Bidirectional histone monoaminylation dynamics regulate neural rhythmicity

General methods (equipment, reagents, chemicals, cell lines)

UV spectrometry was performed on the NanoDrop 2000c (Thermo Fisher Scientific) system. Biochemicals and medium were purchased from Thermo Fisher Scientific or Sigma-Aldrich unless otherwise stated. T4 DNA ligase, DNA polymerase and restriction enzymes were obtained from New England BioLabs. PCR amplifications were performed on the Applied Biosystems Veriti Thermal Cycler using either Taq DNA polymerase (Vazyme Biotech) for routine genotype verification or Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech) for high-fidelity amplification. Site-specific mutagenesis was performed according to standard procedures of the QuickChange Site-Directed Mutagenesis Kit purchased from Stratagene (GE Healthcare) or Mut Express II (Vazyme Biotech). Primer synthesis and DNA sequencing were performed by Integrated DNA Technologies and Genewiz, respectively. PCR amplifications were performed on a Bio-Rad T100TM Thermal Cycler. Centrifugal filtration units were purchased from Millipore, and MINI dialysis units purchased from Pierce. Size-exclusion chromatography was performed on an AKTA FPLC system from GE Healthcare equipped with a P-920 pump and UPC-900 monitor. Sephacryl S-200 columns were obtained from GE Healthcare. All the western blots were performed using the primary antibodies annotated in Supplementary Table 8 and secondary antibodies annotated in Supplementary Table 9 following protocols recommended by the manufacturer. Blots were imaged on an Odyssey CLx Imaging System (Li-Cor). All uncropped and annotated western blots, dot blots and SDS–PAGE gels are included in Supplementary Fig. 1. Amino acid derivatives and coupling reagents were purchased from AGTC Bioproducts. Dimethylformamide (DMF), dichloromethane and triisopropylsilane were purchased from Thermo Fisher Scientific and used without further purification. Hydroxybenzotriazole and O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Thermo Fisher Scientific. Trifluoroacetic acid (TFA) was purchased from Thermo Fisher Scientific. N,N-diisopropylethylamine (DIPEA) was purchased from Thermo Fisher Scientific. Analytical reversed-phase HPLC (RP-HPLC) was performed on the Agilent 1200 series instrument with the Agilent C18 column (5 μm, 4 × 150 mm), used 0.1% TFA in water (HPLC solvent A) and 90% acetonitrile, 0.1% TFA in water (HPLC solvent B) as the mobile phases. Analytical gradients were 0–70% HPLC buffer B over 45 min at a flow rate of 0.5 ml min⁻¹, unless stated otherwise. Preparative scale purifications were conducted on the Agilent LC system. An Agilent C18 preparative column (15–20 μm, 20 × 250 mm) or a semi-preparative column (12 μm, 10 mm × 250 mm) was employed at a flow rate of 20 ml min⁻¹ or 4 ml min⁻¹, respectively. HPLC electrospray ionization MS (HPLC-ESI-MS) analysis was performed on the Agilent 6120 Quadrupole LC/MS spectrometer (Agilent Technologies). All immunoblotting experiments for which quantifications were not included were performed three times. HeLa (CRM-CCL-2) and HEK293T (CRL-3216) cell lines were obtained from the American Type Culture Collection (ATCC). Human tissue culture cell lines (HeLa, HEK293T) were imaged for appropriate morphology and tested negative for mycoplasma contamination.

Expression of TG2 in HEK293T cells

The pShooter pCMV-nuc-myc vector expressing NLS-tagged WT human TG2 was used in our previous research⁴. The catalytically dead mutant TG2(C277A) plasmid was constructed by site-directed mutagenesis using 5′-GTCAAGTATGGCCAGGCCTGGGTCTTCGCCGCC-3′ and 5′-GGCGGCGAAGACCCAGGCCTGGCCATACTTGAC-3′ as primers (the mutation sites are labelled in bold). The gene sequences were confirmed by using the sequencing primer: 5′-GATGACCAGGGTGTGCTGCTG-3′. WT TG2 and the TG2(C277A) mutant were overexpressed in HEK293T cells using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. HEK293T cells (ATCC) were cultured at 37 °C with 5% CO₂ in DMEM medium supplemented with 10% FBS (Sigma-Aldrich), 2 mM l-glutamine and 500 U ml⁻¹ penicillin and streptomycin. The cells were stimulated with 2 μM calcium ionophore (Sigma-Aldrich, A23187) for 6 h at 37 °C before lysis in DPBS buffer (Gibco), and then the expression of TG2 was detected by western blot analyses with anti-TGM2 antibody (CST, 3557).

Knockout and rescue of TG2 expression in HeLa cells

Plasmids psPAX2 and pMD2G (gifts from Q.-E. Wang’s laboratory) were used for viral packing and transfection; TGM2-1_pLentiCRISPR v2 (GenScript; the sequence is provided in the Supplementary Note) was used for gene knockout of TGM2 in HeLa cells. The gRNA sequence of TGM2-1_pLentiCRISPR v2 was CGTCGTGACCAACTACAACT.

The TGM2-1_pLentiCRISPR v2-containing lentivirus was packed in HEK293T cells using Lipofectamine-2000-mediated transfection, where the DNA ratio of TGM2-1_pLentiCRISPR v2, psPAX2 and pMD2G was 4:3:1. Lenti-X concentrators (Clontech, 631231, 631232) were used to collect the packed lentivirus from the clarified supernatants. The viral supernatants were slowly titrated to HeLa cells cultured in DMEM containing 8 μg ml⁻¹ polybrene. Transfected cells were incubated under 37 °C with 5% CO₂ for 24 h before being split and cultured in DMEM containing 50–4,000 ng ml⁻¹ puromycin. Selection medium was replaced every 3-4 days to obtain resistant colonies. TGM2-knockout cell lines were validated using western blot analyses. WT TG2 and the TG2(C277A) mutant were overexpressed in TGM2-knockout HeLa cells using Lipofectamine 2000, as described above, and validated using western blot analyses.

Salt extraction of histones from cells

The extraction of histones from cells was performed according to the previously described high-salt extraction method⁴⁶. In brief, cell lysis solution was prepared using extraction buffer (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 0.2% NP40, protease and phosphatase inhibitors to 1 × from stock). After centrifuging, the pellet was extracted using a no-salt buffer (3 mM EDTA, 0.2 mM EGTA). After discarding the supernatant, the final pellet was extracted using high-salt buffer (50 mM Tris pH 8.0, 2.5 M NaCl, 0.05% NP40) in 4 °C cold room for 1 h. After centrifuging, the supernatant containing extracted histones was collected for further analyses.

Cell fractionation

Cytosolic and nuclear fractions were prepared using NEPER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific) according to the manufacturer’s protocol. Histones were extracted from the pellet using the high-salt extraction protocol, as described above⁴⁶. The purity of fractionation was evaluated using the following antibodies: anti-actin (cytosol), anti-MEK 1/2 (nucleoplasm; not provided in manuscript) and anti-H3 (chromatin).

Pulse–chase experiments and inhibitor treatment

HEK293T and HeLa cells were cultured at 37 °C with 5% CO₂ in DMEM medium supplemented with 10% FBS (Sigma-Aldrich), 2 mM l-glutamine and 500 U ml⁻¹ penicillin and streptomycin. WT TG2 or TG2(C277A) mutant was overexpressed in HEK293T cells. The cultured cells were incubated with 500 μM monoamines (5-PT or histamine) for 6 h before the medium was changed to monoamine-free DMEM. Cells were cultured for an additional 6 h, 12 h or 18 h, after which they were washed with DPBS and collected, and the cytosolic and histone fractions were prepared as previously described^46,47. Samples were separated on a single SDS–PAGE, transferred to a PVDF membrane and blotted with the indicated antibodies (or Cy5 dye) for further analyses. The cells were stimulated with 2 μM calcium ionophore (Sigma-Aldrich, A23187) during incubation with monoamine donors, as described above.

For the inhibitor treatment assays, 100 µM of TG2 inhibitors (ERW1041E or ZDON) were added to cell medium 2 h before adding the corresponding monoamine donors. Cells were incubated for additional 6 h, after which they were collected and histones were extracted and analysed, as described above. The samples were separated on a single SDS–PAGE, transferred to a PVDF membrane and blotted with the indicated antibodies. For the inhibitor-treated in vitro biochemical assays, where 1 μM NCPs were used as substrate and 0.1 μM TG2 was used as catalyst, 1 μM ERW1041E was added to the reaction systems for inhibiting the activity of TG2.

Visualization of H3 serotonylation by CuAAC

HEK293T and HeLa cells were treated with 500 μM 5-PT and stimulated with 2 μM calcium ionophore (Sigma-Aldrich, A23187) during incubation, as described above. The cells were washed by DPBS, collected and then histone factions were extracted through high-salt extraction. Extracted histones were desalinated, lyophilized and then resuspended in DPBS buffer containing 0.4% SDS. A total of 50 μl of freshly dissolved histones was added to a premixed solution containing 3 μl of 10 mM Cy5-azide (Sigma-Aldrich, 777323), 10 μl of a 3:7 mixture of 50 mM CuSO₄ and 100 mM THPTA, and then vortexed. Thereafter, 5 μl of 100 mM freshly made TCEP was added to initiate the click reaction followed by incubation (1–2 h) at 30 °C. Then, 10 μl of 0.5 M EDTA was added to quench the reactions. Excess reagents were removed by MeOH/CHCl₃ protein precipitation or concentration–dilution using a 0.5 ml centrifugal filter (3K, Millipore). The pellets were washed by 500 μl MeOH/H₂O (9:1) before a second centrifugation⁴⁸. The air-dried protein samples were then analysed by SDS–PAGE, followed by in-gel imaging using the Odyssey CLx Imaging System (wavelength 680 nm).

Expression of recombinant WT TG2 and C277A mutant

The pHis-hTGM2 plasmid was gifted from B. I. Lee (Addgene, 100719). The His₈-tagged TG2 C277A mutation was cloned by site-directed mutagenesis using pHis-hTGM2 as the template and the following primer sequences: 5′-GTCAAGTATGGCCAGGCCTGGGTCTTCGCCGCC-3′ and 5′-GGCGGCGAAGACCCAGGCCTGGCCATACTTGAC-3′. The gene sequences were confirmed by using the sequencing primer: 5′-GATGACCAGGGTGTGCTGCTG-3′. The His₈-tagged WT and mutant TG2 proteins were expressed in E. coli Rosetta (DE3) cells with an overnight 0.125 M IPTG induction at 16 °C. The bacterial pellet was lysed by sonication and the lysate was cleared by centrifugation at 12,000 rpm for 30 min. The lysate was loaded onto HisTrap HP Column (GE Healthcare) and eluted on the AKTA FPLC, followed by desalting using Zeba Spin Desalting Columns (7 K MWCO, 10 ml) according to the manufacturer’s protocol. Purified recombinant proteins were analysed by SDS–PAGE and concentrated using stirred ultrafiltration cells (Millipore) according to the manufacturer’s protocol. The concentration of each protein was determined using 280 nm wavelength on a NanoDrop 2000c (Thermo Fisher Scientific).

Peptide synthesis

Standard Fmoc-based solid-phase peptide synthesis (FmocSPPS) was used for the synthesis of peptides in this study. Generally, the peptides were synthesized on ChemMatrix resins with rink amide to generate C-terminal amides. Peptides were synthesized using manual addition of the reagents (using a stream of dry N₂ to agitate the reaction mixture). For amino acid coupling, 5 equiv. Fmoc protected amino acids was pre-activated with 4.9 equiv. HBTU, 5 equiv. hydroxybenzotriazole and 10 equiv. DIPEA in DMF and then reacted with the N-terminally deprotected peptidyl resin. Fmoc deprotection was performed in an excess of 20% (v/v) piperidine in DMF, and the deprotected peptidyl resin was washed thoroughly with DMF to remove trace piperidine. Cleavage from the resin and side-chain deprotection were performed with 95% TFA, 2.5% triisopropylsilane and 2.5% H₂O at room temperature for 1.5 h. The peptides were then precipitated with cold diethyl ether, isolated by centrifugation and dissolved in water with 0.1% TFA followed by RP-HPLC and ESI-MS analyses. Preparative RP-HPLC was used to purify the peptides of interest.

For the synthesis of site-specific monoaminylated H3 peptides (H3_1–10, H3_1–15 and H3_1–21) for biochemical assays in this study, Fmoc-Glu(OAII)-OH was incorporated at position 5 for orthogonal deprotection and further monoaminylation. In brief, the peptides were deprotected by Pd(PPh₃)₄ and PhSiH₃ on resins⁴⁹ and then conjugated with monoamine donors (that is, histamine hydrochloride, serotonin hydrochloride⁴ and acetonide-protected dopamine⁵⁰) through PyAOP and DIEA catalysis⁵¹.

For the synthesis of modified H3 peptide antigens (H3_1–10) in this study (as shown in Extended Data Fig. 4a), (1) Fmoc-Glu(OAII)-OH was incorporated at position 5 and either Fmoc-Lys(Boc)-OH or Fmoc-Lys(Me3)-OH was incorporated at position 4 on 2-Cl trityl resin through iterative FmocSPPS. (2) The deallylation was conducted using Pd(PPh₃)₄ and PhSiH₃, (3) followed by the coupling of Trt-protected histamine (4) and then acidolytic cleavage from the resin as well as global deprotection. Note that both the validated H3Q5his and H3K4me3Q5his antibodies were licensed to Millipore for sale (the catalogue numbers are provided below).

Recombinant histone expression and purification

Recombinant human histones H2A, H2B, H3.2 and H4 were expressed in E. coli BL21 (DE3) or E. coli C41 (DE3), extracted by guanidine hydrochloride and purified by flash reverse chromatography, as previously described⁴⁶. The purified histones were analysed by RP-LC–ESI-MS⁴⁶.

Preparation of histone octamers and 601 DNA

Octamers were prepared as previously described⁴⁶. In brief, recombinant histones were dissolved in unfolding buffer (20 mM Tris-HCl, 6 M GdmCl, 0.5 mM DTT, pH 7.5), and combined with the following stoichiometry: 1.1 equiv. H2A, 1.1 equiv. H2B, 1 equiv. H3.2, 1 equiv. H4. The combined histone solution was adjusted to 1 mg ml⁻¹ concentration and transferred to a dialysis cassette with a 7,000 Da molecular cut-off. Octamers were assembled by dialysis at 4 °C against 3 × 1 l of octamer refolding buffer (10 mM Tris-HCl, 2 M NaCl, 0.5 mM EDTA, 1 mM DTT, pH 7.5) and subsequently purified by size-exclusion chromatography on the Superdex S200 10/300 column. The fractions containing octamers were combined, concentrated, diluted with glycerol to a final 50% (v/v) and stored at −20 °C. The 147 bp 601 DNA fragment was prepared by digestion from a plasmid containing 30 copies of the desired sequence (flanked by blunt EcoRV sites on either site) and purified by PEG-6000 precipitation as described before⁴⁶.

Mononucleosome assembly

Mononucleosome assembly was performed according to the previously described salt dilution method with slight modifications⁴⁶. In brief, the purified WT octamers were mixed with 601 DNA (1:1 ratio) in a 2 M salt solution (10 mM Tris pH 7.5, 2 M NaCl, 1 mM EDTA, 1 mM DTT). After incubation at 37 °C for 15 min, the mixture was gradually diluted (9 × 15 min) at 30 °C by dilution buffer (10 mM Tris pH 7.5, 10 mM NaCl, 1 mM EDTA, 1 mM DTT). The assembled mononucleosomes were concentrated and characterized by native gel electrophoresis (5% acrylamide gel, 0.5× TBE, 120 V, 40 min) using ethidium bromide staining.

In vitro TG2 (de)monoaminylation biochemical assays

TG2 (de)monoaminylation assays were generally performed in the buffer (pH 7.5) containing 50 mM Tris-HCl, 5 mM CaCl₂ and 2 mM DTT (freshly added). For H3 peptide (H3_1–21) (de)monoaminylation and monoamine-replacement, 2 mM peptides were treated with 100 μM TG2 (or cell lysates) at 37 °C in the presence (or absence) of the corresponding monoamines (4 mM) for 2 h and then analysed by LC–MS. For NCP (de)monoaminylation and monoamine replacement, 1 μM NCPs were treated with 0.1 μM TG2 at 37 °C in the presence (or absence) of the corresponding monoamaines (0.5 μM) for 2 h. The (de)monoaminylated NCPs were analysed by SDS–PAGE followed by western blot analysis. H3 was used as the loading control in SDS–PAGE and western blot analyses. Buffer exchange for monoamine-replacement assays was performed using a 0.5 ml centrifugal filter (3K, Millipore) with a 120-fold (v/v) for the removal of excess monoamine from the old reaction buffer systems.

Immunoprecipitation and pull-down of TG2–H3 thioester complexes

To capture the TG2–H3 thioester complex, 50 μM of free H3 proteins were treated with 50 μM of WT TG2 or TG2(C277A) mutant in a buffer (pH 7.0) containing 50 mM Tris-HCl, 5 mM CaCl₂ and 2 mM DTT (freshly added) at 37 °C for 1 h. His₈-tagged TG2 was first pulled-down by BSA-blocked Ni²⁺-NTA agarose beads (Thermo Fisher Scientific). Next, the beads were washed three times with Tris-HCl buffer (pH 7.0), boiled, separated on SDS–PAGE and analysed by western blotting with anti-TGM2 and anti-H3 antibodies to detect the enrichment of H3.

LC–MS/MS validation of histaminylated H3_1–15

Samples were analysed by LC–MS/MS (Dionex 3000 coupled to Q-Exactive mass spectrometer, Thermo Fisher Scientific). Peptides were separated by C-18 reversed phase chromatography (inner diameter, 75 µm, particle size, 3 µm, Nikkyo Technologies) using a gradient increasing from 1% B to 25% B in 16 min (A: 0.1% formic acid, B: 80% acetonitrile in 0.1% formic acid). The mass spectrometer was operated in parallel reaction monitoring (PRM) mode⁴—R35 (MS and MS/MS resolution of 70,000 and 35,000, respectively)⁵² with an AGC target, 5 × 10⁵, maximum injection time of 60 ms and an isolation m/z window of 1.3). MS was acquired from m/z 300 to 1,650 while m/z 100 was set as lowest mass for MS/MS. Charge states 2+ to 5+ of the modified peptide: (ARTKQ(histamine)TARKSTGGKA-NH₂) were targeted. An energy of 25 NCE was used for the peptide in charge states 3+ and 4+ while NCE of 35 was used for the doubly charge version. Extended Data Fig. 3d represents a high-resolution, high-accuracy tandem mass spectra (sample: +histamine/+TG2) of the doubly charged peptide: ARTKQTARKSTGGKA-NH2 modified by histamine at Gln5 (m/z 551.9930 (1.5 ppm)). The full amino acid sequence was accounted for. Selected fragment ions, including y10/b5 and y11/b4 that identify the histamine modified glutamine are annotated.

Identification of H3 glutamine 5 histaminylation from cells

HEK293T cells transfected with WT TG2 or TG2(C277A) mutant plasmids were treated with 500 μM histamine, as described above. Histones were then extracted from collected cell pellets, as described previously⁵³, for further MS analysis. In brief, histones were extracted with chilled 0.2 M sulfuric acid (5:1, sulfuric acid: pellet) and incubated with constant rotation for 4 h at 4 °C, followed by precipitation with 33% trichloroacetic acid overnight at 4 °C. The supernatant was then removed and the tubes were rinsed with ice-cold acetone containing 0.1% hydrochloric acid, centrifuged and rinsed again using 100% ice-cold acetone. After centrifugation, the supernatant was discarded and the pellet was dried using a lyophilizer. The pellet was dissolved in 50 mM ammonium bicarbonate (pH 8.0), and histones were subjected to derivatization using 5 µl of propionic anhydride and 14 µl of ammonium hydroxide (Sigma-Aldrich) to balance the pH at 8.0. The mixture was incubated for 15 min and the procedure was repeated. Histones were then digested with 1 µg of sequencing grade trypsin (Promega) diluted in 50 mM ammonium bicarbonate (1:20, enzyme: sample) overnight at room temperature. Derivatization reaction was repeated to derivatize peptide N termini. The samples were dried by a lyophilizer. Before MS analysis, the samples were desalted using a 96-well plate filter (Orochem) packed with 1 mg of Oasis HLB C-18 resin (Waters). In brief, the samples were resuspended in 100 µl of 0.1% TFA and loaded onto the HLB resin, which was previously equilibrated using 100 µl of the same buffer. After washing with 100 µl of 0.1% TFA, the samples were eluted with a buffer containing 70 µl of 60% acetonitrile and 0.1% TFA and then dried by lyophilizer.

Samples were analysed using nano LC coupled online with MS/MS (nLC–MS/MS). In brief, the samples were resuspended in 10 µl of 0.1% TFA and loaded onto the Dionex RSLC Ultimate 300 (Thermo Fisher Scientific), coupled online with an Orbitrap Fusion Lumos (Thermo Fisher Scientific). Chromatography separation was performed using a two-column system, consisting of a C-18 trap cartridge (300 µm inner diameter, 5 mm length) and a picofrit analytical column (75 µm inner diameter, 25 cm length) packed in-house with reversed-phase Repro-Sil Pur C18-AQ 3 µm resin. Histone peptides were separated using a 30 min gradient from 4 to 30% buffer B (buffer A: 0.1% formic acid; buffer B: 80% acetonitrile + 0.1% formic acid) at a flow rate of 300 ml min⁻¹. The mass spectrometer was set to acquire spectra in a data-independent acquisition mode. The full MS scan was set to 300–1,100 m/z in the orbitrap with a resolution of 120,000 (at 200 m/z) and an AGC target of 5 × 10⁵. MS/MS was performed in the orbitrap with sequential isolation windows of 50 m/z with an AGC target of 2 × 10⁵ and an HCD collision energy of 30.

Targeted MS/MS was performed for the endogenous Q5his peptide (m/z 455.7587) and compared to the MS/MS spectra of a synthetic H3Q5his peptide. Data were manually inspected and the peak intensity was obtained by calculating the area of the extracted ion chromatogram. Histone peptides raw files were imported into EpiProfile 2.0 software⁵⁴ to also quantify acetylated, methylated and phosphorylated peptides to ensure the quality of sample preparations and LC–MS/MS analysis.

ITC

For ITC measurements, synthetic histone peptides and recombinant proteins (CHD1 chromodomain, TAF3 PHD finger, WDR5 WD40 domain, BPTF PHD finger, JMJDA Tudor domain or JARID1A PHD finger; purified, as previously described⁵) were extensively dialysed against ITC buffer: 100 mM NaCl and 20 mM Tris pH 7.5. The titrations were performed using the MicroCal iTC200 system (GE Healthcare) at 25 °C. Each ITC titration consisted of 17 successive injections with 0.4 μl for the first and 2.4 μl for the rest. Peptides were titrated into proteins in all of the experiments. The resultant ITC curves were processed using Origin 7.0 software (OriginLab) according to the ‘one set of sites’ fitting model. ITC statistics are provided in Supplementary Table 1.

Recombinant protein cloning and purification of WDR5 WT, WDR5 mutant and MLL1 complex, the latter of which was used in MALDI-TOF experiments

Full-length WDR5 and truncated WDR5 residues 22 to 334 (WDR5(22–334)) were cloned into pET28b vector for protein purification. Full-length WDR5(K259A) and WDR5(K259E) mutants were generated using a site-directed mutagenesis kit (Agilent). All proteins were expressed in the E. coli BL21 DE3 (Novagen) and induced overnight by 0.2 mM isopropyl β-d-thiogalactoside at 16 °C in the LB medium. The collected cells were suspended in 500 mM NaCl, 20 mM Tris, pH 7.5. After cell lysis and centrifugation, the supernatant was applied to HisTrap column (GE Healthcare). After washing 5 column volumes with the suspension buffer, the protein was eluted with buffer containing 100 mM NaCl, 20 mM Tris pH 7.5, 500 mM imidazole, and cut with Thrombin enzyme overnight. Proteins were further purified by the HiTrap SP (GE Healthcare) cation-exchange column and a HiLoad 16/60 Superdex 75 (GE Healthcare) gel-filtration column using AKTA Purifier 10 systems (GE Healthcare). All proteins were stored in 100 mM NaCl, 20 mM Tris, pH 7.5 at around 10 mg ml⁻¹ in an −80 °C freezer.

Human MLL1 constructs (3745–3969), as well as full-length human WDR5, RBBP5, DPY30 and ASH2L (95–628) proteins, were individually expressed in E. coli BL21 cells. All proteins were induced overnight with 0.2 mM isopropyl β-d-thiogalactoside at 16 °C in LB medium. Cell pellets were suspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM DTT). After cell lysis and centrifugation, the supernatants were purified using HisTrap columns (GE Healthcare) or Glutathione Sepharose 4B beads (GE Healthcare), followed by enzyme digestion to remove tags. All proteins were further purified on HiTrap SP (GE Healthcare) cation-exchange columns or HiTrap Q (GE Healthcare) anion-exchange columns. MLL1, WDR5 and DPY30 were further purified with the HiLoad 10/300 Superdex 75, while ASH2L and RBBP5 were further purified with the HiLoad 10/300 Superdex 200. The buffer for gel-filtration chromatography contained 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM DTT and 10% glycerol. Purified proteins were concentrated to 10–20 mg ml⁻¹ and stored at −80 °C. Note that, for MLL1 complex experiments presented in Fig. 2c and Extended Data Fig. 6a,b, the MLL1 complex was purchased from Active Motif (31423).

MALDI-TOF analysis of MLL1 enzymatic activity

H3K4 methyltransferase assays were conducted by combining 1.2 μM of the MLL(3745–3969)–WDR5–RBBP5–ASH2L–DPY30 complex with 10 μM histone H3 peptide (1–15) and 250 μM methyl-S-adenosyl-methionine in 50 mM Tris, pH 8.5, 50 mM KCl, 5 mM dithiothreitol, 5 mM MgCl₂ and 5% glycerol at 15 °C. The reactions were quenched by the addition of HPLC solvent A (H₂O + 0.1% TFA) and were desalted using C18 ZipTip (Millipore) according to the manufacturer’s protocol before being diluted 1:1 with α-cyano-4- hydroxycinnamic acid (CHCA) matrix in 50% ACN plus 20% acetone with 0.1% TFA and spotted on a MALDI-TOF plate for analysis. The samples were analysed using the Bruker UltrafleXtreme MALDI TOF/TOF mass spectrometer and data were analysed using Bruker Compass flexAnalysis v.3.4.

In vitro enzymatic assays with recombinant MLL1/2/3/4 and SETD1A/B complexes for LC–MS/MS

H3K4 methyltransferase assays were conducted, unless otherwise indicated, by combining 1.2 μM of either MLL1 (Active Motif, 31423), MLL2 (Active Motif, 31498), MLL3 (Active Motif, 31478), MLL4 (Active Motif, 31499), SETD1A (Active Motif, 81341) or SETD1B (Active Motif, 81342) complexes with 10 μM histone H3 peptide (unmodified versus H3Q5his; 1–21) and 100 μM S-adenosyl methionine in 50 mM Tris, pH 7.5, 50 mM KCl, 5 mM DTT, 5 mM MgCl₂ and 5% glycerol at 25 °C for 3 h. The reactions were quenched by the addition of HPLC solvent A (H₂O + 0.1% TFA).

Histone peptide derivatization and digestion

Derivatization of samples was performed as previously described⁵³. In brief, the samples were dissolved in 25 µl of a solution containing 50 mM ammonium bicarbonate, pH 8.0 and 20% acetonitrile. In the fume hood, the samples were mixed with 2 µl of propionic anhydride and 10 µl of ammonium hydroxide (all Sigma-Aldrich) to balance the pH at 8.0. The mixture was incubated at room temperature for 15 min and the procedure was repeated. The samples were digested with 500 ng of sequencing-grade trypsin (Promega) diluted in 50 mM ammonium bicarbonate overnight at room temperature. The derivatization reaction was repeated to derivatize peptide N termini. The samples were then dried in a vacuum centrifuge.

LC–MS/MS acquisition and analysis

Before MS analysis, the samples were desalted using a 96-well plate filter (Orochem) packed with 1 mg of Oasis HLB C-18 resin (Waters). In brief, the samples were resuspended in 100 µl of 0.1% TFA and loaded onto the HLB resin, which was previously equilibrated using 100 µl of the same buffer. After washing with 100 µl of 0.1% TFA, the samples were eluted with a buffer containing 70 µl of 60% acetonitrile and 0.1% TFA and then dried in a vacuum centrifuge.

The samples were loaded onto the Dionex RSLC Ultimate 300 (Thermo Fisher Scientific) system, coupled online with the Orbitrap Fusion Lumos (Thermo Fisher Scientific). Chromatography separation was performed using a two-column system, consisting of a C-18 trap cartridge (300 µm inner diameter, 5 mm length) and a picofrit analytical column (75 µm inner diameter, 25 cm length) packed in-house with reversed-phase Repro-Sil Pur C18-AQ 3 µm resin. The samples were separated using a 45 min gradient from 1 to 30% buffer B (buffer A, 0.1% formic acid; buffer B, 80% acetonitrile + 0.1% formic acid) at a flow rate of 300 nl min⁻¹. The mass spectrometer was set to acquire spectra in a data-independent acquisition mode using isolation windows as previously described⁵⁵. In brief, the full MS scan was set to 300–1,100 m/z in the orbitrap with a resolution of 120,000 (at 200 m/z) and an AGC target of 5 × 10⁵. MS/MS was performed in the orbitrap with sequential isolation windows of 50 m/z with an AGC target of 2 × 10⁵ and an HCD collision energy of 30.

Targeted MS/MS was performed for H3Q5his (m/z 455.7587), H3K4me1Q5his (m/z 462.7665), H3K4me2Q5his (m/z 441.7613) and H3K4me3Q5his (m/z 448.7692) peptides (targeted MS/MS was similarly performed for H3Q5ser, H3K4me1Q5ser, H3K4me2Q5ser and H3K4me3Q5ser, as presented in Extended Data Fig. 6a). Data were manually inspected, and the peak intensity was obtained by calculating the area of the extracted ion chromatogram. To achieve the relative abundance of PTMs, the sum of all different modified forms of a histone peptide was considered as 100%, and the area of the particular peptide was divided by the total area for that histone peptide in all of its modified forms.

Histone tail peptide IPs against recombinant WDR5 and MLL1 complex

Biotinylated unmodified H3, H3Q5ser, H3Q5his, H3K4me3, K4me3Q5his or H3k4me3Q5ser peptides (2 μg; 1–21) were resuspended with 25 μl of prewashed immobilized Streptavidin beads (DynaBeads Streptavidin M-280) in 0.01% DPBS/Triton-X 100, with subsequent incubation (rotating) for 1 h at room temperature. For each IP, 1 μg of full-length recombinant WDR5 (purified as described above) or 1 μg of MLL1 complex (Active Motif, 31423) was added to the beads in 1 ml of binding buffer (250 mM KCl, 25mM HEPES pH 7.5, 5 mM MgCl₂, 0.1% NP-40, 5% glycerol 1 mM DTT and 4% BSA), and each sample was rotated at 4 °C overnight. IPs were then centrifuged for 1 min at 1,000 rpm to pellet the beads. Beads were subsequently washed six times in binding buffer substituted with 500 mM KCl and 0.2% NP-40 (with no BSA). The beads were then washed once in cold DPBS and proteins eluted by boiling for 8 min in 30 μl of denaturing sample buffer before loading onto a gel.

Crystallization and X-ray structure determination

Truncated WDR(22–334) was firstly incubated with H3Q5his peptide at a molar ratio 1:2 for 1 h. Crystallization was performed by the sitting-drop vapour diffusion method under 18 °C by mixing equal volumes (1–2 μl) of protein and reservoir solution. The crystal was obtained at the condition 0.1 M sodium citrate tribasic dihydrate (pH 5.5), 22% polyethylene glycol (PEG) 3350 and 0.1% n-octyl-β-d-glucoside at 18 °C. The crystals were briefly soaked in the cryo-protectant and were flash-frozen in liquid nitrogen for data collection at 100 K. Complex datasets were collected at beamline BL17U at the Shanghai Synchrotron Radiation Facility. All data were indexed, integrated and merged using the HKL2000 software package⁵⁶. The complex structures were solved by molecular replacement using MOLREP⁵⁷. All structures were refined using PHENIX⁵⁸, with iterative manual model building using COOT⁵⁹. Model geometry was analysed with PROCHECK. The electron density of H3Q5 was visible while the histamine modification density was not clear. In the WDR5–H3Q5his structure and MLL3–RBBP5–ASH2L–H3 complex structure (Protein Data Bank (PDB): 5F6K), the model of histamine was built based on the orientation of the H3Q5 residue and restricted within the Ramachandran plot (favoured (95.08%), allowed (4.92%), outliers (9%)). All structural figures were created using PYMOL (http://www.pymol.org/). See Extended Data Table 1.

High-salt extraction of soluble nuclear and chromatin bound fractions

The generation of nuclear soluble extracts (NE) and chromatin bound (CB) fractions was performed as previously described³³ from cells overexpressing 3X-FLAG tagged WDR5 (Addgene, 59974) or a 3× Flag-tagged WDR5(K259A) mutant (generated using a site-directed mutagenesis kit, NEB E0554S, and confirmed by sequencing). Cells were collected, washed in PBS and resuspended in a low-salt buffer (LSB) containing 20 mM HEPES pH 7.9, 25% glycerol, 1.5 mM MgCl₂, 2 mM EDTA, 1 mM DTT and Halt Protease and Phosphatase Inhibitor Cocktail. The cells were then incubated on ice for 15 min to allow swelling. To lyse the cells, non-ionic detergent NP-40 was added (final concentration of 0.75%), and the mixture was gently passed through a 21-gauge needle ten times. The nuclei were collected by centrifugation at 1,100g for 5 min at 4 °C, and the supernatant was collected as the cytoplasmic extract. The nuclei were washed twice with LSB and resuspended in 500 µl of LSB. The pelleted nuclear volume (PNV) was calculated by subtracting 500 µl LSB from the total volume. Nuclei were then repelleted and resuspended in half PNV of LSB. An equal volume of high-salt buffer (20 mM HEPES pH 7.9, 25% glycerol, 1.5 mM MgCl₂, 1.6 M NaCl, 1 mM DTT, Halt Protease and Phosphatase Inhibitor Cocktail) was added dropwise while vortexing at low speed to reach a final NaCl concentration of 400 mM. The samples were incubated at 4 °C with rotation for 1 h before being centrifuged at 21,000g for 10 min at 4 °C. The supernatant was collected as the soluble nuclear extract. The pellet (chromatin fraction) was washed twice with 400 mM NaCl high-salt buffer for 10 min each with shaking, then pelleted and resuspended in 1× SDS–PAGE loading dye (final concentrations: 50 mM Tris-HCl pH 6.8, 3% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.002% bromophenol blue). The samples were boiled at 95 °C for 5 min and cooled on ice three times before the chromatin was sheared by sonication.

Enzymatic assays for antibody validations

For assessments of TG2-mediated transamidation of histamine to histone H3, 0.25 µg guinea pig TG2 (Zedira, T006), 5 mM histamine (Sigma-Aldrich) and 10 µg of recombinant H3.2 were combined with enzymatic buffer containing 250 mM tris-acetate (pH 7.5), 8.75 mM CaCl₂ and 1× protease inhibitor cocktail, followed by incubation for 3 h at room temperature. After incubation, enzymatic reactions were boiled with Laemmli buffer and then run on 4–12% NuPage BisTris gels (Invitrogen) and blotted, as described previously. Enzymatic assays were also performed using serotonin and dopamine to confirm the specificity of our in-house anti-H3Q5his antibody. Enzymatic assays were also performed using reconstituted unmodified (81070) versus K4me3 (31584) mononucleosomes from Active Motif.

Animals

Male and female mice (C57BL/6J; aged 8–10 weeks) were purchased from The Jackson Laboratory. Animals were group housed (2–5 per cage) under a 12 h–12 h light–dark cycle (lights on from 07:00 to 19:00) at constant temperature (23 °C) with ad libitum access to food and water. All animal protocols were approved by the IACUC at the Icahn School of Medicine at Mount Sinai (ISMMS). No wild animals or field collected samples were used in this study. Adequate sample sizes were generally determined based on intersample variability. Throughout the Article, we determined the significance of results based on a general confidence interval of 95%. We do not include specific justifications of sample size within the methods (such as power analyses), as sample sizes were based on extensive laboratory experience with these end points. The sample sizes chosen are consistent with those used by others in the field to achieve statistically significant results comparing stressed versus control animals. Where appropriate, animals were randomly assigned to groups (segregated by viral treatments, or ZT). Tissue samples were not pooled from multiple animals in these studies for western blotting and RNA-seq experiments (that is, each n represents a discrete datapoint). For CUT&RUN, each replicate per antibody was pooled from punches from three animals (per n) for initial processing, with 3 independent biological replicates conducted (n = 3) per antibody. For all viral experiments (RNA-seq, western blotting and behaviour), investigators were blinded to conditions such viral treatment before analysis. No data were excluded from these studies.

Immunoblotting analysis of the brain

Brain tissues were extracted from euthanized mice and immediately frozen whole. Brains were later sectioned using razor blades and a brain block to 1 mm thickness, with tissue punches (1–2 mm) collected for corresponding brain regions. To purify nuclear fractions, punches were homogenized in buffer A containing 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 1 mM EDTA and 1× protease inhibitor cocktail. After homogenization, 0.1% Triton X-100 was added to each homogenate, incubated and rotated at 4 °C for 30 min and then centrifuged for 5 min at 1,300g at 4 °C. Supernatants containing cytosolic fractions were discarded, and the nuclear pellets were resuspended in buffer A to remove any remaining cytosolic contamination, followed by centrifugation for 5 min at 1,300g at 4 °C. After centrifugation, the supernatants were discarded and the pellets were resuspended and sonicated in sample buffer containing 0.3 M sucrose, 5 mM HEPES, 1% SDS and 1× protease inhibitor cocktail. Protein concentrations were measured using the DC protein assay kit (Bio-Rad), and 1–20 µg of protein was loaded onto 4–12% NuPage BisTris gels (Invitrogen) for electrophoresis. Proteins were then transferred to PVDF membranes and blocked for 30 min in 5% milk in PBS + 0.1% Tween-20 (PBST), followed by incubation with primary antibodies overnight at 4 °C. For competition assays, antibodies were pre-incubated with indicated peptides at a 5:1 ratio for 1 h at room temperature before being incubated with the membrane. The following antibodies were used: rabbit anti-H3Q5his (1:200, Millipore, ABE2578), rabbit anti-H3K4me3Q5his (1:500, Millipore, ABE2605), rabbit anti-H3K4me3 (1:1000, Abcam, ab8580) and rabbit anti-H3 (1:50,000, Abcam, ab1791). The next day, the membranes were washed three times in PBST (10 min) and incubated for 1 h with horseradish-peroxidase-conjugated anti-rabbit secondary antibody (Bio-Rad 170-6515; 1:10,000; 1:50,000 for anti-H3 antibody) in 5% milk/PBST at room temperature. After three final washes with PBST, bands were detected using enhanced chemiluminescence (ECL; Millipore). Densitometry was used to quantify protein bands using ImageJ Software (NIH), and proteins were normalized to total H3 or H4. For peptide dot blots, peptides (unmodified versus H3Q5his versus H3Q5ser versus H3Q5dop; 1–10) were dotted as progressive protein concentrations (0.25, 0.5, 1 µg) on a nitrocellulose membrane. Membranes were left to dry at room temperature for 1 h and then blocked in 5% milk/PBST for 1 h. Membranes were treated similar to that described above.

ELISAs

Serotonin and histamine ELISAs were performed using kits from Abcam (ab133053 and ab213975). Mouse brain was collected across the ZT, sliced and punched bilaterally for TMN. TMN punches were resuspended in 100 μl of hypotonic lysis buffer and allowed to swell. Swollen tissue was then denounced homogenized and centrifuged at 20,000 rcf for 10 min to pellet insoluble cellular debris. Once pelleted, the supernatant was collected and used directly as substrate in the ELISA. Both histamine and serotonin ELISAs were performed according to the manufacturer’s instructions. UV-Vis absorbance for all plates were imaged using a spectramax id5 multi-mode microplate reader. Histamine ELISAs were read at 450 nm absorbance while serotonin ELISAs were read at 405 nm. Calculations were performed according to the manufacturer’s instructions.

AAV constructs and viral transduction

AAV H3.3 constructs (empty versus WT versus H3.3(Q5A)-Flag-HA) were generated and validated, as previously described¹². All three vectors contain an IRES-driven GFP fluorescent tag to allow visualization of the injection site during tissue dissection. Animals were anaesthetized with isoflurane (1–3%) and positioned in a stereotaxic frame (Kopf instruments) and 0.5 μl of viral construct was infused bilaterally into TMN using the following coordinates; anterior–posterior (AP) −2.0, medial–lateral (ML) + 0.6, dorsal–ventral (DV) −5.2. After surgery, mice received meloxicam (1 mg per kg) subcutaneously and topical antibiotic treatments for 3 days. All tissue collections or behavioural testing commenced 21 days after surgery to allow for maximal expression of the viral constructs.

Immunohistochemistry

Mice were anaesthetized with ketamine–xylazine (100 and 12 mg per kg) intraperitoneally (i.p.), and then perfused transcardially with cold phosphate-buffered saline (PBS 1×) followed by 4% paraformaldehyde in 1× PBS. Next, brains were post-fixed in 4% paraformaldehyde overnight at 4 °C and then transferred into 30% sucrose/PBS 1× for 2 days. The brains were then cut into serial 40 μm coronal slices. Free-floating TMN slices were washed three times in Tris-buffered saline (TBS 1×), incubated for 30 min in 0.2% Triton X-100/1× TBS, to permeabilize tissue, and then incubated for 1 h at room temperature in blocking buffer (0.3% Triton X-100, 3% donkey serum, 1× TBS). Brain slices were then incubated overnight at room temperature with mouse anti-GFP (1:200; Abcam, ab65856) and HA-488 (1:200; Life Technologies, Alexa Fluor SC-805). The next day, brain slices were washed three times in 1× TBS and then incubated for 2 h at room temperature with a fluorescent-tagged Alexa Fluor 568 anti-mouse secondary antibody (1:500; Life Technologies A11004). Brain sections were then washed three times in 1× TBS, incubated with DAPI (1:10,000, Thermo Fisher Scientific, 62248) for 5 min at room temperature, mounted onto Superfrost Plus slides (Thermo Fisher Scientific) and then coverslipped with Prolong Gold (Invitrogen). Immunofluorescence was visualized using a confocal microscope (Zeiss, LSM 780).

RNA-seq analysis

RNA extractions, library preparation and sequencing

Brain tissues were collected every 4 h across the 24 h zeitgeber in non-virally transduced C57BL/6J mice (aged 8–10 weeks), or 21 days after viral transduction. All brain tissues were immediately frozen after collection. For non-virally transduced brains, tissues were sectioned at 1 mm thick and TMN tissues were collected by tissue punch (1 mm). For virally infused brains, tissues were sectioned at 150 μm on the cryostat, and GFP was illuminated using the NIGHTSEA BlueStar flashlight to microdissect virally infected tissues. Tissues were resuspended in 800 μl of Trizol and homogenized using a small dounce homogenizer (30 strokes loose, 30 strokes tight) at room temperature. Chloroform was added and the aqueous phase isolated. 70% ethanol was added 1:1, and then passed over an RNAeasy minelute column. The Qiagen RNAeasy MicroKit protocol was followed, including all optional steps and DNase treatment. RNA was eluted in 15 μl and quantified using the NanoDrop spectrophotometer. The RNA quality was assessed using a Tapestation RNA screentape (Agilent). Then, 100 μg of total RNA was used as input for library preparation using the Illumina Stranded mRNA Prep, Ligation kit. A total of 14 cycles of PCR amplification was performed, libraries were pooled at an equimolar concentration and sequenced on the Illumina HiSeq 2500 or NovaSeq X+ sequencer by the NYU Genome Technology Center.

RNA-seq data analysis

Raw sequencing reads were demultiplexed using bcl2fastq2 (Illumina, v.2.20). The samples were aligned to the GRCm38 mouse genome using STAR (v.2.7.11b) alignReads in mode BAM SortedByCoordinate⁶⁰. Gene counts were generated using htseq-count (HTSeq v.2.0.5) with the following parameters: –format=bam –minaqual=10 –type=exon –idattr=gene_name –stranded=yes –mode=union using the Ensembl v93 annotation⁶¹. Gene counts were normalized using DEseq2⁶² (v.1.44.0) before analysis using JTKcycle³⁷ (v.3.1) to identify cycling genes from the dataset using the parameters jtkdist (varying depending on replicates), periods(2:6) and jtk.init(periods,4). Genes with P_adj < 0.05 were deemed to be significant and Z scores were computed in R using tidyverse (v.2.0.0). heatmap.2 in R was used to visualize the cycle genes (gplots v.3.1.3.1) across the zeitgeber. Cyclic genes were further assessed using ChEA in Enrichr (https://maayanlab.cloud/Enrichr/, (v.3.2)), which infers transcription factor regulation from integration of previous genome-wide chromatin immunoprecipitation (ChIP) analyses. Further ontology was also conducted using Enrichr. Odds ratios were calculated using the GeneOverlap R package (v.1.26.0). DEseq2 was run to perform pairwise differential expression analyses between H3.3 and GFP viral treated samples (to ensure limited to no significant differences between control groups; number of differentially expressed genes between GFP versus H3.3 WT at: ZT0 = 1, ZT4 = 0, ZT8 = 0, ZT12 = 40, ZT16 = 1, ZT20 = 1) before combining them together to run JTKcycle, as previously described. Differentially expressed genes were defined at FDR < 0.01. Overlap of JTK cycle genes with peak lists from CUT&RUN-seq (see below) was performed in R using dplyr (v.1.1.4). Individual circadian rhythm controlling genes identified in Enrichr were highlighted on heat maps manually.

RT–qPCR

Cell pellets were resuspended in 200 μl Trizol and homogenized using a clean pestle. A total of 600 μl of Trizol was added to the homogenate and allowed to rest at room temperature for 4–5 min. 160 μl chloroform was then added to each tube and mixed vigorously for 15 s, followed by a 3 min rest period. The samples were then centrifuged at 12,000g for 15 min at 4 °C. The top layer was decanted and transferred to a separate Eppendorf tube. 1 volume of 70% ethanol was added to the lysate and mixed thoroughly. The lysate was then transferred to a RNAeasy mini column and centrifuged at 12,000g for 1 min. A master mix of DNase (79256) and RDD buffer were added to the RNAeasy spin column and allowed to incubate at room temperature for 15 min. Then, 350 μl of RW1 was added to the column and centrifuged at 12,000g for 1 min at room temperature. The RNAeasy columns were then transferred to a new 2 ml collection tube and 500 μl of RPE buffer was added to the column and centrifuged at 12,000g for 1 min. Next, 500 μl of 80% ethanol was added to the tube and centrifuged at 12,000g for 1 min. An additional 5 min spin at maximum speed was performed to remove residual ethanol. RNA was eluted in 13 μl of double-distilled H₂O.

To convert RNA into cDNA, 5 μg of total RNA was mixed with 1 μl of 50 μM oligo dT, 1 μl of 10 mM dNTPs and allowed to prime at 65 °C for 5 min, followed by a 1 min incubation of ice. Then, 4 μl of 5× SSIV buffer (18090010), 1 μl of 100 mM DTT, 1 μl of RNAseOUT recombinant RNase inhibitor and 1 μl of Superscript IV reverse transcriptase were added to the sample, and allowed to incubate at 52 °C for 10 min, followed by inactivation by heating at 80 °C for 10 min. Subsequent cDNA was diluted 1:10 and 1 μl per well for RT–qPCR. Data were analysed using the ΔΔC_t method using the 18S RNA gene for normalization. A list of primers used in this study is provided in Supplementary Table 10.

CUT&RUN–seq

Cells

HeLa cells were grown in DMEM with glucose, 10% fetal bovine serum (Sigma-Aldrich) and 1× penicillin–streptomycin (Gibco). Cells were cultured at 37 °C at 5% CO₂. Cells were collected on ice using a cell scraper in the cold room, and washed in PBS. Cells from each 10 cm plate were resuspended in 1 ml of nuclear extract (NE) buffer (20 mM HEPES-KOH, pH 7.9, 10 mM KCl, 0.5 mM spermidine, 0.1% Triton X-100, 20% glycerol and freshly added protease inhibitors (Halt Protease Inhibitor Cocktail, EDTA-free, Thermo Fisher Scientific) and passed through a 21 gauge needle 20 times to lyse the cells. Nuclei were pelleted at 1,100g for 5 min at 4 °C and the supernatant was discarded. Nuclei were washed again in 1 ml NE buffer and counted. In total, 30,000 nuclei were used per biological replicate.

TMN

Brain tissues were collected every 4 h across the 24 h zeitgeber. Tissues were collected after vehicle/zolpidem treatment at ZT = 20. Brain tissues were extracted from euthanized mice and immediately frozen whole in methylbutane (Thermo Fisher Scientific, 277258). Brains were later sectioned using razor blades and a brain block to 1 mm thickness, with 1 mm tissue punches of the TMN bilaterally. For CUT&RUN, each biological replicate per antibody was pooled from three punches animals (per n) for initial processing, with three independent biological replicates conducted (n = 3) per antibody. Each pool of tissue punches was resuspended in 500 μl of NE buffer, and homogenized using 30 strokes of a plastic pestle (Sigma-Aldrich) in a 1.7 ml tube (Eppendorf). Nuclei were pelted at 1,100g for 5 min at 4 °C in a swinging-bucket rotor and supernatant discarded. Nuclei were washed again in 500 μl NE buffer and counted. In total, 30,000 nuclei were used per biological replicate.

CUT&RUN

BioMag Plus Concanavalin A beads (Polysciences) were prepared (15 μl bead slurry per reaction) by washing three times with binding buffer (20 mM HEPES-KOH, pH 7.9, 10 mM KCl, 1 mM CaCl₂, 1 mM MnCl₂), and resuspending in the original volume^63,64,65. 15 μl was aliquoted into a 1.7 ml DNA low-bind tube (Eppendorf) and 300 μl binding buffer was added to each tube. Nuclei from cells or tissues were added (30,000 nuclei in 300 μl of NE buffer) to each tube, and rotated end over end at room temperature for 10 min. The bead-bound nuclei were washed with 1 ml wash buffer (WB: 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 0.1% Tween-20, 0.5 mM spermidine, 0.1% BSA, freshly added protease inhibitors) three times. All washes were done so to minimize pipetting and beads mixed by inversion and light flicking of the tube. Beads were resuspended in 100 μl of antibody buffer (1 ml wash buffer with 2 mM EDTA) and mixed by flicking. Then, 2 μl of antibody (1:50) was added to each tube and mixed by flicking (H3K4me2 (Active Motif, 39141); WDR5 (CST, D9E1I), H3K4me3 (Epicypher, 13-0041), H3Q5his (Millipore, ABE2578), H3K4me3Q5his (Millipore, ABE2570), H3K4me3Q5ser (Millipore, ABE2580)). Bead-bound nuclei were incubated on a mixer (tubes on their side at ~20 degree upward angle) with primary antibodies overnight at 4 °C. Nuclei were washed twice the next day with 1 ml of cold WB. Nuclei were resuspended in 50 μl of cold wash buffer by flicking, and 2.5 μl of pAG-MNase (Epicypher, 15-1016) was added and mixed by flicking and incubated for 1 h at 4 °C on the same mixer. Nuclei were then washed four times with 1 ml ice-cold wash buffer, followed by one wash in 1 ml low-salt rinse buffer (20 mM HEPES, pH 7.5, 0.5 mM spermidine, 0.1% Tween-20 and 0.1% Triton X-100). Nuclei were resuspended in ice-cold calcium incubation buffer (3.5 mM HEPES, pH 7.5, 10 mM CaCl₂, 0.1% Tween-20, 0.1% Triton X-100) and immediately placed into an ice-cold metal block in a 4 °C deli fridge to maintain the temperature. The samples were incubated for 30 min, and then 100 μl of 2× stop buffer (340 mM NaCl, 20 mM EDTA, 5 mM EGTA, 0.1% Tween-20, 0.1% Triton X-100, 25 μg ml⁻¹ RNase A (Thermo Fisher Scientific) and 0.05 ng per 100 μl of E. coli spike-in DNA (Epicypher, 18-1401)) was added, and the beads were mixed by flicking. Nuclei were incubated at 37 °C for 15 min with no shaking to allow for release of chromatin and digestion of RNA. Beads were placed onto a magnet, and the supernatant (200 μl) was collected. DNA was isolated using the Zymo ChIP DNA Clean & Concentrator kit (D5205) and eluted in 30 μl and frozen at −20 °C for library preparation.

CUT&RUN-seq library preparation and sequencing

Library preparation was performed using the NEBnext Ultra II DNA library kit (E7645L) with multiplexed adapters with minor modifications. CUT&RUN DNA underwent end repair and adapter ligation according to the manufacturer’s protocol (1:15 adapter dilution was used). DNA was amplified using 16 PCR cycles with 10 s of extension time per cycle. Libraries were quantified using the Qubit fluorometer (Thermo Fisher Scientific) DNA high sensitivity kit, and the library size distribution was checked using the Tapestation DNA High Sensitivity ScreenTape (Agilent). Libraries were pooled at an equimolar concentration and sequenced on the Illumina NovaSeq 6000 sequencer by the NYU Genome Technology Center.

CUT&RUN qPCR

Ssoadvanced universal SYBR green master mix (1725270) was used according to the manufacturer’s instructions. A master mix containing 2× ssoadvanced mastermix (10 μl per 20 μl reaction) and the desired primers (1 μl of 10 μM forward and reverse primer premixed per 20 μl reaction) and ultra-pure water were premixed to prepare a master mix. Once the master mix was dispensed into a 384-well plate (HSP3801), 1 μl of 1 ng μl⁻¹ CUT&RUN DNA library was dispensed into the desired wells. A QuantStudio5 real-time qPCR instrument was used according to the manufacturer’s instructions. Data were analysed to examine enrichment over IgG controls. A list of primers used in this study is provided in Supplementary Data 7.

CUT&RUN–seq data analysis

Raw sequencing files were demultiplexed using bcl2fastq2 (Illumina, v.2.20). Between 20 and 100 million total reads were achieved for each replicate (average, 42.6 million). The samples were aligned to the hg19 or mm10 genome using bowtie2 (v.2.5.0)⁶⁶, with the following parameters: –local –very-sensitive-local –phred33 -I 10 -X 700 –dovetail –no-unal –no-mixed –no-discordant⁶³. Low-quality reads were filtered out using Samtools (v.1.9) with a cut-off MAPQ score of 30, and only unique reads were retained for further processing⁶⁷. Unique read files for each replicate/timepoint/antibody were merged and used for peak calling using MACS2 (v.3.0.0a6) with the callpeak function and the options -f BAMPE -q 0.05 –broad –broad-cutoff .05, using the corresponding IgG sample as the -c. For visualization, each sample was normalized by scaling the samples based on the E. coli spike-in DNA. Each sample was aligned to the E. coli genome (MG1655), and the uniquely aligned reads were counted. The number of E. coli reads for each replicate between timepoints was compared, with the sample with the lowest number of E. coli reads set at a scaling factor of 1×. The other samples were scaled down by a scaling factor that was computed by dividing the lowest number of E. coli reads by the sample number of E. coli reads. This was done separately for each antibody, as an internal normalization between timepoints across zeitgeber time⁶⁸. The same was done comparing vehicle- and zolpidem-treated animals. Genome coverage tracks (bigwig files) were produced using the deepTools (v.3.5.1) bamCoverage function with the options –binSize 10 –smoothLength 30 –normalizeUsing None –scaleFactor # (derived from E. coli spike in) and using an ENCODE hg19 or mm10 blacklist file (https://doi.org/10.1038/s41598-019-45839-z, v2 for both) to discard regions with consistently non-specific signal⁶⁹. Peak annotation and motif analysis of MACS2⁷⁰ called peaks were performed using HOMER (v.4.11)⁷¹. Heat maps were made using Deeptools (v.3.5.5) computeMatrix and plotHeatmap in reference-point mode, centred over TSSs or peak centres, using binSize 10 and –sortUsing mean, sorted in descending order. TSSs were downloaded from the UCSC (mm10/hg19) table browser using the canonically annotated transcript for each gene. Overlap of various peaks and TSSs was achieved using bedtools intersect (v.2.31)⁷². For generation of average plot profiles (±500 bp of TSS) and input for running JTK cycle on H3K4me3Q5his and H3K4me3Q5ser marks, Deeptools computeMatrix was run centred over TSSs with the parameters -a 500 -b 500 -binSize 100. The resulting matrix and coordinates files were merged in R using dplyr (v.1.1.4), and the average signal over the 1 kb window for each timepoint was computed for the plot profiles. For JTK cycle³⁷, the average signal over the same 1 kb window was computed for all individual biological replicates, and JTK cycle (v.3.1) was run with the options jtkdist(6,3), periods(2:6) and jtk.init(periods,4). Genes with P_adj < 0.05 were further analysed, with Z-scores being computed in R using tidyverse (v.2.0.0) and plotted as a heat map using the function heatmap.2 (gplots v.3.1.3.1).

Sleep manipulation

Mice were individually habituated to locomotor activity monitoring cages and chambers (as described below in the ‘Circadian locomotor activity’ section) for 24 h, and then received either an injection of vehicle or zolpidem (10 mg per kg, i.p.) at 19:00 (the beginning of active phase, ZT12). Animals were placed back into the locomotor activity monitoring cages immediately and activity was measured for 8 h. Animals were immediately euthanized and the brains were collected and frozen for subsequent CUT&RUN–seq experiments.

Circadian locomotor activity

To monitor locomotor activity across sleep–wake cycles, mice were individually placed into clean, transparent home cages with minimal bedding and access to food and water ad libitum. Home cages were placed into larger activity chambers with infrared beams to detect movement across 24 h (clear plexiglass 40 × 40 × 30 cm, Omnitech Electronics), starting at 19:00 (lights off). Activity was monitored through beam breaks, which was collected by Fusion Software (v.5.0) (Omnitech Electronics) software and calculated into 4 h time bins. Animals were monitored 12 h after switching them from their normal light–dark cycle (lights on at 07:00, lights off at 19:00) to dark–dark for 48 h to assess their locomotor behaviour.

Statistics and reproducibility

All in vitro and in cellulo western blotting and MS analyses were repeated independently at least three times with similar results. For western blot comparisons in brain examining rhythmic patterns of expression for H3Q5his, H3K4me3Q5his, HDC and NeuN (all normalized to total H3), nonlinear regression ‘comparison of fits’ analyses were performed between third-order polynomial, cubic trends (alternative hypothesis; that is, rhythmic) versus first-order polynomial, straight line trends (null hypothesis; non-rhythmic). Circadian blots were also assessed using one-way ANOVA with Tukey’s multiple-comparison tests. For behavioural locomotor testing involving two viral treatments and multiple timepoints, repeated-measures two-way ANOVA was performed with subsequent Šidák’s post hoc analyses for multiple comparisons, as well as a posteriori Student’s t-tests (as indicated in the text). For biochemical quantifications of MLL–SETD1 activity (LC–MS/MS), two-way ANOVA was used with Šidák’s multiple-comparison tests. For peptide IP/western blotting experiments with recombinant WDR5 (or MLL1), data were assessed using one-way ANOVA with Tukey’s multiple-comparison tests. All animals used were included as separate ns (samples were not pooled). Significance was determined at P < 0.05. All data are represented as mean ± s.e.m. Statistical analyses were performed in GraphPad Prism 9.

Inclusion and ethics statement

All collaborators associated with this work have fulfilled the criteria for authorship required by Nature Portfolio journals. To obtain authorship, their participation in this study was deemed to be essential for the design and implementation of the work presented. Roles and responsibilities were agreed upon among collaborators ahead or during the research.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.