Histone H3 methylation at lysine 9 (H3K9) is a conserved epigenetic signal, mediating heterochromatin formation by trimethylation, and transcriptional silencing by dimethylation. Defective GLP (Ehmt1) and G9a (Ehmt2) histone lysine methyltransferases, involved in mono and dimethylation of H3K9, confer autistic phenotypes and behavioral abnormalities in animal models. Moreover, EHMT1 loss of function results in Kleefstra syndrome, characterized by severe intellectual disability, developmental delays and psychiatric disorders. We examined the possible role of histone methyltransferases in the etiology of autism spectrum disorders (ASD) and suggest that rare functional variants in these genes that regulate H3K9 methylation may be associated with ASD.

Males and females responses to gestational overnutrition set the stage for subsequent sex-specific differences in adult onset non communicable diseases. Placenta, as a widely recognized programming agent, contibutes to the underlying processes. According to our previous findings, a high-fat diet during gestation triggers sex-specific epigenetic alterations within CpG and throughout the genome, together with the deregulation of clusters of imprinted genes. We further investigated the impact of diet and sex on placental histology, transcriptomic and epigenetic signatures in mice. Both basal gene expression and response to maternal high-fat diet were sexually dimorphic in whole placentas. Numerous genes showed sexually dimorphic expression, but only 11 genes regardless of the diet. In line with the key role of genes belonging to the sex chromosomes, 3 of these genes were Y-specific and 3 were X-specific. Amongst all the genes that were differentially expressed under a high-fat diet, only 16 genes were consistently affected in both males and females. The differences were not only quantitative but remarkably qualitative. The biological functions and networks of genes dysregulated differed markedly between the sexes. Seven genes of the epigenetic machinery were dysregulated, due to effects of diet, sex or both, including the Y- and X-linked histone demethylase paralogues Kdm5c and Kdm5d, which could mark differently male and female epigenomes. The DNA methyltransferase cofactor Dnmt3l gene expression was affected, reminiscent of our previous observation of changes in global DNA methylation. Overall, this striking sexual dimorphism of programming trajectories impose a considerable revision of the current dietary interventions protocols.

Production of knockout mice using targeted embryonic stem cells (ESCs) is a powerful approach for investigating the function of specific genes in vivo. Although the protocol for gene targeting via homologous recombination (HR) in ESCs is already well established, the targeting efficiency varies at different target loci and is sometimes too low. It is known that knockdown of the Bloom syndrome gene, BLM, enhances HR-mediated gene targeting efficiencies in various cell lines. However, it has not yet been investigated whether this approach in ESCs is applicable for successful knockout mouse production. Therefore, we attempted to answer this question. Consistent with previous reports, Blm knockdown enhanced gene targeting efficiencies for three gene loci that we examined by 2.3-4.1-fold. Furthermore, the targeted ESC clones generated good chimeras and were successful in germline transmission. These data suggest that Blm knockdown provides a general benefit for efficient ESC-based and HR-mediated knockout mouse production.

Timing of cell differentiation is strictly controlled and is crucial for normal development and stem cell differentiation. However, underlying mechanisms regulating differentiation timing are fully unknown. Here, we show a molecular mechanism determining differentiation timing from mouse embryonic stem cells (ESCs). Activation of protein kinase A (PKA) modulates differentiation timing to accelerate the appearance of mesoderm and other germ layer cells, reciprocally correlated with the earlier disappearance of pluripotent markers after ESC differentiation. PKA activation increases protein expression of G9a, an H3K9 methyltransferase, along with earlier H3K9 dimethylation and DNA methylation in Oct3/4 and Nanog gene promoters. Deletion of G9a completely abolishes PKA-elicited acceleration of differentiation and epigenetic modification. Furthermore, G9a knockout mice show prolonged expressions of Oct3/4 and Nanog at embryonic day 7.5 and delayed development. In this study, we demonstrate molecular machinery that regulates timing of multilineage differentiation by linking signaling with epigenetics.

Immunoglobulin E (IgE) plays a central role in the pathogenesis of Type I hypersensitivity through interaction with a high-affinity receptor (FcεRIα). For therapeutic applications, substantial attention has been focused recently on the blockade of the IgE interaction with FcεRIα. While exploring better options for preventing allergic diseases, we found that the Fab fragment of the rat anti-murine IgE antibody (Fab-6HD5) strongly inhibited passive cutaneous anaphylaxis (PCA) in vivo, as well as spleen tyrosine kinase (Syk) activity and β-hexosaminidase release from basophilic leukemia cells in vitro. The in vivo effects of Fab-6HD5 pre-administration were maintained over a long period of time for at least 10 days. Using flow cytometry analysis, we also found that Fab-6HD5 did not recognize the IgE Cε3 domain containing specific binding sites for FcεRIα. Furthermore, deletion-mapping studies revealed that Fab-6HD5 recognized conformational epitopes on the Cε2 domain of IgE. Given that the Cε2 domain plays a key role in stabilizing the interaction of IgE with FcRIα, our results suggest that the specific binding of Fab-6HD5 to the Cε2 domain prevents allergic reactions through destabilizing the preformed IgE-FcεRIα complex on rat mast cells. Although the present study was performed using animal models, these findings support the idea that a certain antibody directed against IgE CH domains may contribute to preventing allergic diseases through interacting with IgE-FcεRIα complex.

In most animals, avoiding pathogenic bacteria is crucial for better health and a long life span. For this purpose, animals should be able to quickly sense the presence or uptake of pathogens. The intestine could be a candidate organ to induce escape behaviors; however, the intestinal signaling mechanism for acute regulation of neuronal activity is not well understood. Here, we show that adult Caenorhabditis elegans can respond to the pathogenic bacterium Pseudomonas aeruginosa within 30 min of exposure. This behavior was much faster than previously observed avoidance behaviors in response to P. aeruginosa. By genetic screening, we isolated a mutant defective in this quick avoidance behavior and found that the novel F-box protein FBXC-58 is involved. FBXC-58 is expressed in several tissues, but defective avoidance was rescued by expression of the protein in the intestine. Interestingly, we also found that some but not all mutants in the p38-MAPK and insulin-like signaling pathways, which function in the immune response to pathogens in the intestine, were defective in the quick avoidance behavior to P. aeruginosa. These results suggest that a novel signaling pathway in the intestine exists to regulate neuronal activity for a quick behavioral response.

Recently, advances in next-generation sequencing technologies have enabled genome-wide analyses of epigenetic modifications; however, it remains difficult to analyze the states of histone modifications at a single-cell resolution in living multicellular organisms because of the heterogeneity within cellular populations. Here we describe a simple method to visualize histone modifications on the specific sequence of target locus at a single-cell resolution in living Caenorhabditis elegans, by combining the LacO/LacI system and a genetically-encoded H4K20me1-specific probe, "mintbody". We demonstrate that Venus-labeled mintbody and mTurquoise2-labeled LacI can co-localize on an artificial chromosome carrying both the target locus and LacO sequences, where H4K20me1 marks the target locus. We demonstrate that our visualization method can precisely detect H4K20me1 depositions on the her-1 gene sequences on the artificial chromosome, to which the dosage compensation complex binds to regulate sex determination. The degree of H4K20me1 deposition on the her-1 sequences on the artificial chromosome correlated strongly with sex, suggesting that, using the artificial chromosome, this method can reflect context-dependent changes of H4K20me1 on endogenous genomes. Furthermore, we demonstrate live imaging of H4K20me1 depositions on the artificial chromosome. Combined with ChIP assays, this mintbody-LacO/LacI visualization method will enable analysis of developmental and context-dependent alterations of locus-specific histone modifications in specific cells and elucidation of the underlying molecular mechanisms.

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.

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 β (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.

To study the biological function of uncharacterized proteins, specific antibodies are in high demand. However, the production of desirable antibodies such as highly specific or high affinity is not always successful. Furthermore, even if commercially available antibodies exist, the cost, quality, and accessibility often differ from country to country. In comparison, epitope tags are reliable and economical options since good antibodies against major epitope tags are commercially available. Although exogenously expressed epitope-tagged protein appears as a timely method, the excessive protein production may not faithfully recapitulate its biology. Thanks to the recent advances in genome editing by CRISPR-Cas9, HDR-mediated endogenous protein tagging has become an accessible approach for many labs. However, currently the synthesis of long (>100 bp), chemically modified oligos can be time-consuming and costly. To develop a reliable, simple, and cost-effective epitope-tagging method that requires minimal materials and apparatus, we focus on an approach utilizing two non-chemically modified shorter-annealed oligos (semi-dsODNs) mediated HDR for epitope tags insertion. We also use a cationic lipid chemical, polyethyleneimine (PEI), for plasmid delivery to minimize the cost and materials used while a considerable success rate could be achieved. • This protocol provides a more economical way to generate CRISPR-Cas9 mediated gene knock-in. • This protocol provides a simplified design of semi-dsODN without chemical modification on the oligos. • This protocol provides a simplified experimental procedure. In vitro assembled Cas9 complex and electroporation are not required.

Haploinsufficiency of EHMT1, which encodes histone H3 lysine 9 (H3K9) methyltransferase G9a-like protein (GLP), causes Kleefstra syndrome (KS), a complex disorder of developmental delay and intellectual disability. Here, we examined whether postnatal supply of GLP can reverse the neurological phenotypes seen in Ehmt1 Δ/+ mice as a KS model. Ubiquitous GLP supply from the juvenile stage ameliorated behavioral abnormalities in Ehmt1 Δ/+ mice. Postnatal neuron-specific GLP supply was not sufficient for the improvement of abnormal behaviors but still reversed the reduction of H3K9me2 and spine number in Ehmt1 Δ/+ mice. Interestingly, some inflammatory genes, including IL-1β (Il1b), were upregulated and activated microglial cells increased in the Ehmt1 Δ/+ brain, and such phenotypes were also reversed by neuron-specific postnatal GLP supply. Il1b inactivation canceled the microglial and spine number phenotypes in the Ehmt1 Δ/+ mice. Thus, H3K9me2 and some neurological phenotypes are reversible, but behavioral abnormalities are more difficult to improve depending on the timing of GLP supply.

Liquid-liquid phase separation (LLPS) of proteins and DNAs has been recognized as a fundamental mechanism for the formation of intracellular biomolecular condensates. Here, we show the role of the constituent DNA components, i.e., the phosphate groups, deoxyribose sugars, and nucleobases, in LLPS with a polycationic peptide, linker histone H1, a known key regulator of chromatin condensation. A comparison of the phase behavior of mixtures of H1 and single-stranded DNA-based oligomers in which one or more of the constituent moieties of DNA were removed demonstrated that not only the electrostatic interactions between the anionic phosphate groups of the oligomers and the cationic residues of H1, but also the interactions involving nucleobases and deoxyriboses (i) promoted the generation of spherical liquid droplets via LLPS as well as (ii) increased the density of DNA and decreased its fluidity within the droplets under low-salt conditions. Furthermore, we found the formation of non-spherical assemblies with both mobile and immobile fractions at relatively higher concentrations of H1 for all the oligomers. The roles of the DNA components that promote phase separation and modulate droplet characteristics revealed in this study will facilitate our understanding of the formation processes of the various biomolecular condensates containing nucleic acids, such as chromatin organization.

The Drosophila behavior/human splicing protein family is involved in numerous steps of gene regulation. In humans, this family consists of three proteins: SFPQ, PSPC1, and NONO. Hemizygous loss-of-function (LoF) variants in NONO cause a developmental delay with several complications (e.g., distinctive facial features, cardiac symptoms, and skeletal symptoms) in an X-linked recessive manner. Most of the reported variants have been LoF variants, and two missense variants have been reported as likely deleterious but with no functional validation. We report three individuals from two families harboring an identical missense variant that is located in the nuclear localization signal, NONO: NM_001145408.2:c.1375C > G p.(Pro459Ala). All of them were male and the variant was inherited from their asymptomatic mothers. Individual 1 was diagnosed with developmental delay and cardiac phenotypes (ventricular tachycardia and dilated cardiomyopathy), which overlapped with the features of reported individuals having NONO LoF variants. Individuals 2 and 3 were monozygotic twins. Unlike in Individual 1, developmental delay with autistic features was the only symptom found in them. A fly experiment and cell localization experiment showed that the NONO variant impaired its proper intranuclear localization, leading to mild LoF. Our findings suggest that deleterious NONO missense variants should be taken into consideration when whole-exome sequencing is performed on male individuals with developmental delay with or without cardiac symptoms.

DNA methylation, repressive histone modifications, and PIWI-interacting RNAs are essential for controlling retroelement silencing in mammalian germ lines. Dysregulation of retroelement silencing is associated with male sterility. Although retroelement silencing mechanisms have been extensively studied in mouse germ cells, little progress has been made in humans. Here, we show that the Krüppel-associated box domain zinc finger proteins are associated with DNA methylation of retroelements in human primordial germ cells. Further, we show that the hominoid-specific retroelement SINE-VNTR-Alus (SVA) is subjected to transcription-directed de novo DNA methylation during human spermatogenesis. The degree of de novo DNA methylation in SVAs varies among human individuals, which confers significant inter-individual epigenetic variation in sperm. Collectively, our results highlight potential molecular mechanisms for the regulation of retroelements in human male germ cells.

Protein methylation occurs predominantly on lysine and arginine residues, but histidine also serves as a methylation substrate. However, a limited number of enzymes responsible for this modification have been reported. Moreover, the biological role of histidine methylation has remained poorly understood to date. Here, we report that human METTL18 is a histidine methyltransferase for the ribosomal protein RPL3 and that the modification specifically slows ribosome traversal on Tyr codons, allowing the proper folding of synthesized proteins. By performing an in vitro methylation assay with a methyl donor analog and quantitative mass spectrometry, we found that His245 of RPL3 is methylated at the τ-N position by METTL18. Structural comparison of the modified and unmodified ribosomes showed stoichiometric modification and suggested a role in translation reactions. Indeed, genome-wide ribosome profiling and an in vitro translation assay revealed that translation elongation at Tyr codons was suppressed by RPL3 methylation. Because the slower elongation provides enough time for nascent protein folding, RPL3 methylation protects cells from the cellular aggregation of Tyr-rich proteins. Our results reveal histidine methylation as an example of a ribosome modification that ensures proteome integrity in cells.

Epigenetic mechanisms are essential to establish and safeguard cellular identities in mammals. They dynamically regulate the expression of genes, transposable elements and higher-order chromatin structures. Consequently, these chromatin marks are indispensable for mammalian development and alterations often lead to disease, such as cancer. Bivalent promoters are especially important during differentiation and development. Here we used a genetic screen to identify new regulators of a bivalent repressed gene. We identify BEND3 as a regulator of hundreds of bivalent promoters, some of which it represses, and some of which it activates. We show that BEND3 is recruited to a CpG-containg consensus site that is present in multiple copies in many bivalent promoters. Besides having direct effect on the promoters it binds, the loss of BEND3 leads to genome-wide gains of DNA methylation, which are especially marked at regions normally protected by the TET enzymes. DNA hydroxymethylation is reduced in Bend3 mutant cells, possibly as consequence of altered gene expression leading to diminished alpha-ketoglutarate production, thus lowering TET activity. Our results clarify the direct and indirect roles of an important chromatin regulator, BEND3, and, more broadly, they shed light on the regulation of bivalent promoters.

Point mutations within the TERT promoter are the most recurrent somatic noncoding mutations identified across different cancer types, including glioblastoma, melanoma, hepatocellular carcinoma, and bladder cancer. They are most abundant at -146C > T and -124C > T, and rarer at -57A > C, with the latter originally described as a familial case, but subsequently shown also to occur somatically. All three mutations create de novo E26-specific (ETS) binding sites and result in activation of the TERT gene, allowing cancer cells to achieve replicative immortality. Here, we used a systematic proteomics screen to identify transcription factors preferentially binding to the -146C > T, -124C > T, and -57A > C mutations. Although we confirmed binding of multiple ETS factors to the mutant -146C > T and -124C > T sequences, we identified E4F1 as a -57A > C-specific binder and ZNF148 as a TERT wild-type (WT) promoter binder that showed reduced interaction with the -124C > T allele. Both proteins are activating transcription factors that bind specifically to the -57A > C and WT (at position 124) TERT promoter sequence in corresponding cell lines, and up-regulate TERT transcription and telomerase activity. Our work describes new regulators of TERT gene expression with possible roles in cancer.

Here, we found that the PR domain protein Prdm8 serves as a key regulator of the length of the multipolar phase by controlling the timing of morphological transition. We used a mouse line with expression of Prdm8-mVenus reporter and found that Prdm8 is predominantly expressed in the middle and upper intermediate zone during both the late and terminal multipolar phases. Prdm8 expression was almost coincident with Unc5D expression, a marker for the late multipolar phase, although the expression of Unc5D was found to be gradually down-regulated to the point at which mVenus expression was gradually up-regulated. This expression pattern suggests the possible involvement of Prdm8 in the control of the late and terminal multipolar phases, which controls the timing for morphological transition. To test this hypothesis, we performed gain- and loss-of-function analysis of neocortical development by using in utero electroporation. We found that the knockdown of Prdm8 results in premature change from multipolar to bipolar morphology, whereas the overexpression of Prdm8 maintained the multipolar morphology. Additionally, the postnatal analysis showed that the Prdm8 knockdown stimulated the number of early born neurons, and differentiated neurons located more deeply in the neocortex, however, majority of those cells could not acquire molecular features consistent with laminar location. Furthermore, we found the candidate genes that were predominantly utilized in both the late and terminal multipolar phases, and these candidate genes included those encoding for guidance molecules. In addition, we also found that the expression level of these guidance molecules was inhibited by the introduction of the Prdm8 expression vector. These results indicate that the Prdm8-mediated regulation of morphological changes that normally occur during the late and terminal multipolar phases plays an important role in neocortical development.

In eukaryotic nuclei, DNA is wrapped around an octamer of core histones to form nucleosomes, and chromatin fibers are thought to be stabilized by linker histones of the H1 type. Higher eukaryotes express multiple variants of histone H1; chickens possess six H1 variants. Here, we generated and analyzed the phenotype of a complete deletion of histone H1 genes in chicken cells. The H1-null cells showed decreased global nucleosome spacing, expanded nuclear volumes, and increased chromosome aberration rates, although proper mitotic chromatin structure appeared to be maintained. Expression array analysis revealed that the transcription of multiple genes was affected and was mostly downregulated in histone H1-deficient cells. This report describes the first histone H1 complete knockout cells in vertebrates and suggests that linker histone H1, while not required for mitotic chromatin condensation, plays important roles in nucleosome spacing and interphase chromatin compaction and acts as a global transcription regulator.

The proper tissue-specific regulation of gene expression is essential for development and homeostasis in metazoans. However, the illegitimate expression of normally tissue-restricted genes-like testis- or placenta-specific genes-is frequently observed in tumors; this promotes transformation, but also allows immunotherapy. Two important questions are: how is the expression of these genes controlled in healthy cells? And how is this altered in cancer? To address these questions, we used an unbiased approach to test the ability of 350 distinct genetic or epigenetic perturbations to induce the illegitimate expression of over 40 tissue-restricted genes in primary human cells. We find that almost all of these genes are remarkably resistant to reactivation by a single alteration in signaling pathways or chromatin regulation. However, a few genes differ and are more readily activated; one is the placenta-expressed gene ADAM12, which promotes invasion. Using cellular systems, an animal model, and bioinformatics, we find that a non-canonical but druggable TGF-β/KAT2A/TAK1 axis controls ADAM12 induction in normal and cancer cells. More broadly, our data show that illegitimate gene expression in cancer is an heterogeneous phenomenon, with a few genes activatable by simple events, and most genes likely requiring a combination of events to become reactivated.