A loss-of-function variant in SUV39H2 identified in autism-spectrum disorder causes altered H3K9 trimethylation and dysregulation of protocadherin ß-cluster genes in the developing brain.

Recent evidence has documented the potential roles of histone-modifying enzymes in autism-spectrum disorder (ASD). Aberrant histone H3 lysine 9 (H3K9) dimethylation resulting from genetic variants in histone methyltransferases is known for neurodevelopmental and behavioral anomalies. However, a systematic examination of H3K9 methylation dynamics in ASD is lacking. Here we resequenced nine genes for histone methyltransferases and demethylases involved in H3K9 methylation in individuals with ASD and healthy controls using targeted next-generation sequencing. We identified a novel rare variant (A211S) in the SUV39H2, which was predicted to be deleterious. The variant showed strongly reduced histone methyltransferase activity in vitro. In silico analysis showed that the variant destabilizes the hydrophobic core and allosterically affects the enzyme activity. The Suv39h2-KO mice displayed hyperactivity and reduced behavioral flexibility in learning the tasks that required complex behavioral adaptation, which is relevant for ASD. The Suv39h2 deficit evoked an elevated expression of a subset of protocadherin ß (Pcdhb) cluster genes in the embryonic brain, which is attributable to the loss of H3K9 trimethylation (me3) at the gene promoters. Reduced H3K9me3 persisted in the cerebellum of Suv39h2-deficient mice to an adult stage. Congruently, reduced expression of SUV39H1 and SUV39H2 in the postmortem brain samples of ASD individuals was observed, underscoring the role of H3K9me3 deficiency in ASD etiology. The present study provides direct evidence for the role of SUV39H2 in ASD and suggests a molecular cascade of SUV39H2 dysfunction leading to H3K9me3 deficiency followed by an untimely, elevated expression of Pcdhb cluster genes during early neurodevelopment.

First outbreak of Haemophilus influenzae clone ST422 with low susceptibility to quinolones in paediatric patients in Japan.

Introduction. Recently, a Haemophilus influenzae clone with low susceptibility to quinolones emerged in paediatric patients in Japan. Isolates of this clone survived for a long time when exposed to the therapeutic concentration of quinolones, despite being classified as 'susceptible' under the criteria of the Clinical and Laboratory Standards Institute. In the present study, we report the first outbreak of this clone in paediatric patients in 2018.Aim. Our aim was to characterise the first outbreak of an H. influenzae clone with low susceptibility to quinolones.Methodology. All H. influenzae isolates (n=62), collected at a Japanese teaching hospital in 2018, were characterized by both antimicrobial susceptibility tests and multilocus sequence typing. In addition, the similarity in genetic backgrounds was analysed by PFGE.Results. Among all the isolates (n=62), quinolone low-susceptible isolates accounted for 19.4 % (n=12). Seven out of 12 isolates were identified as sequence type 422 (ST422) and showed more than 90 % similarity to each other by PFGE analysis. All ST422 isolates exhibited identical amino acid substitutions in both quinolone resistance-determining regions in GyrA and ParC. In addition, all these isolates were from paediatric patients who had been referred by different primary care clinics and had no relationship to each other.Conclusion. In this study, we describe an outbreak of a quinolone low-susceptible ST422 clone in paediatric patients in Japan. Because ST422 isolates have already been reported in at least five other countries, it has the potential to spread worldwide.

H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly.

The methylation of histone H3 at lysine 9 (H3K9me), performed by the methyltransferase Clr4/SUV39H, is a key event in heterochromatin assembly. In fission yeast, Clr4, together with the ubiquitin E3 ligase Cul4, forms the Clr4 methyltransferase complex (CLRC), whose physiological targets and biological role are currently unclear. Here, we show that CLRC-dependent H3 ubiquitylation regulates Clr4's methyltransferase activity. Affinity-purified CLRC ubiquitylates histone H3, and mass spectrometric and mutation analyses reveal that H3 lysine 14 (H3K14) is the preferred target of the complex. Chromatin immunoprecipitation analysis shows that H3K14 ubiquitylation (H3K14ub) is closely associated with H3K9me-enriched chromatin. Notably, the CLRC-mediated H3 ubiquitylation promotes H3K9me by Clr4, suggesting that H3 ubiquitylation is intimately linked to the establishment and/or maintenance of H3K9me. These findings demonstrate a cross-talk mechanism between histone ubiquitylation and methylation that is involved in heterochromatin assembly.

Emergence of Haemophilus influenzae with low susceptibility to quinolones and persistence in tosufloxacin treatment.

BACKGROUND: The use of non-ß-lactam agents has increased in Japan due to the prevalence of ß-lactam-resistant pathogens. This study aimed to clarify the recent trend of antimicrobial susceptibility and molecular epidemiological features in Haemophilus influenzae. METHODS: Fifty-seven Haemophilus influenzae isolated from a Japanese teaching hospital in 2017 were characterised, and the data were compared with those of a previous study. The MICs were determined using the broth dilution method. Genetic backgrounds were compared by multilocus sequence typing. The bactericidal activity of tosufloxacin at, or near, the therapeutic Cmax was determined in vitro, with susceptible isolates and quinolone low-susceptible isolates by time-kill assay. RESULTS: The results of the susceptibility tests showed that >90% of isolates were susceptible to cephalosporins and carbapenems, whereas ampicillin-susceptible and clarithromycin-susceptible isolates decreased. Regarding quinolones, low-susceptible isolates were noted in 2017, although all isolates were judged as susceptible. All low-susceptible isolates had an amino acid substitution in GyrA, and two isolates had an additional substitution in ParC. These isolates had different genetic backgrounds. Furthermore, the time-kill kinetic assay using the Cmax of tosufloxacin indicated that the low-susceptible isolates could persist for at least 8hours. CONCLUSIONS: This study revealed that Haemophilus influenzae has demonstrated multidrug low-susceptibility in recent years. The low-susceptible isolates had genetic diversity, meaning that resistance occurred independently.

Isolation of multidrug-resistant Haemophilus influenzae harbouring multiple exogenous genes from a patient diagnosed with acute sinusitis.

In paediatric patients, ß-lactams and macrolides are widely used to treat acute otitis media and sinusitis, which are often caused by either Streptococcus pneumoniae or Haemophilus influenzae. However, resistant isolates have emerged and are becoming more prevalent. H. influenzae generally acquires antimicrobial resistance by mutation or by expression of ß-lactamase. In this study, we isolated H. influenzae from a paediatric patient diagnosed with acute sinusitis. This strain harboured multiple exogenous resistance genes: blaTEM-1, mef(A) and tet(M). DNA sequencing suggested that both mef(A) and tet(M) had been transferred from S. pneumoniae or another Streptococcus. This typical outpatient had not been exposed to excessive levels of antibiotics and had no underlying diseases, strongly suggesting that this type of resistant isolate could become more prevalent.

Preparation of Cell Lysates of Fission Yeast for Immunoprecipitation.

Immunoprecipitation is one of the most important and widely used techniques for the detection and purification of a protein of interest. Thanks to highly specific interaction between antigen and antibody, a target protein is purified and concentrated effectively. To obtain reasonable amounts of a target protein, it is crucially important to prepare total cell lysates in which the target protein is present in a soluble form. Here, we describe methods to prepare total cell lysates of fission yeast, which are then used directly for immunoprecipitation. We also describe some tips to select reagents for preparing buffers having a substantial impact on protein solubility, because there is essentially no reagent that can accommodate the full range of proteins having different characteristics.

Impact of nucleic acid and methylated H3K9 binding activities of Suv39h1 on its heterochromatin assembly.

SUV39H is the major histone H3 lysine 9 (H3K9)-specific methyltransferase that targets pericentric regions and is crucial for assembling silent heterochromatin. SUV39H recognizes trimethylated H3K9 (H3K9me3) via its chromodomain (CD), and enriched H3K9me3 allows SUV39H to target specific chromosomal regions. However, the detailed targeting mechanisms, especially for naïve chromatin without preexisting H3K9me3, are poorly understood. Here we show that Suv39h1's CD (Suv39h1-CD) binds nucleic acids, and this binding is important for its function in heterochromatin assembly. Suv39h1-CD had higher binding affinity for RNA than DNA, and its ability to bind nucleic acids was independent of its H3K9me3 recognition. Suv39h1 bound major satellite RNAs in vivo, and knockdown of major satellite RNAs lowered Suv39h1 retention on pericentromere. Suv39h1 mutational studies indicated that both the nucleic acid-binding and H3K9me-binding activities of Suv39h1-CD were crucial for its pericentric heterochromatin assembly. These results suggest that chromatin-bound RNAs contribute to creating SUV39H's target specificity.

Mitochondrial localization of fission yeast manganese superoxide dismutase is required for its lysine acetylation and for cellular stress resistance and respiratory growth.

Manganese-dependent superoxide dismutase (MnSOD) is localized in the mitochondria and is important for oxidative stress resistance. Although transcriptional regulation of MnSOD has been relatively well studied, much less is known about the protein's posttranslational regulation. In budding yeast, MnSOD is activated after mitochondrial import by manganese ion incorporation. Here we characterize posttranslational modification of MnSOD in the fission yeast Schizosaccharomyces pombe. Fission yeast MnSOD is acetylated at the 25th lysine residue. This acetylation was diminished by deletion of N-terminal mitochondrial targeting sequence, suggesting that MnSOD is acetylated after import into mitochondria. Mitochondrial localization of MnSOD is not essential for the enzyme activity, but is crucial for oxidative stress resistance and growth under respiratory conditions of fission yeast. These results suggest that, unlike the situation in budding yeast, S. pombe MnSOD is already active even before mitochondrial localization; nonetheless, mitochondrial localization is critical to allow the cell to cope with reactive oxygen species generated inside or outside of mitochondria.

Marine antifungal theonellamides target 3beta-hydroxysterol to activate Rho1 signaling.

Linking bioactive compounds to their cellular targets is a central challenge in chemical biology. Here we report the mode of action of theonellamides, bicyclic peptides derived from marine sponges. We generated a chemical-genomic profile of theonellamide F using a collection of fission yeast strains in which each open reading frame (ORF) is expressed under the control of an inducible promoter. Clustering analysis of the Gene Ontology (GO) terms associated with the genes that alter drug sensitivity suggested a mechanistic link between theonellamide and 1,3-beta-D-glucan synthesis. Indeed, theonellamide F induced overproduction of 1,3-beta-D-glucan in a Rho1-dependent manner. Subcellular localization and in vitro binding assays using a fluorescent theonellamide derivative revealed that theonellamides specifically bind to 3beta-hydroxysterols, including ergosterol, and cause membrane damage. The biological activity of theonellamides was alleviated in mutants defective in ergosterol biosynthesis. Theonellamides thus represent a new class of sterol-binding molecules that induce membrane damage and activate Rho1-mediated 1,3-beta-D-glucan synthesis.

Methylation of ribosomal protein L42 regulates ribosomal function and stress-adapted cell growth.

Lysine methylation is one of the most common protein modifications. Although lysine methylation of histones has been extensively studied and linked to gene regulation, that of non-histone proteins remains incompletely understood. Here, we show a novel regulatory role of ribosomal protein methylation. Using an in vitro methyltransferase assay, we found that Schizosaccharomyces pombe Set13, a SET domain protein encoded by SPAC688.14, specifically methylates lysine 55 of ribosomal protein L42 (Rpl42). Mass spectrometric analysis revealed that endogenous Rpl42 is monomethylated at lysine 55 in wild-type S. pombe cells and that the methylation is lost in Delta set13 mutant cells. Delta set13 and Rpl42 methylation-deficient mutant S. pombe cells showed higher cycloheximide sensitivity and defects in stress-responsive growth control compared with wild type. Genetic analyses suggested that the abnormal growth phenotype was distinct from the conserved stress-responsive pathway that modulates translation initiation. Furthermore, the Rpl42 methylation-deficient mutant cells showed a reduced ability to survive after entering stationary phase. These results suggest that Rpl42 methylation plays direct roles in ribosomal function and cell proliferation control independently of the general stress-response pathway.

A novel series of vectors for chromosomal integration in fission yeast.

A series of fission yeast targeting vectors that can be used for wild-type strains having no selectable markers have been designed. The functions of one of three marker genes, lys1(+), arg1(+), and his3(+), involved in amino acid synthesis, are impaired by integration of the fragments generated by restriction enzyme digestion of the plasmids. Successful integration of the fragments into the targeted loci can be readily verified by their requirement for amino acids, or by the PCR diagnostic analysis. Since these selection markers are not used commonly in fission yeast, these plasmids are likely to facilitate studies that require the co-expression of genes such as co-localization and co-immunoprecipitation experiments, by employing them in combination with most of the previously reported markers.

A series of promoters for constitutive expression of heterologous genes in fission yeast.

Inducible/repressible promoters are useful for the maintenance of toxic genes or timely expression. For ectopic expression of cloned genes in the fission yeast Schizosaccharomyces pombe, the thiamine-regulatable nmt1 promoter has been widely used, since the transcriptional activity of this promoter can be controlled by thiamine. However, this property sometimes limits a certain type of research, since the expression inevitably requires cells to be cultivated under the conditions that induce promoter activation. To allow constitutive expression of heterologous genes, we cloned three promoters of cam1+, tif51+ and ef1a-c+. Construction of a series of vectors comprising these promoters and their introduction into the fission yeast cells demonstrated that the activity was different among these promoters but was not affected by cultured media commonly used in fission yeast. Therefore, a promoter with appropriate strength would be selectable from these promoters, depending on the genes to be expressed.

Global analysis of gel mobility of proteins and its use in target identification.

SDS-PAGE is a basic method that has long been used for separation of proteins according to their molecular sizes. Despite its simplicity, it provides information on characteristics of proteins beyond their molecular masses because gel mobility of proteins often reflects their physicochemical properties and post-translational modifications. Here we report on a global analysis of gel mobility of the proteome, which we term the "mobilitome," covering 93.4% of the fission yeast proteome. To our surprise, more than 40% of proteins did not migrate to their calculated positions. Statistical analyses revealed that the discrepancy was largely dependent on the hydrophobicity of proteins. This experimental data set, with a high coverage rate of real mobility, made it feasible to identify proteins detected on the gel without using any specialized techniques. This approach enabled us to detect previously unknown post-translational modifications of a protein; for example, we revealed that eIF5A is novel substrate of a Sir2-related deacetylase Hst2. Furthermore, we concomitantly identified twelve acetylated and eight methylated proteins using specific anti-acetylated and anti-methylated lysine antibodies, most of which had not been known to be subject to the modifications. Thus, we propose the general usefulness of the mobilitome and electrophoresis-based methodology for the identification and characterization of proteins detected on the gel.

ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe.

Cloning of the entire set of an organism's protein-coding open reading frames (ORFs), or 'ORFeome', is a means of connecting the genome to downstream 'omics' applications. Here we report a proteome-scale study of the fission yeast Schizosaccharomyces pombe based on cloning of the ORFeome. Taking advantage of a recombination-based cloning system, we obtained 4,910 ORFs in a form that is readily usable in various analyses. First, we evaluated ORF prediction in the fission yeast genome project by expressing each ORF tagged at the 3' terminus. Next, we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein. Furthermore, using leptomycin B, an inhibitor of the nuclear export protein Crm1, we identified 285 proteins whose localization is regulated by Crm1.

pDUAL, a multipurpose, multicopy vector capable of chromosomal integration in fission yeast.

A novel series of plasmid vectors named pDUAL have been developed. These vectors enable one to introduce not only multicopies of genes with episomal maintenance but also a single copy with chromosomal integration into the fission yeast, Schizosaccharomyces pombe. The multicopy plasmids can be easily converted to fragments for chromosomal integration by digestion of the plasmids with a certain restriction endonuclease before transformation of the yeast cells. The resultant fragments, lacking the autonomously replicating sequence, are designed for targeting into the chromosomal leu1 locus by homologous recombination. Whether the transformants are the results of episomal maintenance of the plasmid or homologous gene targeting can be readily checked by their requirement for uracil or leucine, or by the PCR diagnostic analysis. Furthermore, we propose the use of pDUAL derivatives for PCR-based chromosomal tagging of a gene to introduce several tags into 5'-terminus of a gene, employing a set of primers. Using these all-in-one vectors, a suitable mode of expression of a cloned gene can be selected for individual analysis without any complicated subcloning processes.