Mice

Mice were C57BL/6 background, details available on request. Male mice were used for all experiments. All mice were kept in specific pathogen-free conditions and fed ad lib. Mice for the ChIP and RNA-seq experiments were housed at the Babraham Institute Biological Service Unit. All experimental protocols at Babraham Institute were approved by the Babraham Research Campus local ethical review committee and the Home Office (PPL 80/2488 and 70/8994). The antibiotics treatment experiments were performed at the University of Campinas. Male C57BL/6 mice at age 8–12 weeks were provided by the Multidisciplinary Centre for Biological Investigation (CEIMB) and all the experimental procedures were approved by the Ethics Committee on Animal Use of the Institute of Biology, University of Campinas (protocol number 3742-1). No sample size calculation was used as the minimum number of mice used was 3 or 4. Mice of the same age and breed were randomly put in the experimental groups. The order of samples from groups was mixed on collection. Sample size is reported in exact numbers and no samples are excluded from the analysis. No blinding was conducted.

Antibiotic treatment of mice

Mice received 200 µl of a mixture of antibiotics (5 mg/ml of neomicin, 5 mg/ml of gentamicin, 5 mg/ml of ampicillin, 5 mg/ml of metronidazole, and 2.5 mg/ml of vancomycin, Sigma Aldrich) daily for 3 days by gavage. The weight of the animals was monitored throughout the experiment. At the end of treatment period, feces were collected and snap-frozen in liquid nitrogen. After that, the animals were anesthetized using a mix of ketamine and xylazine (300 and 30 mg/kg, respectively) and the blood was collected by cardiac puncture. The blood was maintained at room temperature (RT) for 30 min and then centrifuged (3000×g, 8 min). The serum was collected and frozen at −80 °C. After euthanizing the animals by cervical dislocation, the entire intestine was harvested and the small intestine, colon, and cecum were isolated.

Determination of fecal bacterial load

Stool samples were collected on the third day after treatment by gavage with a mix of antibiotics or placebo. Fifty milligrams of the samples were used for extraction of the microbial genomic DNA using the InvitrogenTM PureLinkTM Microbiome DNA Purification kit (Thermo Fisher Scientific, MA, USA). Bacterial DNA was quantified by real-time PCR using primers complementary to 16S rDNA of Eubacteria (sense ACT CCT ACG GGA GGC AGC AGT; anti-sense ATT ACC GCG GCT GCT GGC)40. To determine the fecal bacterial load, a standard curve with serial dilutions was employed using genomic DNA extracted from Escherichia coli grown in vitro. Results obtained were normalized by the control condition (untreated mice).

SCFA measurements

Colonic luminal content samples were weighed into 1.5 ml tubes, crushed and homogenized in 100 µl of distilled water. Subsequently, 40 mg of sodium chloride, 20 mg of citric acid, 40 µl of 1 M hydrochloric acid, and 200 µl of butanol were added. The tubes were vortexed for 2 min and centrifuged at 18,000×g for 15 min. The supernatant was transferred to microtubes, and 1 µl was injected into the gas chromatograph. For serum measurements, 20 mg of sodium chloride, 10 mg of citric acid, 20 µl of 1 M hydrochloric acid, and 100 µl of butanol were added to 100 µl of serum samples. Tubes were vortexed and centrifuged as previously described and 1 µl was injected into the gas chromatograph. To quantify SCFAs, a calibration curve for the concentration range of 0.015–1 mg/ml was constructed. SCFAs measurements were performed following a recently published protocol41: chromatographic analyses were performed using an Agilent 6850 system with ExChrom software, equipped with a 7683B automatic liquid sampler, a flame ionization detector (FID) (Agilent Technologies, USA), and a fused-silica capillary RTX-WAX (Restec Corporation, U.S.) with dimensions of 60 m × 0.25 mm internal diameter (i.d.) coated with a 0.15-µm thick layer of polyethylene glycol. The initial oven temperature was 100 °C (hold 2 min), which was increased to 200 °C at a rate of 15 °C/min (hold 5 min). The FID temperature was maintained at 260 °C, and the flow rates of H 2 , air, and the make-up gas N 2 were 35, 350, and 25 ml/min, respectively. Sample volumes of 1 µl were injected at 260 °C using a split ratio of approximately 25:1. Nitrogen was used as the carrier gas at 25 ml/min. The runtime for each analysis was 12.95 min.

Small intestinal and colon epithelium extraction

Animals were sacrificed by cervical dislocation or exposure to CO 2 . Dissected small intestines and colons were opened longitudinally and washed three times with ice cold Hank's balanced salt solution without Ca2+/Mg2+ (HBSS). Intestinal and colon epithelium were dissociated with 30 mM EDTA/HBSS on ice with shaking for 30 min for small intestine and 1 h for colon; 50 ml Falcon tubes containing the tissue were then shaken vigorously by hand (2–3 shakes/second) for 5 min. The colon epithelium was incubated for an additional 10 min on ice in 30 mM EDTA/HBSS and further shaken for 5 min. Mucus and sub-mucosa were removed by dripping the material through a 100 μm followed by a 70 μm cell strainer. The extracted cells were pelleted at 475×g at 4 °C for 10 min. The cells were washed with ice cold HBSS and re-pelleted as above for further use. For the H3K18cr ChIP, three colons were combined.

Purification and enzymatic digestion of histones

For isolating a crypt-enriched fraction, villi were removed by shaking the small intestine pieces for 10 s after incubation for 5 min on ice in HBSS–30 mM EDTA and before the additional incubation in HBSS–30 mM EDTA for 15 min. Histones were then acid extracted following a protocol published in ref. 42: one small intestine/colon was used per extraction followed by MS analysis. Small intestine epithelium, colon epithelium, and a crypt-enriched fraction were homogenized in lysis buffer (10% sucrose; 0.5 mM EGTA, pH 8.0; 15 mM NaCl; 60 mM KCl; 15 mM HEPES; 0.5% Triton; 0.5 mM PMSF; 1 mM DTT; 5 mM NaF; 5 mM Na 3 VO 4 ; 5 mM Na-butyrate, cocktail of protease inhibitors (Sigma)). Nuclei were separated from the cytoplasm by centrifugation on sucrose cushions, washed in cold PBS, and then extracted in 0.4 N HCl overnight (o.n.) at 4 °C. Core histones, together with linker histones protein, were dialyzed against 100 mM ice-cold acetic acid. The concentration of purified samples was measured using the Bradford protein assay. Approximately 10 µg were separated on SDS-PAGE and bands corresponding to the histones H3 were excised and in-gel digested43. Briefly, gel bands were cut in pieces and destained with repeated washes in 50% acetonitrile (ACN) in H 2 O, alternated with dehydration steps in 100% ACN. Gel pieces were then in-gel chemically alkylated by incubation with D6-acetic anhydride (Sigma 175641) in 1 M NH 4 HCO 3 and CH 3 COONa solution as catalyzer. After 3 h at 37 °C with high shaking in a thermomixer, chemically modified gel slices were washed with NH 4 HCO 3 , alternated with ACN at increasing percent (from 50 to 100%). In-gel digestion was performed with 100 ng/µl trypsin (Promega V5113) in 50 mM NH 4 HCO 3 at 37 °C overnight. Chemical acetylation occurs on unmodified and monomethylated lysines and prevents trypsin digestion at these residues, thus producing a pattern of digestion similar to that obtained with the Arg-C protease (the so-called “Arg-C like” in-gel digestion pattern). The resulting histone peptides display an optimal length for MS detection and enhanced hydrophobicity that increases their separation at ultra-pressure chromatographic regimes.

Finally, digested peptides were collected and extracted using 5% formic acid alternated with ACN 100%. Digested peptides were desalted and concentrated using a combination of reverse-phase C18/C “sandwich” system and strong cation exchange (SCX) chromatography on hand-made micro-columns (StageTips44, 45). Eluted peptides were lyophilized, suspended in 1% TFA in H 2 O, and then subjected to LC-MS/MS.

LC-MS/MS

The peptide mixtures were analyzed by online nano-flow LC-MS/MS using an EASY-nLC 1000 (Thermo Fisher Scientic) connected to a QExactive (Thermo Fisher Scientific) through a nano-electrospray ion source. The nano-LC system was operated in one column setup with a 25-cm analytical column (75 µm inner diameter, 350 µm outer diameter) packed with C18 resin (ReproSil, Pur C18AQ 1.9 m, Dr. Maisch, Germany) configuration. Solvent A was 0.1% formic acid (FA) in ddH 2 O and solvent B was 80% ACN with 0.1% FA. Samples were injected in an aqueous 1% TFA solution at a flow rate of 500 nl/min. Peptides were separated with a gradient of 0–40% solvent B for 100 min, followed by a gradient of 40–60% in 5 min, and 60–95% over 5 min at a flow rate of 250 nl/min. The Q-Exactive instrument was operated in the data-dependent acquisition (DDA) to automatically switch between full scan MS and MS/MS acquisition. Survey full scan MS spectra (from m/z 300–1150) were analyzed in the Orbitrap detector with resolution R = 60,000 at m/z 200. The 10 most intense peptides were sequentially isolated to a target value of 3 × 106 and fragmented by high-energy collisional dissociation (HCD) with a normalized collision energy setting of 27%. The maximum allowed ion accumulation times were 20 ms for full scans and 50 ms for MS/MS and the target value for MS/MS was set to 1 × 106. Standard mass spectrometric conditions for all experiments were as follows: spray voltage, 2.4 kV; no sheath and auxiliary gas flow.

MS data analysis and relative abundance profiling

Acquired RAW data were analyzed by MaxQuant(MQ) software v1.5.2.8, using the Andromeda search engine46. Uniprot Mouse database (70,902 entries) was used for peptide identification. Enzyme specificity was set to Arg-C. Estimated false discovery rate of all peptide identifications was set at a maximum of 1% (Decoy database-based approach). Mass tolerance for searches was set to a maximum of 6 parts per million (ppm) for peptide masses and 20 ppm for HCD fragment ion masses. A maximum of three missed cleavages was allowed. In the search, we focused on lysine methylation and acylation, including as variable modifications: D3-acetylation (+45.0294 Da), D3-acetylation (+45.0294 Da) plus monomethylation (+14.016 Da), dimethylation (+28.031 Da), trimethylation (+42.046 Da), acetylation (+42.010 Da), and crotonylation (+68.074 Da). The use of high-accuracy criteria for HCD fragment ions tolerance (20 ppm) guarantee the capability to discriminate among other possible forms of acylation (e.g., lysine butyrylation (70.0418 Da) and β-hydroxybutyrylation (86.0367 Da)), therefore they were not included in the search. MaxQuant search results were exported and peptides with Andromeda score <60 and localization probability score <0.75 were removed. Filtered data were subjected to manual inspection and validation using the viewer.exe module integrated in MQ software47. Extracted ion chromatograms were constructed for each precursor based on the m/z value, using a mass tolerance of 10 ppm with a mass precision up to four decimals. For each histone-modified peptide, the relative abundance percentage (RA%) was estimated by dividing the area under the curve (AUC) of each modified peptide over the sum of the areas corresponding to all observed isoforms of that peptide, including the unmodified forms48. Significant changes among crypt and colon versus small intestine fraction have been calculated with a two-way ANOVA test using the Perseus software49. p-Value < 0.01 (1% FDR) were considered as significant.

Antibodies and western blot analysis

Anti-lysine crotonyl antibody (PTM-501, 1:5000), anti-crotonyl-histone H3 lys18 (anti-H3K18cr, PTM-517, 1:5000), and anti-butyryl-histone H3 lys18 (anti-H3K18bt, PTM-306, 1:5000) were from PTM BIOLABS; anti-tri-methyl H3 lys4 antibody was from Active Motif (anti-H3K4me3, Cat.39159); anti-crotonyl-histone H4 lys8 antibody (anti-H4K8cr, ab201075), anti-acetyl-histone H3 lys18 (anti-H3K18ac, ab1191, 1:10000), anti-histone H4 (ab31827, 1:40000) anti-histone H3 (ab1791, 1:40000), and anti-LaminB1 (ab16048, 1:5000) were from Abcam; mouse monoclonal anti-HDAC1 (Clone 2E10, 1:5000) from Millipore, monoclonal anti-HDAC2 (C-8) (sc9959, 1:5000) from SantaCruz, and mouse polyclonal HDAC3 (BD61124, 1:5000) from BD biosciences.

Western blots were washed with tris-borate-sodium-0.05% Tween-20 (TBS-T) and developed using enhanced chemiluminescence (ECL); uncropped western blots are shown in Supplementary Figures 12–14.

Cryosections and immunofluorescence staining

Cryosections were prepared from adult murine tissue, fixed for 90 min at RT with 4% formaldehyde in phosphate buffered saline (PBS). After 2 × 5 min washes with 1× PBS, samples were incubated in 30% sucrose overnight at 4 °C and embedded in Cryomatrix (ThermoFisher 6769006). Frozen blocks were cut with the Leica CM1860 cryotome to 8 μm sections and attached to Superfrost Plus slides (ThermoFisher 4951PLUS4). Before staining, sections were brought to RT, dried for 3 min at 60 °C, permeabilized for 20 min at RT in 1% Triton-X-100/PBS, and unmasked for 30 min at 95 °C in a citrate based-unmasking buffer (VECTOR H3300) followed by 3 × 5 min washes in PBS. Slides were blocked for 1 h in 5% FBS/PBS (FBS: fetal bovine serum) at RT followed by primary antibody (pan-crotonyl PTM Biolabs Inc. #PTM501, 1 µg/ml final conc.) incubation in blocking solution for 1 h at RT. After 4 × 5 min washes in 1× PBS, secondary antibody (AlexaFluor 488 Invitrogen A11008, 1 µg/ml final conc.) was applied for 1 h at RT in blocking solution supplemented with DAPI. After 4 × 5 min washes in 1× PBS, samples were mounted in Vectashield H-1000 and sealed. Controls without the primary antibody were processed accordingly. Imaging was performed with the Zeiss780 confocal microscope using 20× air and 63× oil immersion objectives at optimal resolution settings. Z-stacks of whole sections were imaged and further processed to maximum projections with ImageJ software. For optimal print results background correction and contrast enhancement with up to 3% pixel saturation were performed with ImageJ.

Cell culture and cell cycle analysis

Human colon carcinoma cells (HCT116) were a gift from Simon Cook's lab (Babraham Institute) who obtained them from Bert Vogelstein, John Hopkins University, Baltimore. This cell line is not in the database of commonly misidentified cell lines (ICLAC). They were grown in DMEM media containing glucose and pyruvate, 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. For the cell cycle analysis, HCT116 cells were blocked at G1 with 15 nM of abemaciclib (LY2835219, Seleck Chemicals) for 48 h and released by washing 2× with PBS and adding fresh medium. Class I HDAC inhibition was with 5 μM MS275 for 48 h. Cells were washed 2× with PBS and fresh medium supplemented with 1 μM MS275 was added. Cells were harvested at indicated intervals after release and one half was analyzed by western blotting. The remainder were used to determine cell cycle profiles with propidium iodide staining using the BD Pharmingen PI/RNAse staining buffer on LSRII Flow Cytometer (BD Biosciences) and Cell Cycle tool on FlowJo 10.0.8. HDAC1 over-expression was performed by transfecting HCT116 cells using Lipofectamine® 2000 Transfection Reagent following manufacturer's protocol using 50 μL of reagent and 12 μg of p181 pK7-HDAC1 (GFP) plasmid DNA (gift from Ramesh Shivdasani, Addgene plasmid # 1105450) or an N-terminal deletion mutant in a 60-mm dish for 16 h.

Whole cell extract preparations

For whole cell extract preparation, cells were detached with trypsin, washed in PBS, and boiled in Laemmli sample buffer (Biorad) for 5 min. The extracts were briefly sonicated to remove high molecular weight DNA before loading on an SDS-polyacrylamide gel for electrophoresis.

Intestinal organoid seeding and cultures

Small intestinal crypts were derived from wildtype C57BL/6 mice using a slightly modified protocol from reference51. In brief, collected small intestines were opened longitudinally and the majority of villi removed by gentle scraping with a coverslip. The tissue was cut to 3–5 mm pieces, washed five times with cold PBS, and vigorous shaking. After 30 min incubation on ice with 2 mM EDTA/ PBS, the remaining villi were removed with short shaking and tissue pieces were incubated for additional 30 min in 5 mM EDTA/PBS on ice. After another short shake, crypts were passed through a 40-μm cell strainer and pelleted at 425×g 1500 rpm at 4 °C for 10 min. The pellet was washed with cold PBS and 100–200 crypts were suspended in 50 μl Red-phenol-free Matrigel (BD Biosciences) droplets. After polymerization, complete medium containing advanced DMEM/F12 (Sigma), 2 mM Glutamax (Invitrogen), 10 mM HEPES (Gibco), 100 U/ml penicillin/streptomycin (Invitrogen), 1 mM N-acetyl-cysteine (Sigma), 1× B27 supplement (Invitrogen), 1× N 2 supplement (Invitrogen), 50 ng/ml mouse EGF (Peprotech), 100 ng/ml mouse Noggin (Peprotech), and 10% human R-spondin-1-conditioned medium from R-spondin-1-transfected HEK293T cells (Cultrex) was added to the cultures. Medium was changed every 3 days and organoids passaged after 7–10 days.

ChIP-seq of extracted colon epithelium

The colon epithelium cell pellet was resuspended in 10 ml of PBS-1% formaldehyde and fixation was carried out for 10 min, at RT with gentle agitation. The reaction was quenched by the addition of glycine (0.125 M final concentration) and the cells were pelleted at 475×g at 4 °C for 10 min, washed once with PBS, re-pelleted and either snap-frozen in liquid nitrogen for storage at −80 °C or processed further immediately. The cell pellet was resuspended and incubated for 10 min in 500 μl 50 mM Tris-HCl, pH 8.0, 10 mM EDTA 1% SDS (ChIP lysis buffer) on ice. Sonication of the chromatin to 100–500 bp fragment size range in polystyrene tubes was performed with a water-cooled Bioruptor (Diagenode), high power, 4 °C, 12 cycles, 30 s on, 30 s off. The sonicated material was transferred to a 1.5-ml tube, incubated for 30–45 min on ice, and pelleted at 20,800×g for 10 min, 4 °C to precipitate SDS. We pooled three colons to perform three ChIP experiments, using 20–25 μg equivalent of DNA for each experiment. For immunoprecipitation, the chromatin was diluted 1:10 with ChIP dilution buffer (16.7 mM Tris-HCl, pH 8.0, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton-X-100), 5 μg anti-H3K18cr antibody (PTM-517) or H3K4me3 antibody (Active Motif Cat.39159) per 20 μg DNA was added and this was incubated overnight on a rotating wheel at 4 °C. One percent input chromatin was collected and kept on ice. Immunoprecipitation of chromatin complexes was with Protein A-coated Dynabeads (Novex, Cat.10001D); 20–30 μl of bead suspension were washed two times with ChIP dilution buffer and the antibody–chromatin mix was added to the beads. Immunoprecipitation was for 2 h at 4 °C on a rotating wheel. Following this incubation, tubes were spun briefly and bound material was separated from unbound using a magnetic stand on ice.

All washes were performed at 4 °C for 5 min on a rotating wheel using 20× volumes with respect to the beads volume used. Beads were washed 1× with low-salt wash buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton-X-100, 0.1 % SDS), 2× with high-salt wash buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% Triton-X-100, 0.1 % SDS), and 1× with 10 mM Tris-HCl, 1 mM EDTA (1× TE). Elution of DNA from beads was with 200 μl of freshly prepared elution buffer (0.1 M NaHCO 3 , 1% SDS) at 65 °C for 30 min in a thermomixer at 1000 rpm. Supernatant was separated using a magnetic stand and transferred to a fresh tube. After bringing all inputs to 200 μl with elution buffer, both chromatin and input samples were reverse cross-linked by adding 8 μl of 5 M NaCl followed by an incubation at 65 °C overnight at 300 rpm in a thermomixer. Proteinase K Solution (Ambion, Cat:AM2548) was added to samples to a final concentration of 0.25 mg/ml and incubated for 2 h at 65 °C, 300 rpm. Chipped DNA was purified with QIAquick PCR purification kit (Qiagen, Cat.28104) and quantified using QubitTM 3.0 fluorimeter. Library preparation was performed from 5 ng of purified DNA using the NEBNext® UltraTM II DNA Library Prep Kit for Illumina® with the following modifications: Illumina Tru-Seq adaptors were used and library amplification was performed with the KAPA PCR Amplification kit (KAPA, Cat. KK2501) using 11 cycles. Libraries were sequenced on a HiSeq2500 sequencer (Illumina) according to manufacturer's instructions.

ChIP-seq and ChIP-qPCR of HCT116 cell extracts

HCT116 cells were treated with either 5 µM MS275 or DMSO for 18 h. Cells were trypsinized and fixed as indicated in the ChIP-seq section above. Fixed cells were re-suspended in Sonication buffer (150 mM NaCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.1% Triton, 1% SDS complemented with 10 mM sodium butyrate and protease inhibitor cocktail (P8340, Sigma)) and sonicated as described above for 15 cycles. After centrifugation at 14,000×g for 10 min, supernatant was diluted 10× in ChIP dilution buffer; 1% input chromatin was collected and kept on ice and 30 µg equivalent of DNA per sample was incubated overnight on a rotating wheel at 4 °C with 5 µg of anti-H3K18cr antibody (PTM-517, PTM Biolabs) or 0.3 µg of anti-H3K18ac antibody (ab1191, Abcam). Twenty microliters of Magnetic ProtA/G Beads (Millipore) were added to the samples and incubated on a rotating wheel for 3 h at 4 °C. Antibody-bound beads were washed as described above. Chip DNA was eluted at 65 °C for 30 min in 200 µl of elution buffer. De-crosslinking and DNA elution of both ChIP and input samples was performed as described in the ChIP-seq section. Real time qPCR analysis was carried out on input and ChIP DNA samples using the SYBR® Green PCR Master Mix (Applied Biosystems) and run on a BioRad CFX96 qPCR system. Each experiment has been carried out two times (biological replicates) and each sample has been run in triplicate (technical replicate). One percent of starting chromatin was used as input and data were analyzed accordingly. Primers were used at a final concentration of 250 nM with 62 °C as annealing/extension temperature and are listed in Table 2.

Table 2 Primers used in ChIP-qPCR Full size table

RNA-seq

RNA was extracted from both HCT116 cells (four biological replicates) and mouse colon epithelium (three mice as biological replicates) using the RNeasy Plus Mini Kit (Qiagen), following the manufacturer's instructions. Extracted RNA was quantified with Nanodrop and the quality assessed on a Bioanalyzer (Agilent). Library preparation was performed from 500 ng of RNA using the NEB Next® UltraTM Directional RNA Library Prep Kit for Illumina® and the NEBNext® Poly(A) mRNA magnetic isolation module. Illumina Tru-Seq adaptors were used and library amplification was performed with the KAPA PCR Amplification kit (KAPA, Cat. KK2501) using 14 cycles. Libraries were sequenced on a HiSeq2500 sequencer (Illumina) according to the manufacturer's instructions.

Bioinformatic analysis

RNA-seq: Sequencing reads were adaptor trimmed using Trim Galore! (version 0.4.2) and mapped to the mouse (GRCm38/mm10) reference genome with HiSat2 (version 2.0.5). Analysis of RNA-seq data was performed with SEQMONK version 1.36.0 on filtered reads with a MAPQ score of >60 for uniquely mapped reads. Read counts were quantified using the RNA-seq quantitation pipeline implemented in Seqmonk, quantifying only probes with at least one read. Probe read values were corrected for transcript length and divided into percentile bins according to their average expression levels of three replicates.

ChIP-seq analysis: For mouse colon and HCT116 H3K18cr, two replicates and for H3K4me3 three biological replicates were used. Sequencing reads were adaptor trimmed using Trim Galore! (version 0.3.8 and 0.4.2) and mapped to the mouse (GRCm38/mm10) or human (GRCh38) reference genomes with Bowtie2 (version 2.0.4.). Analysis of ChIP-seq data was performed with SEQMONK version 1.36.0 on filtered reads with a MAPQ score of >42, duplicate reads were always removed. For Fig. 2b, the genome was segmented in 1000 bp non-overlapping 'probes' and read counts were quantified for each probe and normalized to the largest datastore. For Fig. 2c, H3K4me3 peaks were identified using the MACS peak finder embedded in the SEQMONK program with the ChIP-seq data of H3K4me3 from colon epithelial cells and INPUT as reference. Selected fragment size was 300 bp and p-value significance threshold was 10−5. Reads were quantitated in and ±5 kbp around the MACS peaks. For Fig. 2d, read counts were quantified over the TSS (using a window of ±1 kb upstream of genes) and probes that had more than 100 reads in input were removed in both input and ChIP. For Fig. 2e, read counts were quantified over the TSS (using a window of ±0.5 kb upstream of transcripts) and were overlapped with RNA-seq data. KEGG pathway analysis was with DAVID 6.8 with electronic annotations excluded.

ChIP-qPCR data analysis

Prior to analysis, a logarithmic transformation of the data was carried out. Subsequently, we carried out a two-way ANOVA test followed by Holm-Sidak's multiple comparison test. Data were analyzed using the Graphpad Prism Software.

In vitro enzymatic assays of histone decrotonylation and deacetylation

Recombinant human histone H3.1 (NEB) was acetylated or crotonylated in vitro using the recombinant catalytic domain of p300 (human, ENZO) in presence of acetyl-CoA or crotonyl-CoA (Sigma-Aldrich), respectively. The reaction was carried out with 5.65 μM histone H3.1 and 0.66 μM P300 catalytic domain in 50 mM Tris-HCL pH 8, 50 mM KCl, 0.1 mM EDTA, 0.01 % Tween-20, 10% glycerol, 1 mM DTT, and 87 μM crotonyl-CoA or acetyl-CoA at 30 °C for 2 h. The reaction was stopped by heating at 65 °C for 5 min and the histones were diluted with two volumes of HDAC buffer (25 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM MgCl 2 , 1 μM ZnSO 4 ) for either decrotonylation or deacetylation reactions. Decrotonylation/deacetylation was typically performed with 1.75 μM modified histones and 0.12 μM HDAC1 (recombinant, human, Active Motif) or 0.18 μM HDAC2 (recombinant, human, ABCAM) at 30 °C for 2 h. Recombinant HDAC3/Ncor1 complex was from ENZO Life Sciences. All HDACs were produced in insect cells and purified. After the reaction, the histone modifications were identified by western blot. The pixel intensities of the western blot bands were quantified with the Image J software.

For the enzyme kinetic analysis, histone H3 was acetylated or crotonylated in vitro as described above. Dot blot western analysis with synthetic H3 peptides that were specifically crotonylated or acetylated at K18 indicated full crotonylation and acetylation of the histone H3 at K18 under these conditions, therefore, we assume that the histone had been fully crotonylated or acetylated. Different dilutions of modified histones were prepared in HDAC buffer (25 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM MgCl 2 , 1 μM ZnSO 4 ) for either decrotonylation or deacetylation reactions. Decrotonylation/deacetylation was performed with 1.41 to 0.19 μM modified histone H3 and 0.03 μM HDAC1 (recombinant, human, Active Motif) at 30 °C and stopped at 95 °C for 1 min. Five different modified histone concentrations and five different time points were performed in triplicate for the decrotonylation and deacetylation reactions.

Each reaction was spotted in quadruplicate and left to dry before being rinsed with transfer buffer and TBS-T. Western blot was performed using anti-H3K18ac and anti-H3K18cr antibodies. Spots were quantified using image J and spot intensity was converted to substrate concentration by multiplying each relative value by the concentration of modified histone in the reaction mix. Substrate concentration against time were plotted for each histone concentration and a linear regression was fitted for the first 30 s to 1 min of the reaction, as appropriate. The rate of each reaction set was plotted as replicates against substrate concentration. GraphPad Prism Version 7 software was used to calculate K m , V max , and K cat .

Colorimetric deacetylation assay

Synthesis of BOC-Lys(acetyl)-AMC and BOC-Lys(crotonyl)-AMC and the colorimetric assay was essentially as in ref. 2 with some modifications: for the synthesis of crotonyl-N-hydroxysuccinimide, N-hydroxysuccinimide (149 mg, 6 equivalents, eq) was dissolved in 2.5 ml anhydrous dichloromethane to which diisopropylethylamine (167 mg, 6 eq) was added. The solution was cooled in an ice bath and trans-crotonoyl chloride (135 mg, 6 eq) was added and the reaction stirred at 20 °C for 3 h. Ethyl acetate (5 ml) was added and the solution washed with 5 ml brine, dried over magnesium sulfate and concentrated. The crude product was dissolved in 3 ml dry ACN and the appropriate amount used in the next step. Acetyl-N-hydroxysuccinimide was prepared in the same way but using acetyl chloride rather than crotonoyl chloride. For the synthesis of BOC-Lys (crotonyl)-AMC, Boc-Lys-AMC acetate salt (100 mg, 1 eq) purchased from Bachem AG was dissolved in 4 ml water:ACN (1:1). Sodium bicarbonate (19.95 mg, 1.1 eq) was added in 1 ml water followed by trans-crotonyl-N-hydroxysuccinimide ester (2 eq) in 2 ml ACN. The reaction was left for 18 h and then concentrated to remove the ACN. The product was extracted into ethyl acetate, dried over anhydrous magnesium sulfate and concentrated. The product was purified on silica in dichloromethane:methanol (15:1). BOC-Lys(acetyl)-AMC was prepared in the same way but using acetyl-N-hydroxysuccinimide rather than crotonyl-N-hydroxysuccinimide.

The enzymatic reaction was set up in a 96-well plate in triplicate in a 51-μl volume with 0.035 μM HDAC1 and 0.2 mM BOC-Lys(acetyl)-AMC/BOC-Lys(crotonyl)-AMC in HDAC buffer (see above) at 30 °C for 2 h. After this, 5 μl of a 1 mg/ml trypsin solution in 1 mM HCl was added and the mixture was incubated 1 h at 37 °C. Fluorescence was read with a Pherastar plate reader at 360 nm excitation/450 nm emission.

Data availability

Next-generation sequencing data have been submitted to GEO under accession code GSE96035. The authors declare that all other data supporting the findings of this study are within the manuscript and its supplementary files or are available from the corresponding authors upon request.