocyclic compound binding 39 histone binding 40 histone demethylase activity (H4−K20 specific) DNA binding 41 nucleoplasm 42 nuclear lumen 43 intracellular organelle lumen nucleus 44 chromatin 45 intracellular organelle part 46 chromosome 47 protein−DNA complex cytosol 48 cytoplasm 49 50 51 0 20 40 60 80 52 −log10(p−value) 53 54 55 ACS Paragon Plus Environment 56 57 58

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Figure 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

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1 2 3 4 5 6 7 TSA+NAM 8 NaCr 9 10 α-HA 11 12α-actin 13 14 15 16 17 18 19 20 21 22 23 Ctrl 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 TSA 45 NaCr 46 α-Flag 47 48 49 α-GAPDH 50 51 52 53 54 55 56 57 58


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 HeLa cell 15 treated with NaCr 16 Ctrl 48H 17 Digest with 18 19 K trypsin 20 Cr 21 22 bRP 23 24 25 26 Ab enrich 27 28 K K K K K K crotonylated 29 Cr Cr Cr Cr Cr Cr peptides 30 31 32 33 LC-MS/MS analysis 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 ACS Paragon Plus Environment 56 57 58

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Large-Scale Identification of Protein Crotonylation Reveals Its Role in

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