Crucial role of iron in epigenetic rewriting during adipocyte differentiation mediated by JMJD1A and TET2 activity.

Iron metabolism is closely associated with the pathogenesis of obesity. However, the mechanism of the iron-dependent regulation of adipocyte differentiation remains unclear. Here, we show that iron is essential for rewriting of epigenetic marks during adipocyte differentiation. Iron supply through lysosome-mediated ferritinophagy was found to be crucial during the early stage of adipocyte differentiation, and iron deficiency during this period suppressed subsequent terminal differentiation. This was associated with demethylation of both repressive histone marks and DNA in the genomic regions of adipocyte differentiation-associated genes, 　including Pparg, which encodes PPARγ, the master regulator of adipocyte differentiation. In addition, we identified several epigenetic demethylases to be responsible for iron-dependent adipocyte differentiation, with the histone demethylase jumonji domain-containing 1A and the DNA demethylase ten-eleven translocation 2 as the major enzymes. The interrelationship between repressive histone marks and DNA methylation was indicated by an integrated genome-wide association analysis, and was also supported by the findings that both histone and DNA demethylation were suppressed by either the inhibition of lysosomal ferritin flux or the knockdown of iron chaperone poly(rC)-binding protein 2. In summary, epigenetic regulations through iron-dependent control of epigenetic enzyme activities play an important role in the organized gene expression mechanisms of adipogenesis.

Aberrant activation of estrogen receptor-α signaling in Mettl14-deficient uteri impairs embryo implantation.

The precise control of endometrial receptivity is crucial for successful embryo implantation, which is strictly regulated by the ovarian steroid hormones estrogen and progesterone. Despite our improved understanding of the genetic regulation of implantation downstream of the action of hormones, we do not know much about the epigenetic regulation that occurs during early pregnancy. To investigate the role of the N6-methyladenosine (m6A) RNA modification in embryo implantation, we generated mice with conditional deletion of Mettl14, a core component of the m6A writer complex, in the uterus. These mice were infertile due to implantation failure. We showed that Mettl14-deficient uteri had aberrant upregulation of estrogen receptor α (ERα) signaling and ERα phosphorylation, but progesterone receptor (PGR) signaling was largely unaffected. Additionally, Mettl14 deletion led to abnormal activation of the innate immune pathway in Mettl14-deficient uteri. This effect was accompanied by the infiltration of immune cells, such as macrophages and dendritic cells, into the basal region of the endometrial epithelium. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) showed that genes involved in the innate immune response had decreased m6A peaks in Mettl14-deficient mice. These results suggest that Mettl14 plays a crucial role in successful implantation by precisely regulating both ERα signaling and innate immunity in the uterus.

Multiplexed genome editing by in vivo electroporation of Cas9 ribonucleoproteins effectively induces endometrial carcinoma in mice.

Synergistic effects among multiple gene mutations are involved in cancer development and progression. However, developing genetically modified mouse models to analyze various combinations of mutations is extremely labor-intensive and time-consuming. To address these problems, we developed a novel method for in vivo multiplexed genome editing of the murine uterus to model human endometrial carcinoma (EMC). To do this, we injected a CRISPR-Cas9 ribonucleoprotein complex into the uterine cavity of adult female mice, followed by electroporation. Evaluation of reporter mice demonstrated that genome editing occurred specifically in uterine epithelial cells, which are the origin of EMCs. Simultaneous targeting of Pten/Trp53/Lkb1, or targeting of Pten/Lkb1 along with the Ctnnb1ΔEx3 mutation, resulted in efficient generation of invasive tumors in wild-type females within 3 months. This novel method will enable rapid and easy validation of many combinations of gene mutations that lead to endometrial carcinogenesis.

A Lipid Nanoparticle-Based Method for the Generation of Liver-Specific Knockout Mice.

Knockout mice are useful tools that can provide information about the normal function of genes, including their biochemical, developmental, and physiological roles. One problem associated with the generation of knockout mice is that the loss of some genes of interest produces a lethal phenotype. Therefore, the use of conditioned knockout mice, in which genes are disrupted in specific organs, is essential for the elucidation of disease pathogenesis and the verification of drug targets. In general, conditional knockout mice are produced using the Cre/loxP system; however, the production of the large numbers of Cre/flox knockout and control mice required for analysis requires substantial time and effort. Here, we describe the generation of liver-specific conditional knockout mice via the introduction of lipid nanoparticles encapsulating Cre mRNA into the liver of floxed mice. This technique does not require the production of offspring by mating floxed mice and is therefore more convenient than the conventional method. The results presented here demonstrate that the LNP-based method enables liver-specific gene knockout in a short period of time.

DNA methylation analysis of multiple imprinted DMRs in Sotos syndrome reveals IGF2-DMR0 as a DNA methylation-dependent, P0 promoter-specific enhancer.

Haploinsufficiency of NSD1, which dimethylates histone H3 lysine 36 (H3K36), causes Sotos syndrome (SoS), an overgrowth syndrome. DNMT3A and DNMT3B recognizes H3K36 trimethylation (H3K36me3) through PWWP domain to exert de novo DNA methyltransferase activity and establish imprinted differentially methylated regions (DMRs). Since decrease of H3K36me3 and genome-wide DNA hypomethylation in SoS were observed, hypomethylation of imprinted DMRs in SoS was suggested. We explored DNA methylation status of 28 imprinted DMRs in 31 SoS patients with NSD1 defect and found that hypomethylation of IGF2-DMR0 and IG-DMR in a substantial proportion of SoS patients. Luciferase assay revealed that IGF2-DMR0 enhanced transcription from the IGF2 P0 promoter but not the P3 and P4 promoters. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) revealed active enhancer histone modifications at IGF2-DMR0, with high enrichment of H3K4me1 and H3 lysine 27 acetylation (H3K27ac). CRISPR-Cas9 epigenome editing revealed that specifically induced hypomethylation at IGF2-DMR0 increased transcription from the P0 promoter but not the P3 and P4 promoters. NSD1 knockdown suggested that NSD1 targeted IGF2-DMR0; however, IGF2-DMR0 DNA methylation and IGF2 expression were unaltered. This study could elucidate the function of IGF2-DMR0 as a DNA methylation dependent, P0 promoter-specific enhancer. NSD1 may play a role in the establishment or maintenance of IGF2-DMR0 methylation during the postimplantation period.

Synergistic Upregulation of Target Genes by TET1 and VP64 in the dCas9-SunTag Platform.

Overexpression of a gene of interest is a general approach used in both basic research and therapeutic applications. However, the conventional approach involving overexpression of exogenous genes has difficulty achieving complete genome coverage, and is also limited by the cloning capacity of viral vectors. Therefore, an alternative approach would be to drive the expression of an endogenous gene using an artificial transcriptional activator. Fusion proteins of dCas9 and a transcription activation domain, such as dCas9-VP64, are widely used for activation of endogenous genes. However, when using a single sgRNA, the activation range is low. Consequently, tiling of several sgRNAs is required for robust transcriptional activation. Here we describe the screening of factors that exhibit the best synergistic activation of gene expression with TET1 in the dCas9-SunTag format. All seven factors examined showed some synergy with TET1. Among them, VP64 gave the best results. Thus, simultaneous tethering of VP64 and TET1 to a target gene using an optimized dCas9-SunTag format synergistically activates gene expression using a single sgRNA.

Arid1b Haploinsufficiency Causes Abnormal Brain Gene Expression and Autism-Related Behaviors in Mice.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with core symptoms that include poor social communication, restricted interests, and repetitive behaviors. Several ASD mouse models exhibit impaired social interaction, anxiety-like behavior, and elevated perseveration. Large-scale whole exome sequencing studies identified many genes putatively associated with ASD. Like chromodomain helicase DNA binding protein 8 (CHD8), the most frequently mutated gene in individuals with ASD, the candidate gene AT-rich interaction domain 1B (ARID1B) encodes a chromatin remodeling factor. Arid1b heterozygous knockout (hKO) mice exhibited ASD-like traits related to social behavior, anxiety, and perseveration, in addition to associated features reported in some cases of ASD, such as reduced weight, impaired motor coordination, and hydrocephalus. Hydrocephalus was present in 5 of 91 hKO mice, while it was not observed in wild-type littermates (0 of 188). Genome-wide gene expression patterns in Arid1b hKO mice were similar to those in ASD patients and Chd8-haploinsufficient mice, an ASD model, and to developmental changes in gene expression in fast-spiking cells in the mouse brain. Our results suggest that Arid1b haploinsufficiency causes ASD-like phenotypes in mice.

Posttranscriptional regulation of histone lysine methyltransferase GLP in embryonic male mouse germ cells.

The epigenetic status of germ cells changes dynamically during development. In this study, we analyzed the dynamics of histone H3 lysine 9 dimethylation (H3K9me2), a highly conserved mark of epigenetic silencing, and the expression of two lysine methyltransferases, G9a/Ehmt2/KMT1C and GLP/Ehmt1/KMT1D, in murine male embryonic germ cells after sex determination. Our previous studies established that G9a and GLP are the primary enzymes for H3K9me2 and predominantly exist as a G9a-GLP heteromeric complex that appears to be a functional H3K9 methyltransferase in vivo. During the period from Embryonic Day (E) 13.5 to E18.5 in mice, gonadal H3K9me2 levels were substantially lower in germ cells than in cells of nongerm lineage. Immunohistochemical analysis showed that during this phase in development, GLP level, but not G9a level, was also significantly lower in male germ cells. However, GLP mRNA was present in E13 and E16 male germ cells, with levels similar to those in cells of nongerm lineage. Interestingly, GLP is upregulated in embryonic male germ cells deficient for Nanos2, which encodes a germ cell-specific RNA-binding protein. Our data suggest that GLP protein expression is posttranscriptionally regulated in murine embryonic male germ cells after sex determination and that low H3K9me2 level results from the absence of GLP (severe reduction of the G9a-GLP heteromeric complex).

Generation of an ICF syndrome model by efficient genome editing of human induced pluripotent stem cells using the CRISPR system.

Genome manipulation of human induced pluripotent stem (iPS) cells is essential to achieve their full potential as tools for regenerative medicine. To date, however, gene targeting in human pluripotent stem cells (hPSCs) has proven to be extremely difficult. Recently, an efficient genome manipulation technology using the RNA-guided DNase Cas9, the clustered regularly interspaced short palindromic repeats (CRISPR) system, has been developed. Here we report the efficient generation of an iPS cell model for immunodeficiency, centromeric region instability, facial anomalies syndrome (ICF) syndrome using the CRISPR system. We obtained iPS cells with mutations in both alleles of DNA methyltransferase 3B (DNMT3B) in 63% of transfected clones. Our data suggest that the CRISPR system is highly efficient and useful for genome engineering of human iPS cells.

miR-29 represses the activities of DNA methyltransferases and DNA demethylases.

Members of the microRNA-29 (miR-29) family directly target the DNA methyltransferases, DNMT3A and DNMT3B. Disturbances in the expression levels of miR-29 have been linked to tumorigenesis and tumor aggressiveness. Members of the miR-29 family are currently thought to repress DNA methylation and suppress tumorigenesis by protecting against de novo methylation. Here, we report that members of the miR-29 family repress the activities of DNA methyltransferases and DNA demethylases, which have opposing roles in control of DNA methylation status. Members of the miR-29 family directly inhibited DNA methyltransferases and two major factors involved in DNA demethylation, namely tet methylcytosine dioxygenase 1 (TET1) and thymine DNA glycosylase (TDG). Overexpression of miR-29 upregulated the global DNA methylation level in some cancer cells and downregulated DNA methylation in other cancer cells, suggesting that miR-29 suppresses tumorigenesis by protecting against changes in the existing DNA methylation status rather than by preventing de novo methylation of DNA.

CRISPR/Cas9.

CRISPR/Cas9 is the genome-editing technology that is most widely used around the world. Its widespread adoption is largely due to its simplicity and ease of use. Here, we introduce the construction of vectors and genome editing of the target gene in cells using the CRISPR/Cas9 system.

Regulation of Gene Expression Using dCas9-SunTag Platforms.

Regulating gene expression is important for basic research and therapeutic applications. The epigenome is a record of genetic modifications such as DNA methylation and histone modifications, and epigenetic changes can play a key role in modifying gene expression. With the advent of genome editing technologies, it has become possible to manipulate the epigenome of specific genomic regions to control gene expression. In particular, CRISPR-Cas9 systems have been used widely for epigenome editing due to their high efficiency, versatility, specificity, and ease of use. Here, we describe a protocol for the upregulation of specific genes using the dCas9-SunTag system.

Generation of Epigenetic Disease Model Mice by Targeted Demethylation of the Epigenome.

Epigenetic regulatory mechanisms play an important role in gene silencing and genome stability; therefore, epigenetic mutations cause a variety of diseases. Analysis of the epigenome by next-generation sequencers has revealed many epigenetic mutations in various diseases such as cancer, obesity, diabetes, autism, allergies, immune diseases, and imprinting diseases. Unfortunately, it has been difficult to identify the causative epigenetic mutations because there has been no method to generate animals with target-specific epigenetic mutations. However, it has become possible to generate such animals due to the recent development of epigenome editing technology. Here, we introduce the generation of epigenome-edited mice by target-specific DNA demethylation.

Genome-wide analysis of DNA methylation and expression of microRNAs in breast cancer cells.

DNA methylation of promoters is linked to transcriptional silencing of protein-coding genes, and its alteration plays important roles in cancer formation. For example, hypermethylation of tumor suppressor genes has been seen in some cancers. Alteration of methylation in the promoters of microRNAs (miRNAs) has also been linked to transcriptional changes in cancers; however, no systematic studies of methylation and transcription of miRNAs have been reported. In the present study, to clarify the relation between DNA methylation and transcription of miRNAs, next-generation sequencing and microarrays were used to analyze the methylation and expression of miRNAs, protein-coding genes, other non-coding RNAs (ncRNAs), and pseudogenes in the human breast cancer cell lines MCF7 and the adriamycin (ADR) resistant cell line MCF7/ADR. DNA methylation in the proximal promoter of miRNAs is tightly linked to transcriptional silencing, as it is with protein-coding genes. In protein-coding genes, highly expressed genes have CpG-rich proximal promoters whereas weakly expressed genes do not. This is only rarely observed in other gene categories, including miRNAs. The present study highlights the epigenetic similarities and differences between miRNA and protein-coding genes.

Efficient generation of epigenetic disease model mice by epigenome editing using the piggyBac transposon system.

BACKGROUND: Epigenome-edited animal models enable direct demonstration of disease causing epigenetic mutations. Transgenic (TG) mice stably expressing epigenome-editing factors exhibit dramatic and stable changes in target epigenome modifications. Successful germline transmission of a transgene from founder mice to offspring will yield a sufficient number of epigenome-edited mice for phenotypic analysis; however, if the epigenetic mutation has a detrimental phenotypic effect, it can become difficult to obtain the next generation of animals. In this case, the phenotype of founder mice must be analyzed directly. Unfortunately, current TG mouse production efficiency (TG founders per pups born) is relatively low, and improvements would increase the versatility of this technology. RESULTS: In the current study, we describe an approach to generate epigenome-edited TG mice using a combination of both the dCas9-SunTag and piggyBac (PB) transposon systems. Using this system, we successfully generated mice with demethylation of the differential methylated region of the H19 gene (H19-DMR), as a model for Silver-Russell syndrome (SRS). SRS is a disorder leading to growth retardation, resulting from low insulin-like growth factor 2 (IGF2) gene expression, often caused by epimutations at the H19-IGF2 locus. Under optimized conditions, the efficiency of TG mice production using the PB system was approximately threefold higher than that using the conventional method. TG mice generated by this system showed demethylation of the targeted DNA region and associated changes in gene expression. In addition, these mice exhibited some features of SRS, including intrauterine and postnatal growth retardation, due to demethylation of H19-DMR. CONCLUSIONS: The dCas9-SunTag and PB systems serve as a simple and reliable platform for conducting direct experiments using epigenome-edited founder mice.

The Dnmt3b splice variant is specifically expressed in in vitro-manipulated blastocysts and their derivative ES cells.

Manipulation of preimplantation embryos in vitro, such as in vitro fertilization (IVF), in vitro culture (IVC), intracytoplasmic sperm injection (ICSI), somatic cell nuclear transfer (SCNT) and other assisted reproduction technologies (ART), has contributed to the development of infertility treatment and new animal reproduction methods. However, such embryos often exhibit abnormal DNA methylation patterns in imprinted genes and centromeric satellite repeats. These DNA methylation patterns are established and maintained by three DNA methyltransferases: Dnmt1, Dnmt3a and Dnmt3b. Dnmt3b is responsible for the creation of methylation patterns during the early stage of embryogenesis and consists of many alternative splice variants that affect methylation activity; nevertheless, the roles of these variants have not yet been identified. In this study, we found an alternatively spliced variant of Dnmt3b lacking exon 6 (Dnmt3bΔ6) that is specific to mouse IVC embryos. Dnmt3bΔ6 also showed prominent expression in embryonic stem (ES) cells derived from in vitro manipulated embryos. Interestingly, IVC blastocysts were hypomethylated in centromeric satellite repeat regions that could be susceptible to methylation by Dnmt3b. In vitro methylation activity assays showed that Dnmt3bΔ6 had lower activity than normal Dnmt3b. Our findings suggest that Dnmt3bΔ6 could induce a hypomethylation status especially in in vitro manipulated embryos.

MeCP2-dependent repression of an imprinted miR-184 released by depolarization.

Both fragile X syndrome and Rett syndrome are commonly associated with autism spectrum disorders and involve defects in synaptic plasticity. MicroRNA is implicated in synaptic plasticity because fragile X mental retardation protein was recently linked to the microRNA pathway. DNA methylation is also involved in synaptic plasticity since methyl CpG-binding protein 2 (MeCP2) is mutated in patients with Rett syndrome. Here we report that expression of miR-184, a brain-specific microRNA repressed by the binding of MeCP2 to its promoter, is upregulated by the release of MeCP2 after depolarization. The restricted release of MeCP2 from the paternal allele results in paternal allele-specific expression of miR-184. Our finding provides a clue to the link between the microRNA and DNA methylation pathways.

Calcium-Free and Cytochalasin B Treatment Inhibits Blastomere Fusion in 2-Cell Stage Embryos for the Generation of Floxed Mice via Sequential Electroporation.

The generation of conditional knockout mice using the Cre-loxP system is advantageous for the functional analysis of genes. Flanked by two loxP sites (floxed) mice can be directly obtained from fertilized eggs by the CRISPR/Cas9 genome editing system. We previously reported that sequential knock-in (KI) of each loxP site by electroporation (EP) at the 1- and 2-cell embryonic stages increases the number of mice with floxed alleles compared with simultaneous KI. However, EP at the 2-cell stage frequently induced blastomere fusion. These fused embryos cannot develop to term because they are tetraploidized. In this study, we examined the following three conditions to inhibit blastomere fusion by EP at the 2-cell stage: (1) hypertonic treatment, (2) Calcium (Ca2+)-free treatment, and (3) actin polymerization inhibition. Hypertonic treatment of 2-cell stage embryos prevented blastomere fusion and facilitated blastocyst development; however, KI efficiency was decreased. Ca2+-free treatment and actin polymerization inhibition by cytochalasin B (CB) reduced fusion rate, and did not have negative effects on development and KI efficiency. These results suggest that Ca2+-free and CB treatment at the 2-cell stage is effective to generate floxed mice in combination with a sequential EP method.

Successful generation of epigenetic disease model mice by targeted demethylation of the epigenome.

BACKGROUND: Epigenetic modifications, including DNA methylation, play an important role in gene silencing and genome stability. Consequently, epigenetic dysregulation can cause several diseases, such as cancer, obesity, diabetes, autism, and imprinting disorders. RESULTS: We validate three methods for the generation of epigenome-edited mice using the dCas9-SunTag and single-chain variable fragment-TET1 catalytic domain. We generate model mice for Silver-Russell syndrome (SRS), an imprinting disorder, by target-specific DNA demethylation in the H19 differentially methylated region. Like SRS patients, these mice show H19 upregulation and Igf2 downregulation, leading to severe intrauterine and postnatal growth retardation. CONCLUSION: This is the first report of an imprinting disease model animal generated by targeted demethylation of specific loci of the epigenome in fertilized eggs. Epigenome-edited animals are also useful for exploring the causative epimutations in epigenetic diseases.

Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing.

Recently, we reported PPARα-dependent DNA demethylation of the Fgf21 promoter in the postnatal mouse liver, where reduced DNA methylation is associated with enhanced gene expression after PPARα activation. However, there is no direct evidence for the effect of site-specific DNA methylation on gene expression. We employed the dCas9-SunTag and single-chain variable fragment (scFv)-TET1 catalytic domain (TET1CD) system to induce targeted DNA methylation of the Fgf21 promoter both in vitro and in vivo. We succeeded in targeted DNA demethylation of the Fgf 21 promoter both in Hepa1-6 cells and PPARα-deficient mice, with increased gene expression response to PPARα synthetic ligand administration and fasting, respectively. This study provides direct evidence that the DNA methylation status of a particular gene may determine the magnitude of the gene expression response to activation cues.