Optimized Protocol for the Regulation of DNA Methylation and Gene Expression Using Modified dCas9-SunTag Platforms.

DNA methylation, one of the most studied epigenetic modifications, regulates many biological processes. Dysregulation of DNA methylation is implicated in the etiology of several diseases, such as cancer and imprinting diseases. Accordingly, technologies designed to manipulate DNA methylation at specific loci are considered worthwhile and many epigenome editing technologies have been developed, which were based on ZF, TALE, and CRISPR-dCas9. Here, we describe a protocol for the application of a modified dCas9-SunTag system, which increased the efficiency of targeted demethylation and gene activation at specific DNA loci. The original SunTag system consists of 10 copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve more efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, an enzyme that demethylates DNA, we changed the linker length to 22 amino acids. Moreover, we describe the co-recruitment of TET1 and VP64 for efficient gene activation. Since we showed the manipulation of DNA methylation at specific loci and gene activation, its application could lead to its future use in the clinic.

Improving cell-specific recombination using AAV vectors in the murine CNS by capsid and expression cassette optimization.

The production of cell-type- and age-specific genetically modified mice is a powerful approach for unraveling unknown gene functions. Here, we present a simple and timesaving method that enables adeno-associated virus (AAV)-mediated cell-type- and age-specific recombination in floxed mice. To achieve astrocyte-specific recombination in floxed Ai14 reporter mice, we intravenously injected blood-brain barrier-penetrating AAV-PHP.eB vectors expressing Cre recombinase (Cre) using the astrocyte-specific mouse glial fibrillary acidic protein (mGfaABC1D) promoter. However, we observed nonspecific neuron-predominant transduction despite the use of an astrocyte-specific promoter. We speculated that subtle but continuous Cre expression in nonastrocytic cells triggers recombination, and that excess production of Cre in astrocytes inhibits recombination by forming Cre-DNA aggregates. Here, we resolved this paradoxical event by dividing a single AAV into two mGfaABC1D-promoter-driven AAV vectors, one expressing codon-optimized flippase (FlpO) and another expressing flippase recognition target-flanked rapidly degrading Cre (dCre), together with switching the neuron-tropic PHP.eB capsid to astrocyte-tropic AAV-F. Moreover, we found that the FlpO-dCre system with a target cell-tropic capsid can also function in neuron-targeting recombination in floxed mice.

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.

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.

The N6-methyladenosine methyltransferase METTL16 enables erythropoiesis through safeguarding genome integrity.

During erythroid differentiation, the maintenance of genome integrity is key for the success of multiple rounds of cell division. However, molecular mechanisms coordinating the expression of DNA repair machinery in erythroid progenitors are poorly understood. Here, we discover that an RNA N6-methyladenosine (m6A) methyltransferase, METTL16, plays an essential role in proper erythropoiesis by safeguarding genome integrity via the control of DNA-repair-related genes. METTL16-deficient erythroblasts exhibit defective differentiation capacity, DNA damage and activation of the apoptotic program. Mechanistically, METTL16 controls m6A deposition at the structured motifs in DNA-repair-related transcripts including Brca2 and Fancm mRNAs, thereby upregulating their expression. Furthermore, a pairwise CRISPRi screen revealed that the MTR4-nuclear RNA exosome complex is involved in the regulation of METTL16 substrate mRNAs in erythroblasts. Collectively, our study uncovers that METTL16 and the MTR4-nuclear RNA exosome act as essential regulatory machinery to maintain genome integrity and erythropoiesis.

Protein kinase Cγ in cerebellar Purkinje cells regulates Ca2+-activated large-conductance K+ channels and motor coordination.

The cerebellum, the site where protein kinase C (PKC) was first discovered, contains the highest amount of PKC in the central nervous system, with PKCγ being the major isoform. Systemic PKCγ-knockout (KO) mice showed impaired motor coordination and deficient pruning of surplus climbing fibers (CFs) from developing cerebellar Purkinje cells (PCs). However, the physiological significance of PKCγ in the mature cerebellum and the cause of motor incoordination remain unknown. Using adeno-associated virus vectors targeting PCs, we showed that impaired motor coordination was restored by re-expression of PKCγ in mature PKCγ-KO mouse PCs in a kinase activity-dependent manner, while normal motor coordination in mature Prkcgfl/fl mice was impaired by the Cre-dependent removal of PKCγ from PCs. Notably, the rescue or removal of PKCγ from mature PKCγ-KO or Prkcgfl/fl mice, respectively, did not affect the CF innervation profile of PCs, suggesting the presence of a mechanism distinct from multiple CF innervation of PCs for the motor defects in PKCγ-deficient mice. We found marked potentiation of Ca2+-activated large-conductance K+ (BK) channel currents in PKCγ-deficient mice, as compared to wild-type mice, which decreased the membrane resistance, resulting in attenuation of the electrical signal during the propagation and significant alterations of the complex spike waveform. These changes in PKCγ-deficient mice were restored by the rescue of PKCγ or pharmacological suppression of BK channels. Our results suggest that PKCγ is a critical regulator that negatively modulates BK currents in PCs, which significantly influences PC output from the cerebellar cortex and, eventually, motor coordination.

The Ser19Stop single nucleotide polymorphism (SNP) of human PHYHIPL affects the cerebellum in mice.

The HapMap Project is a major international research effort to construct a resource to facilitate the discovery of relationships between human genetic variations and health and disease. The Ser19Stop single nucleotide polymorphism (SNP) of human phytanoyl-CoA hydroxylase-interacting protein-like (PHYHIPL) gene was detected in HapMap project and registered in the dbSNP. PHYHIPL gene expression is altered in global ischemia and glioblastoma multiforme. However, the function of PHYHIPL is unknown. We generated PHYHIPL Ser19Stop knock-in mice and found that PHYHIPL impacts the morphology of cerebellar Purkinje cells (PCs), the innervation of climbing fibers to PCs, the inhibitory inputs to PCs from molecular layer interneurons, and motor learning ability. Thus, the Ser19Stop SNP of the PHYHIPL gene may be associated with cerebellum-related diseases.

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.

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.

Anephrogenic phenotype induced by SALL1 gene knockout in pigs.

To combat organ shortage in transplantation medicine, a novel strategy has been proposed to generate human organs from exogenous pluripotent stem cells utilizing the developmental mechanisms of pig embryos/foetuses. Genetically modified pigs missing specific organs are key elements in this strategy. In this study, we demonstrate the feasibility of using a genome-editing approach to generate anephrogenic foetuses in a genetically engineered pig model. SALL1 knockout (KO) was successfully induced by injecting genome-editing molecules into the cytoplasm of pig zygotes, which generated the anephrogenic phenotype. Extinguished SALL1 expression and marked dysgenesis of nephron structures were observed in the rudimentary kidney tissue of SALL1-KO foetuses. Biallelic KO mutations of the target gene induced nephrogenic defects; however, biallelic mutations involving small in-frame deletions did not induce the anephrogenic phenotype. Through production of F1 progeny from mutant founder pigs, we identified mutations that could reliably induce the anephrogenic phenotype and hence established a line of fertile SALL1-mutant pigs. Our study lays important technical groundwork for the realization of human kidney regeneration through the use of an empty developmental niche in pig foetuses.

Editing of DNA Methylation Using dCas9-Peptide Repeat and scFv-TET1 Catalytic Domain Fusions.

DNA methylation, one of the most studied epigenetic modifications, regulates many biological processes. Dysregulation of DNA methylation is implicated in the etiology of several diseases, such as cancer and imprinting diseases. Accordingly, technologies designed to manipulate DNA methylation at specific loci are very important, and many epigenome editing technologies have been developed, based on zinc finger proteins, TALEs, and CRISPR/dCas9 targeting. We describe a protocol to induce and assess DNA demethylation on a target gene. It is based on a modification of the dCas9-SunTag system for efficient, targeted demethylation at specific DNA loci. The original SunTag system consists of ten copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, an enzyme that demethylates DNA, we changed the linker length to 22 amino acids.

Genome Engineering Using Haploid Embryonic Stem Cells.

Haploidy is a useful feature for the study of gene function because disruption of one allele in haploid cells, which contain only a single set of chromosomes, can cause loss-of-function phenotypes. Recent success in generating haploid embryonic stem (ES) cells from several mammalian species, including human, provides a new platform for simple genetic manipulation of the mammalian genome. The genome-editing potential of the CRISPR/Cas system is enhanced by the use of haploid ES cells. For example, CRISPR/Cas has been used for high-efficiency generation of multiple knockouts and knockins in haploid ES cells, with potential application in genome-wide screening. In addition, live mice can be successfully obtained by nuclear transplantation of haploid ES cells into oocytes, providing a novel approach for the generation of gene-modified animals.

TWIST1 induces MMP3 expression through up-regulating DNA hydroxymethylation and promotes catabolic responses in human chondrocytes.

The objective was to investigate the levels of TWIST1 in normal and OA cartilage and examine its role in regulating gene expression in chondrocytes. Human cartilage tissues and chondrocytes were obtained at autopsy from normal knee joints and from OA-affected joints at the time of total knee arthroplasty. TWIST1 expression was increased in human OA knee cartilage compared to normal knee cartilage. TWIST1 induced matrix metalloproteinase 3 (MMP3) expression without direct binding to MMP3 promoter and increased the 5-hydroxymethylcytosine (5hmC) level at the MMP3 promoter. The effect of TWIST1 on expression of TET family (TET1, 2 and 3) was measured in stable TWIST1 transfected TC28 cells, and TET1 expression was up-regulated. TWIST1 dependent upregulation of Mmp3 expression was suppressed in Tet triple KO fibroblast derived from mouse ES cells. Increased TWIST1 expression is a feature of OA-affected cartilage. We identified a novel mechanism of catabolic reaction where TWIST1 up-regulates MMP3 expression by enriching 5hmC levels at the MMP3 promoter via TET1 induction. These findings implicate TWIST1 as an important factor regulating OA related gene expression. Clarifying epigenetic mechanisms of 5hmC induced by TWIST1 is a critical molecule to understanding OA pathogenesis.

Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions.

Despite the importance of DNA methylation in health and disease, technologies to readily manipulate methylation of specific sequences for functional analysis and therapeutic purposes are lacking. Here we adapt the previously described dCas9-SunTag for efficient, targeted demethylation of specific DNA loci. The original SunTag consists of ten copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, which induces demethylation, we changed the linker length to 22 amino acids. The system attains demethylation efficiencies >50% in seven out of nine loci tested. Four of these seven loci showed demethylation of >90%. We demonstrate targeted demethylation of CpGs in regulatory regions and demethylation-dependent 1.7- to 50-fold upregulation of associated genes both in cell culture (embryonic stem cells, cancer cell lines, primary neural precursor cells) and in vivo in mouse fetuses.

Regulation of CpG methylation by Dnmt and Tet in pluripotent stem cells.

Vertebrate genomes are highly methylated at cytosine residues in CpG sequences. CpG methylation plays an important role in epigenetic gene silencing and genome stability. Compared with other epigenetic modifications, CpG methylation is thought to be relatively stable; however, it is sometimes affected by environmental changes, leading to epigenetic instability and disease. CpG methylation is reversible and regulated by DNA methyltransferases and demethylases including ten-eleven translocation. Here, we discuss CpG methylation instability and the regulation of CpG methylation by DNA methyltransferases and ten-eleven translocation in pluripotent stem cells.

5-Hydroxymethylcytosine Marks Sites of DNA Damage and Promotes Genome Stability.

5-hydroxymethylcytosine (5hmC) is a DNA base created during active DNA demethylation by the recently discovered TET enzymes. 5hmC has essential roles in gene expression and differentiation. Here, we demonstrate that 5hmC also localizes to sites of DNA damage and repair. 5hmC accumulates at damage foci induced by aphidicolin and microirradiation and colocalizes with major DNA damage response proteins 53BP1 and γH2AX, revealing 5hmC as an epigenetic marker of DNA damage. Deficiency for the TET enzymes eliminates damage-induced 5hmC accumulation and elicits chromosome segregation defects in response to replication stress. Our results indicate that the TET enzymes and 5hmC play essential roles in ensuring genome integrity.

p53 suppresses tetraploid development in mice.

Mammalian tetraploid embryos die in early development because of defects in the epiblast. Experiments with diploid/tetraploid chimeric mice, obtained via the aggregation of embryonic stem cells, clarified that while tetraploid cells are excluded from epiblast derivatives, diploid embryos with tetraploid extraembryonic tissues can develop to term. Today, this method, known as tetraploid complementation, is usually used for rescuing extraembryonic defects or for obtaining completely embryonic stem (ES) cell-derived pups. However, it is still unknown why defects occur in the epiblast during mammalian development. Here, we demonstrated that downregulation of p53, a tumour suppressor protein, rescued tetraploid development in the mammalian epiblast. Tetraploidy in differentiating epiblast cells triggered p53-dependent cell-cycle arrest and apoptosis, suggesting the activation of a tetraploidy checkpoint during early development. Finally, we found that p53 downregulation rescued tetraploid embryos later in gestation.

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.

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.