Abstract

For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur1. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism.

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Data availability

The X-ray structures (coordinates and structure factor files) of SETD3 with bound actin peptide have been submitted to the PDB under accession numbers 6MBJ (P21), 6MBK (two complexes in P212121) and 6MBL (one complex in P212121). Source Data for this study are provided included in the online version of the paper for Figs. 4d, 5b, e, f, i, k and Extended Data Figs. 7e, 9c, h. Gel source data are provided as Supplementary Fig. 1 for cropped images shown in Fig. 1a–e, 2a, c, 3a–g, 4c, 5g, j and Extended Data Fig. 1b. Mass spectrometry data associated with Fig. 1c and Extended Data Fig. 1h are provided in Supplementary Tables 1. Mass spectrometry data associated with Fig. 3i are provided in Supplementary Tables 2. Additional requests for data can be made to the corresponding authors.

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Acknowledgements

We thank J. Drozak and colleagues for sharing their independent identification of SETD3 as the actin-His73 methyltransferase22 and members of the Gozani laboratory for critical reading of the manuscript. This work was supported in part by grants from the NIH to O.G. (R01 GM079641), J.E.C. (DP2 AI104557 and U19 AI109662), X.C. (R01 GM114306), J.A.S. (GM33289), and a CPRIT grant to X.C. (RR160029). J.E.E. received support from Stanford ChEM-H. J.E.C. is supported by an AAF Scholar Award.

Reviewer information

Nature thanks S. Richard and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author contributions

A.W.W. performed biochemical and molecular experiments, and was responsible for experimental design and execution, data analysis and manuscript preparation. A.W.W. performed and analysed mass spectrometry experiments with help from S.L., T.-M.L. and J.E.E. C.M.N. and J.D. performed mouse experiments with help from Y.S.O. K.M.C. and J.G.V.-M. performed histopathology. J.M. and T.C. analysed plasma amino acids. S.D., J.R.H., X.Z. and X.C. performed kinetic experiments and determined X-ray structures. D.S., D.V.T. and C.L. provided myosin and analysed actin–myosin interactions, supervised by K.M.R. and J.A.S. O.G., J.E.C. and X.C. supervised the research, interpreted data and prepared the manuscript.

Author information

Author notes

  1. These authors contributed equally: Alex W. Wilkinson, Jonathan Diep, Shaobo Dai

  2. These authors jointly supervised this work: Xiaodong Cheng, Jan E. Carette, Or Gozani

Affiliations

  1. Department of Biology, Stanford University, Stanford, CA, USA

    • Alex W. Wilkinson
    • , Shuo Liu
    • , Tie-Mei Li
    •  & Or Gozani
  2. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

    • Jonathan Diep
    • , Yaw Shin Ooi
    •  & Jan E. Carette
  3. Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    • Shaobo Dai
    • , John R. Horton
    • , Xing Zhang
    •  & Xiaodong Cheng
  4. Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA

    • Dan Song
    • , Chao Liu
    • , Darshan V. Trivedi
    • , Katherine M. Ruppel
    •  & James A. Spudich
  5. Department of Comparative Medicine, Stanford University School of Medicine, Stanford, CA, USA

    • José G. Vilches-Moure
    • , Kerriann M. Casey
    •  & Claude M. Nagamine
  6. Stanford Healthcare, Palo Alto, CA, USA

    • Justin Mak
  7. Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA

    • Tina Cowan
  8. Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA

    • Joshua E. Elias

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Competing interests

O.G. is a co-founder of EpiCypher Inc. and Athelas Therapeutics Inc.

Corresponding authors

Correspondence to Xiaodong Cheng or Jan E. Carette or Or Gozani.

Extended data figures and tables

  1. Extended Data Fig. 1 Identification of actin as a SETD3 substrate.

    a, Top, domain structure of SETD3 containing an N-terminal SET domain and a C-terminal domain homologous to plant Rubisco LSMT. Bottom, alignment of the homologous methyltransferases human SETD3 and human SETD6. Red box, putative catalytic tyrosine. b, Methylation reactions as in Fig. 1a with non-radiolabelled SAM and analysed by western blot with indicated antibodies. Total histone H3 is shown as a loading control. c, SETD3 localizes to the cytoplasm. Representative immunofluorescence images of GFP or GFP–SETD3 localization in HeLa cells (left) are merged with DAPI counterstaining (right). Scale bars, 7 μm. dg, Biochemical enrichment of a candidate SETD3 substrate. d, Schematic of biochemical strategy to identify the methylated band indicated in Fig. 1b. e, In vitro methylation reactions using cell extracts separated by size-exclusion chromatography as a substrate. Reactions were performed with either wild-type (WT) SETD3 or a putative catalytic mutant (Y312A). Reactions were analysed as in Fig. 1. Arrowhead, candidate substrate. f, Ion-exchange chromatography separates candidate substrates. Fractions positive for SETD3-specific methylation by size-exclusion chromatography were further separated by ion-exchange chromatography and either the flow through (FT) or the pooled fractions containing a notable silver-stained band (arrowhead) at the size of the candidate substrate were used as substrate in an in vitro methylation reaction. g, Flow through or pooled eluent from ion-exchange chromatography were used as substrate for methylation reactions as in e. Arrowhead, protein band in eluent that was analysed by mass spectrometry. h, Top candidate substrates with molecular weights (MW) that were similar to the size of the SETD3-dependent band from in vitro reactions with cell extracts. Candidates identified by mass spectrometry are ranked by abundance determined by MS/MS count (Supplementary Table 1). i, In vitro methylation reactions with SETD3 on actin, recombinant histone H3 (rH3), FOXM1 or a no substrate as a control. Top, 3H-SAM is the methyl donor and methylation visualized by autoradiography. Bottom, Coomassie staining of proteins in the reaction. j, In vitro methylation reactions with the indicated enzymes and substrates. All experiments were repeated at least three times with similar results.

  2. Extended Data Fig. 2 SETD3 does not methylate β-actin lysines.

    GST–β-actin was expressed in bacteria, and cleaved using PreScission Protease. Cleaved β-actin was used in an in vitro methylation assay with SETD3 and deuterated SAM. Spectra are representative of experiments independently performed three times with similar results. MS/MS spectra with identified ions for unmethylated lysine residues produced with indicated proteases. a, K18 (trypsin) (identified N-terminal GPLGS amino acids are residual, vector-specific amino acids from the original GST fusion protein). b, Lys50 (trypsin). c, Lys61 (trypsin). d, Lys68 (trypsin). e, Lys84 (chymotrypsin). f, Lys113 (trypsin). g, Lys118 (Glu-C). h, Lys191 (trypsin). i, Lys213 (trypsin). j, Lys215 (Glu-C). k, Lys238 (chymotrypsin). l, Lys284/K291 (chymotrypsin). m, Lys315 and Lys326 (trypsin). n, Lys326 and Lys328 (trypsin). o, Lys336 (trypsin). p, Lys373 (trypsin).

  3. Extended Data Fig. 3 SETD3 generates actin-His73(3-me).

    ac, MS/MS identifies His73 of actin as a SETD3-methylated residue. a, Top, methylated tryptic peptide with indicated b and y ions. Modifications: me, methylation; ox, oxidation. Bottom, m/z for b and y ions identified in MS/MS spectra from methylation reactions with (+SETD3) or without (−SETD3) using deuterated SAM as the methyl donor (+17.03 Da mass shift). Peptides containing His73 are indicated by an asterisk. b, c, In vitro methylation reactions with (b) and without (c, negative control) SETD3 and β-actin with deuterated SAM were analysed by mass spectrometry. Left, MS/MS spectra of tryptic peptides containing His73. Right, b and y ions identified from oxidized peptides in each spectrum are indicated. d, Dot blot loading control for Fig. 2c of biotinylated ACTB peptides (amino acids 66–80) that are unmodified at His73 or methylated in the N3 (3-me) or N1 (1-me) position. Serial dilutions of the peptides were visualized by streptavidin–HRP and chemiluminescence. eg, Methylation reaction kinetics for SETD3 and the actin peptide. e, Time course of SETD3 with His(1-me) or His(3-me) modified peptides as substrate. Inset, unmodified peptide using 80-fold lower enzyme concentrations compared to the concentrations used for modified peptides. f, g, Steady-state kinetics of SETD3 for the human β-actin peptide (residues 66–80) (f) and SAM (g). Kinetic measurements were performed using a bioluminescence methyltransferase assay, MTase-Glo (Promega). Data are mean ± s.d. of biological triplicates for each data point. ag, Data are representative of three independent experiments with similar results.

  4. Extended Data Fig. 4 Methylation of the conserved actin-His73 residue is present in diverse organisms.

    a, Summary of actin histidine methylation on the conserved His73 residue among model organisms. Abundance of methylation is reported as a percentage of peptide that is methylated. b, Chromatograms of MH3+ ions (m/z ± 10 p.p.m.) for methylated and unmethylated versions of the indicated peptides. The unmethylated actin peptide that was analysed for each species is shown above the corresponding chromatograms. When α-actin peptides were detected, associated chromatograms and quantification are provided on the right. The area of indicated peaks was normalized to the sum of the area between peptides (with or without methylation) and the percentage abundance is labelled. Chromatograms that quantify actin histidine methylation in human and mouse can be found in Extended Data Figs. 6, 7. N.D., not detected. Quantification represents data from two independent experiments with similar results.

  5. Extended Data Fig. 5 Structural details of SETD3–actin peptide interactions.

    a, Overall structure of SETD3 with a V-shaped cleft constructed by the SET domain (green) and an LSMT-like domain (cyan). Helices are shown as cylinders and strands as arrows. The actin peptide is shown as a stick model. b, View of the target histidine through the channel from the SAH-binding pocket. c, Omit electron density of FoFc, contoured at 3.0σ above the mean, is shown for omitting cofactor SAH and the actin peptide used for co-crystallization. d, Details of inter- and intramolecular interactions between SETD3 (green) and actin peptide (yellow). e, Trp79 of actin bound in a hydrophobic surface pocket of SETD3. f, Thr77 and Asn78 of actin form hydrogen bonds with Asn153 and Gln254 of SETD3. g, Val76 of actin is in van der Waals contact with SETD3 residues His323 and Arg315, which in turn interacts with Glu72 of actin. h, Ile75 of actin is in van der Waals contact with main-chain Cα of Asn255 and Gln256 of SETD3. i, Gly74 of actin is located in the amino end of a SETD3 helix. The imidazole ring of His73 (the substrate target) is parallel with the aromatic ring of Tyr312 of SETD3. j, SETD3 Arg315 bridges between the carboxylate oxygen of Glu72 and the main-chain carbonyl oxygen of His73 of actin. k, Ile71 of actin is accommodated in a surface hydrophobic pocket of SETD3. l, Tyr69 and Pro70 of actin interact with a stretch of SETD3 residues from Ile283 to Leu289. m, Tyr69 of actin packs against Pro258 of SETD3 and Lys68 of actin interacts with Glu290 of SETD3. n, Leu67 and Pro70 of actin form an intramolecular interaction and both interact with Ile283 and Thr284 of SETD3. o, Alignment of the amino acids from all six human actin isoforms corresponding to β-actin amino acids 66–80. Variant amino acids are highlighted in yellow. p, The cofactor (SAH) binding site includes residues Arg253, Tyr312, Asn277 and His278 of SETD3.

  6. Extended Data Fig. 6 SETD3 is required for actin methylation in cells.

    a, Peptide dot blot spotted with biotinylated β-actin peptides (amino acids 66–80) containing His73, His73(3-me) or His73(1-me). Blots were probed with a His73(3-me)-specific antibody or streptavidin as a loading control. bd, Chromatograms for quantification of actin-His73me in human cells. Stoichiometry of oxidized β/γ-actin His73 peptide (YPIEHGIVTNWDDM(ox)EK) with and without methylation after purification from cells. MH3+ m/z: unmethylated, 654.968 ± 10 p.p.m.; methylated, 659.6401 ± 10 p.p.m. α-Actin peptide was not detected in these cells. Quantification performed as in Extended Data Fig. 4. b, Chromatograms of HT1080 cells treated with CRISPR–Cas9 that is targeted with a control sgRNA or SETD3-specific sgRNAs. c, Chromatograms of actin His73 methylation in wild-type HeLa cells or clonal HeLa SETD3 knockout cells. N.D., not detected. d, Chromatograms of actin His73 methylation in HT1080 cells treated with CRISPR–Cas9 targeted with either a control or SETD3-specific sgRNA and complemented with CRISPR-resistant SETD3(WT), SETD3(NHY) or control plasmids. Experiments were independently performed three times with similar results.

  7. Extended Data Fig. 7 SETD3 is required for actin-His73 methylation in mice.

    ad, Chromatograms for the quantification of actin histidine methylation in mouse tissues. a, Chromatograms to determine the abundance of histidine methylation of the β/γ-actin His73 peptide (YPIEHGIVTNWDDM(ox)EK) with and without methylation after purification from brain tissue of mice with the indicated genotypes. MH3+ m/z: unmethylated, 654.968 ± 10 p.p.m.; methylated, 659.640 ± 10 p.p.m. α-Actin peptide was not detected in these cells. Quantification performed as in Extended Data Fig. 4. b, Chromatograms to determine the abundance of histidine methylation on α-actin H75 peptide (YPIEHGIITNWDDM(ox)EK) from skeletal muscle as in a. MH3+ m/z: unmethylated, 659.640 ± 10 p.p.m.; methylated, 664.312 ± 10 p.p.m. The β/γ-actin peptide was not detected in these cells. c, Chromatograms to determine the abundance of β/γ-actin His73 methylation from uterine tissue as in a. d, Chromatograms to determine the abundance of α-actin H75 methylation from uterine tissue as in b. Quantification was independently performed three times with similar results. e, Quantitative amino acid panel from mouse blood serum. Quantitative profiling of amino acids in plasma is used clinically to diagnose metabolic disorders. 3-methyl histidine (indicated by an asterisk) is one of the amino acids measured in the panel, and actin is thought to be the primary source of this metabolite. Amino acid levels from Setd3−/− (KO, n = 8) mice were normalized to levels of amino acids from animals with normal His73 methylation levels (total, n = 12; Setd3+/+ (WT), n = 5; Setd3+/− (HET), n = 7). Standard error of the difference between two means is shown for the indicated n. Source data

  8. Extended Data Fig. 8 SETD3-dependent actin-His73 methylation modestly regulates polymerization.

    a, b, Purification of actin with and without His73 methylation. a, Coomassie-stained gel of the actin purified from HeLa cell lines described in Fig. 3f for actin(+me) or actin(−me), and used in the biochemical assays described in Fig. 4a, b and in ce. b, Mass spectrometry quantification of actin His73 methylation from a. Representative data from an experiment performed at least three times with similar results. c, Methylation does not alter actin depolymerization rates. Actin polymerized as in Fig. 4a was diluted to 0.02 μM and depolymerization was monitored by fluorescence normalized to initial values. d, Elongation of the indicated monomeric actin (1 μM, 0.1 μM pyrene–actin) measured in the presence of 2 μM phalloidin–actin seeds made with methylated (circles) or unmethylated (triangles) actin. e, Arp2/3 complex-induced actin polymerization performed in the presence of 100 nM WASP VCA and 5 nM Arp2/3 complex with 1 μM of the indicated monomeric actin and 0.1 μM pyrene–actin. ce, Mean values plotted with s.e.m. from three independent biological replicates. f, SETD3 promotes cell migration. Representative images of cell migration assays performed three times with similar results using cells from Fig. 4c. A circular void of cells was created at the start of the assay (0 h, dashed red circle). After 24 h of migration, cells were fixed and stained with DAPI. Scale bars, 100 μm.

  9. Extended Data Fig. 9 Analysis of SETD3 and actin-His73me in parturition and uterine smooth muscle contraction.

    a, b, Histology of muscle tissues from Setd3+/+ and Setd3−/− mice. a, Haematoxylin and eosin staining of aorta, colon, heart, tongue and hind limb muscle from Setd3+/+ and Setd3−/− mice. b, Tongue and hind limb (striated muscle) sections from a were re-imaged without a condenser to highlight the sarcoplasmic striations characteristic of this muscle type. Scale bars, 20 μm. Images from three independent experiments gave similar results. c, Quantification of pups per litter for Setd3+/+ (n = 12), Setd3+/− (n = 43), Setd3−/− (n = 26) mothers. ***P value ≤ 0.001. d, e, Labour induction at 19 d.p.c. does not rescue dystocia of Setd3−/− pregnant mice. d, Schematic of prostaglandin treatment protocol. The PGF2α cocktail was administered at 0, 3 and 8 h on day 19 with euthanasia and quantification of fetuses at 32 h after the first treatment. e, Quantification of the births and post-term fetuses in utero for the indicated genotypes at the time shown in the schematic. Note: controls delivered before treatment commenced. f, Collagen contraction assay as in Fig. 5h with indicated reconstitution cell lines from Fig. 5j performed with three independent biological replicates. g, h, Actin-His73me does not notably alter myosin activity. g, In vitro actin motility assay. Gliding velocities of actin filaments prepared from actin described in Fig. 4 were measured using human β-cardiac sS1 myosin or a hyperactive mutant (H251N). Data are mean ± s.e.m. velocities from four different experiments. h, Actin-activated ATPase of human β-cardiac sS1 using actin as in g. The Michaelis–Menten equation was fitted to the ATPase data for wild-type actin (solid line) and knockout actin (dashed line). Points are means from two independent experiments. For gel source data, see Supplementary Fig. 1. Source data

  10. Extended Data Table 1 Summary of X-ray data collection from the SER-CAT beamline (22-ID) at a wavelength of 1 Å and refinement statistics

Supplementary information

  1. Supplementary Figure 1

    Source gel data for this study: Fig. 1a, 1d, 1e, 2a, 2c, 3a-f,g, 4c, 5g, 5j; Extended Data Fig. 1b.

  2. Reporting Summary

  3. Supplementary Table 1

    Mass spectrometry identifications of candidate SETD3 substrates after ion-exchange chromatography.

  4. Supplementary Table 2

    SETD3-dependent H/K/R-methylated peptides identified from HeLa cells. Peptide ratios are presented as transformed and log2-transformed. Forward experiment: HeLa WT, light; HeLa SETD3 KO, heavy. Reverse experiment: HeLa WT, heavy; HeLa SETD3 KO, light.

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https://doi.org/10.1038/s41586-018-0821-8

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