Characterization of SETD3 methyltransferase-mediated protein methionine methylation.

Most characterized protein methylation events encompass arginine and lysine N-methylation, and only a few cases of protein methionine thiomethylation have been reported. Newly discovered oncohistone mutations include lysine-to-methionine substitutions at positions 27 and 36 of histone H3.3. In these instances, the methionine substitution localizes to the active-site pocket of the corresponding histone lysine methyltransferase, thereby inhibiting the respective transmethylation activity. SET domain-containing 3 (SETD3) is a protein (i.e. actin) histidine methyltransferase. Here, we generated an actin variant in which the histidine target of SETD3 was substituted with methionine. As for previously characterized histone SET domain proteins, the methionine substitution substantially (76-fold) increased binding affinity for SETD3 and inhibited SETD3 activity on histidine. Unexpectedly, SETD3 was active on the substituted methionine, generating S-methylmethionine in the context of actin peptide. The ternary structure of SETD3 in complex with the methionine-containing actin peptide at 1.9 Å resolution revealed that the hydrophobic thioether side chain is packed by the aromatic rings of Tyr312 and Trp273, as well as the hydrocarbon side chain of Ile310 Our results suggest that placing methionine properly in the active site-within close proximity to and in line with the incoming methyl group of SAM-would allow some SET domain proteins to selectively methylate methionine in proteins.

An engineered variant of SETD3 methyltransferase alters target specificity from histidine to lysine methylation.

Most characterized SET domain (SETD) proteins are protein lysine methyltransferases, but SETD3 was recently demonstrated to be a protein (i.e. actin) histidine-N3 methyltransferase. Human SETD3 shares a high structural homology with two known protein lysine methyltransferases-human SETD6 and the plant LSMT-but differs in the residues constituting the active site. In the SETD3 active site, Asn255 engages in a unique hydrogen-bonding interaction with the target histidine of actin that likely contributes to its >1300-fold greater catalytic efficiency (kcat/Km ) on histidine than on lysine. Here, we engineered active-site variants to switch the SETD3 target specificity from histidine to lysine. Substitution of Asn255 with phenylalanine (N255F), together with substitution of Trp273 with alanine (W273A), generated an active site mimicking that of known lysine methyltransferases. The doubly substituted SETD3 variant exhibited a 13-fold preference for lysine over histidine. We show, by means of X-ray crystallography, that the two target nitrogen atoms-the N3 atom of histidine and the terminal ϵ-amino nitrogen of lysine-occupy the same position and point toward and are within a short distance of the incoming methyl group of SAM for a direct methyl transfer during catalysis. In contrast, SETD3 and its Asn255 substituted derivatives did not methylate glutamine (another potentially methylated amino acid). However, the glutamine-containing peptide competed with the substrate peptide, and glutamine bound in the active site, but too far away from SAM to be methylated. Our results provide insight into the structural parameters defining the target amino acid specificity of SET enzymes.

Enterovirus pathogenesis requires the host methyltransferase SETD3.

Enteroviruses (EVs) comprise a large genus of positive-sense, single-stranded RNA viruses whose members cause a number of important and widespread human diseases, including poliomyelitis, myocarditis, acute flaccid myelitis and the common cold. How EVs co-opt cellular functions to promote replication and spread is incompletely understood. Here, using genome-scale CRISPR screens, we identify the actin histidine methyltransferase SET domain containing 3 (SETD3) as critically important for viral infection by a broad panel of EVs, including rhinoviruses and non-polio EVs increasingly linked to severe neurological disease such as acute flaccid myelitis (EV-D68) and viral encephalitis (EV-A71). We show that cytosolic SETD3, independent of its methylation activity, is required for the RNA replication step in the viral life cycle. Using quantitative affinity purification-mass spectrometry, we show that SETD3 specifically interacts with the viral 2A protease of multiple enteroviral species, and we map the residues in 2A that mediate this interaction. 2A mutants that retain protease activity but are unable to interact with SETD3 are severely compromised in RNA replication. These data suggest a role of the viral 2A protein in RNA replication beyond facilitating proteolytic cleavage. Finally, we show that SETD3 is essential for in vivo replication and pathogenesis in multiple mouse models for EV infection, including CV-A10, EV-A71 and EV-D68. Our results reveal a crucial role of a host protein in viral pathogenesis, and suggest targeting SETD3 as a potential mechanism for controlling viral infections.

Structural basis for the target specificity of actin histidine methyltransferase SETD3.

SETD3 is an actin histidine-N3 methyltransferase, whereas other characterized SET-domain enzymes are protein lysine methyltransferases. We report that in a pre-reactive complex SETD3 binds the N3-protonated form (N3-H) of actin His73, and in a post-reactive product complex, SETD3 generates the methylated histidine in an N1-protonated (N1-H) and N3-methylated form. During the reaction, the imidazole ring of His73 rotates ~105°, which shifts the proton from N3 to N1, thus ensuring that the target atom N3 is deprotonated prior to the methyl transfer. Under the conditions optimized for lysine deprotonation, SETD3 has weak lysine methylation activity on an actin peptide in which the target His73 is substituted by a lysine. The structure of SETD3 with Lys73-containing peptide reveals a bent conformation of Lys73, with its side chain aliphatic carbons tracing along the edge of imidazole ring and the terminal ε-amino group occupying a position nearly identical to the N3 atom of unmethylated histidine.

SETD3 is an actin histidine methyltransferase that prevents primary dystocia.

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.

Molecular and Neural Functions of Rai1, the Causal Gene for Smith-Magenis Syndrome.

Haploinsufficiency of Retinoic Acid Induced 1 (RAI1) causes Smith-Magenis syndrome (SMS), which is associated with diverse neurodevelopmental and behavioral symptoms as well as obesity. RAI1 encodes a nuclear protein but little is known about its molecular function or the cell types responsible for SMS symptoms. Using genetically engineered mice, we found that Rai1 preferentially occupies DNA regions near active promoters and promotes the expression of a group of genes involved in circuit assembly and neuronal communication. Behavioral analyses demonstrated that pan-neural loss of Rai1 causes deficits in motor function, learning, and food intake. These SMS-like phenotypes are produced by loss of Rai1 function in distinct neuronal types: Rai1 loss in inhibitory neurons or subcortical glutamatergic neurons causes learning deficits, while Rai1 loss in Sim1+ or SF1+ cells causes obesity. By integrating molecular and organismal analyses, our study suggests potential therapeutic avenues for a complex neurodevelopmental disorder.

A PWWP Domain of Histone-Lysine N-Methyltransferase NSD2 Binds to Dimethylated Lys-36 of Histone H3 and Regulates NSD2 Function at Chromatin.

The readout of histone modifications plays a critical role in chromatin-regulated processes. Dimethylation at Lys-36 on histone H3 (H3K36me2) is associated with actively transcribed genes, and global up-regulation of this modification is associated with several cancers. However, the molecular mechanism by which H3K36me2 is sensed and transduced to downstream biological outcomes remains unclear. Here we identify a PWWP domain within the histone lysine methyltransferase and oncoprotein NSD2 that preferentially binds to nucleosomes containing H3K36me2. In cells, the NSD2 PWWP domain interaction with H3K36me2 plays a role in stabilizing NSD2 at chromatin. Furthermore, NSD2's ability to induce global increases in H3K36me2 via its enzymatic activity, and consequently promote cellular proliferation, is compromised by mutations within the PWWP domain that specifically abrogate H3K36me2-recognition. Together, our results identify a pivotal role for NSD2 binding to its catalytic product in regulating its cellular functions, and suggest a model for how this interaction may facilitate epigenetic spreading and propagation of H3K36me2.

Structural Basis for the Unique Multivalent Readout of Unmodified H3 Tail by Arabidopsis ORC1b BAH-PHD Cassette.

DNA replication initiation relies on the formation of the origin recognition complex (ORC). The plant ORC subunit 1 (ORC1) protein possesses a conserved N-terminal BAH domain with an embedded plant-specific PHD finger, whose function may be potentially regulated by an epigenetic mechanism. Here, we report structural and biochemical studies on the Arabidopsis thaliana ORC1b BAH-PHD cassette which specifically recognizes the unmodified H3 tail. The crystal structure of ORC1b BAH-PHD cassette in complex with an H3(1-15) peptide reveals a strict requirement for the unmodified state of R2, T3, and K4 on the H3 tail and a novel multivalent BAH and PHD readout mode for H3 peptide recognition. Such recognition may contribute to epigenetic regulation of the initiation of DNA replication.

The PZP Domain of AF10 Senses Unmodified H3K27 to Regulate DOT1L-Mediated Methylation of H3K79.

AF10, a DOT1L cofactor, is required for H3K79 methylation and cooperates with DOT1L in leukemogenesis. However, the molecular mechanism by which AF10 regulates DOT1L-mediated H3K79 methylation is not clear. Here we report that AF10 contains a "reader" domain that couples unmodified H3K27 recognition to H3K79 methylation. An AF10 region consisting of a PHD finger-Zn knuckle-PHD finger (PZP) folds into a single module that recognizes amino acids 22-27 of H3, and this interaction is abrogated by H3K27 modification. Structural studies reveal that H3 binding triggers rearrangement of the PZP module to form an H3(22-27)-accommodating channel and that the unmodified H3K27 side chain is encased in a compact hydrogen-bond acceptor-lined cage. In cells, PZP recognition of H3 is required for H3K79 dimethylation, expression of DOT1L-target genes, and proliferation of DOT1L-addicted leukemic cells. Together, our results uncover a pivotal role for H3K27-via readout by the AF10 PZP domain-in regulating the cancer-associated enzyme DOT1L.

SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer.

Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.

Histone-binding domains: strategies for discovery and characterization.

Chromatin signaling dynamics fundamentally regulate eukaryotic genomes. The reversible covalent post-translational modification (PTM) of histone proteins by chemical moieties such as phosphate, acetyl and methyl groups constitutes one of the primary chromatin signaling mechanisms. Modular protein domains present within chromatin-regulatory activities recognize or "read" specifically modified histone species and transduce these modified species into distinct downstream biological outcomes. Thus, understanding the molecular basis underlying PTM-mediated signaling at chromatin requires knowledge of both the modification and the partnering reader domains. Over the last ten years, a number of innovative approaches have been developed and employed to discover reader domain binding events with histones. Together, these studies have provided crucial insight into how chromatin pathways influence key cellular programs. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.

A molecular wound response program associated with regeneration initiation in planarians.

Planarians are capable of regenerating any missing body part and present an attractive system for molecular investigation of regeneration initiation. The gene activation program that occurs at planarian wounds to coordinate regenerative responses remains unknown. We identified a large set of wound-induced genes during regeneration initiation in planarians. Two waves of wound-induced gene expression occurred in differentiated tissues. The first wave includes conserved immediate early genes. Many second-wave genes encode conserved patterning factors required for proper regeneration. Genes of both classes were generally induced by wounding, indicating that a common initial gene expression program is triggered regardless of missing tissue identity. Planarian regeneration uses a population of regenerative cells (neoblasts), including pluripotent stem cells. A class of wound-induced genes was activated directly within neoblasts, including the Runx transcription factor-encoding runt-1 gene. runt-1 was required for specifying different cell types during regeneration, promoting heterogeneity in neoblasts near wounds. Wound-induced gene expression in neoblasts, including that of runt-1, required SRF (serum response factor) and sos-1. Taken together, these data connect wound sensation to the activation of specific cell type regeneration programs in neoblasts. Most planarian wound-induced genes are conserved across metazoans, and identified genes and mechanisms should be important broadly for understanding wound signaling and regeneration initiation.

The Zn finger protein Iguana impacts Hedgehog signaling by promoting ciliogenesis.

Hedgehog signaling is critical for metazoan development and requires cilia for pathway activity. The gene iguana was discovered in zebrafish as required for Hedgehog signaling, and encodes a novel Zn finger protein. Planarians are flatworms with robust regenerative capacities and utilize epidermal cilia for locomotion. RNA interference of Smed-iguana in the planarian Schmidtea mediterranea caused cilia loss and failure to regenerate new cilia, but did not cause defects similar to those observed in hedgehog(RNAi) animals. Smed-iguana gene expression was also similar in pattern to the expression of multiple other ciliogenesis genes, but was not required for expression of these ciliogenesis genes. iguana-defective zebrafish had too few motile cilia in pronephric ducts and in Kupffer's vesicle. Kupffer's vesicle promotes left-right asymmetry and iguana mutant embryos had left-right asymmetry defects. Finally, human Iguana proteins (dZIP1 and dZIP1L) localize to the basal bodies of primary cilia and, together, are required for primary cilia formation. Our results indicate that a critical and broadly conserved function for Iguana is in ciliogenesis and that this function has come to be required for Hedgehog signaling in vertebrates.