Role of Mammalian DNA Methyltransferases in Development.

DNA methylation at the 5-position of cytosine (5mC) plays vital roles in mammalian development. DNA methylation is catalyzed by DNA methyltransferases (DNMTs), and the two DNMT families, DNMT3 and DNMT1, are responsible for methylation establishment and maintenance, respectively. Since their discovery, biochemical and structural studies have revealed the key mechanisms underlying how DNMTs catalyze de novo and maintenance DNA methylation. In particular, recent development of low-input genomic and epigenomic technologies has deepened our understanding of DNA methylation regulation in germ lines and early stage embryos. In this review, we first describe the methylation machinery including the DNMTs and their essential cofactors. We then discuss how DNMTs are recruited to or excluded from certain genomic elements. Lastly, we summarize recent understanding of the regulation of DNA methylation dynamics in mammalian germ lines and early embryos with a focus on both mice and humans.

RNA 5-methylcytosine marks mitochondrial double-stranded RNAs for degradation and cytosolic release.

Mitochondria are essential regulators of innate immunity. They generate long mitochondrial double-stranded RNAs (mt-dsRNAs) and release them into the cytosol to trigger an immune response under pathological stress conditions. Yet the regulation of these self-immunogenic RNAs remains largely unknown. Here, we employ CRISPR screening on mitochondrial RNA (mtRNA)-binding proteins and identify NOP2/Sun RNA methyltransferase 4 (NSUN4) as a key regulator of mt-dsRNA expression in human cells. We find that NSUN4 induces 5-methylcytosine (m5C) modification on mtRNAs, especially on the termini of light-strand long noncoding RNAs. These m5C-modified RNAs are recognized by complement C1q-binding protein (C1QBP), which recruits polyribonucleotide nucleotidyltransferase to facilitate RNA turnover. Suppression of NSUN4 or C1QBP results in increased mt-dsRNA expression, while C1QBP deficiency also leads to increased cytosolic mt-dsRNAs and subsequent immune activation. Collectively, our study unveils the mechanism underlying the selective degradation of light-strand mtRNAs and establishes a molecular mark for mtRNA decay and cytosolic release.

Base-resolution m5C profiling across the mammalian transcriptome by bisulfite-free enzyme-assisted chemical labeling approach.

5-methylcytosine (m5C) is a prevalent RNA modification crucial for gene expression regulation. However, accurate and sensitive m5C sites identification remains challenging due to severe RNA degradation and reduced sequence complexity during bisulfite sequencing (BS-seq). Here, we report m5C-TAC-seq, a bisulfite-free approach combining TET-assisted m5C-to-f5C oxidation with selective chemical labeling, therefore enabling direct base-resolution m5C detection through pre-enrichment and C-to-T transitions at m5C sites. With m5C-TAC-seq, we comprehensively profiled the m5C methylomes in human and mouse cells, identifying a substantially larger number of confident m5C sites. Through perturbing potential m5C methyltransferases, we deciphered the responsible enzymes for most m5C sites, including the characterization of NSUN5's involvement in mRNA m5C deposition. Additionally, we characterized m5C dynamics during mESC differentiation. Notably, the mild reaction conditions and preservation of nucleotide composition in m5C-TAC-seq allow m5C detection in chromatin-associated RNAs. The accurate and robust m5C-TAC-seq will advance research into m5C methylation functional investigation.

Selective chemical tracking of Dnmt1 catalytic activity in live cells.

Enzymatic methylation of cytosine to 5-methylcytosine in DNA is a fundamental epigenetic mechanism involved in mammalian development and disease. DNA methylation is brought about by collective action of three AdoMet-dependent DNA methyltransferases, whose catalytic interactions and temporal interplay are poorly understood. We used structure-guided engineering of the Dnmt1 methyltransferase to enable catalytic transfer of azide tags onto DNA from a synthetic cofactor analog, Ado-6-azide, in vitro. We then CRISPR-edited the Dnmt1 locus in mouse embryonic stem cells to install the engineered codon, which, following pulse internalization of the Ado-6-azide cofactor by electroporation, permitted selective azide tagging of Dnmt1-specific genomic targets in cellulo. The deposited covalent tags were exploited as "click" handles for reading adjoining sequences and precise genomic mapping of the methylation sites. The proposed approach, Dnmt-TOP-seq, enables high-resolution temporal tracking of the Dnmt1 catalysis in mammalian cells, paving the way to selective studies of other methylation pathways in eukaryotic systems.

Mechanism and function of oxidative reversal of DNA and RNA methylation.

The importance of eukaryotic DNA methylation [5-methylcytosine (5mC)] in transcriptional regulation and development was first suggested almost 40 years ago. However, the molecular mechanism underlying the dynamic nature of this epigenetic mark was not understood until recently, following the discovery that the TET proteins, a family of AlkB-like Fe(II)/α-ketoglutarate-dependent dioxygenases, can oxidize 5mC to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Since then, several mechanisms that are responsible for processing oxidized 5mC derivatives to achieve DNA demethylation have emerged. Our biochemical understanding of the DNA demethylation process has prompted new investigations into the biological functions of DNA demethylation. Characterization of two additional AlkB family proteins, FTO and ALKBH5, showed that they possess demethylase activity toward N(6)-methyladenosine (m(6)A) in RNA, indicating that members of this subfamily of dioxygenases have a general function in demethylating nucleic acids. In this review, we discuss recent advances in this emerging field, focusing on the mechanism and function of TET-mediated DNA demethylation.

RNA 5-Methylcytosine Facilitates the Maternal-to-Zygotic Transition by Preventing Maternal mRNA Decay.

The maternal-to-zygotic transition (MZT) is a conserved and fundamental process during which the maternal environment is converted to an environment of embryonic-driven development through dramatic reprogramming. However, how maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein, through genome-wide profiling of RNA 5-methylcytosine (m5C) modification in zebrafish early embryos, we found that m5C-modified maternal mRNAs display higher stability than non-m5C-modified mRNAs during MZT. We discovered that Y-box binding protein 1 (Ybx1) preferentially recognizes m5C-modified mRNAs through π-π interactions with a key residue, Trp45, in Ybx1's cold shock domain (CSD), which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Together with the mRNA stabilizer Pabpc1a, Ybx1 promotes the stability of its target mRNAs in an m5C-dependent manner. Our study demonstrates an unexpected mechanism of RNA m5C-regulated maternal mRNA stabilization during zebrafish MZT, highlighting the critical role of m5C mRNA modification in early development.

Programming crystallization kinetics of self-assembled DNA crystals with 5-methylcytosine modification.

Self-assembled DNA crystals offer a precise chemical platform at the ångström-scale for DNA nanotechnology, holding enormous potential in material separation, catalysis, and DNA data storage. However, accurately controlling the crystallization kinetics of such DNA crystals remains challenging. Herein, we found that atomic-level 5-methylcytosine (5mC) modification can regulate the crystallization kinetics of DNA crystal by tuning the hybridization rates of DNA motifs. We discovered that by manipulating the axial and combination of 5mC modification on the sticky ends of DNA tensegrity triangle motifs, we can obtain a series of DNA crystals with controllable morphological features. Through DNA-PAINT and FRET-labeled DNA strand displacement experiments, we elucidate that atomic-level 5mC modification enhances the affinity constant of DNA hybridization at both the single-molecule and macroscopic scales. This enhancement can be harnessed for kinetic-driven control of the preferential growth direction of DNA crystals. The 5mC modification strategy can overcome the limitations of DNA sequence design imposed by limited nucleobase numbers in various DNA hybridization reactions. This strategy provides a new avenue for the manipulation of DNA crystal structure, valuable for the advancement of DNA and biomacromolecular crystallography.

Epigenetic marks or not? The discovery of novel DNA modifications in eukaryotes.

DNA modifications add another layer of complexity to the eukaryotic genome to regulate gene expression, playing critical roles as epigenetic marks. In eukaryotes, the study of DNA epigenetic modifications has been confined to 5mC and its derivatives for decades. However, rapid developing approaches have witnessed the expansion of DNA modification reservoirs during the past several years, including the identification of 6mA, 5gmC, 4mC, and 4acC in diverse organisms. However, whether these DNA modifications function as epigenetic marks requires careful consideration. In this review, we try to present a panorama of all the DNA epigenetic modifications in eukaryotes, emphasizing recent breakthroughs in the identification of novel DNA modifications. The characterization of their roles in transcriptional regulation as potential epigenetic marks is summarized. More importantly, the pathways for generating or eliminating these DNA modifications, as well as the proteins involved are comprehensively dissected. Furthermore, we briefly discuss the potential challenges and perspectives, which should be taken into account while investigating novel DNA modifications.

DNA methylation: a historical perspective.

In 1925, 5-methylcytosine was first reported in bacteria. However, its biological importance was not intuitive for several decades. After this initial lag, the ubiquitous presence of this methylated base emerged across all domains of life and revealed a range of essential biological functions. Today, we are armed with the knowledge of the key factors that establish, maintain, and remove DNA methylation and have access to a staggering and rapidly growing number of base-resolution methylation maps. Despite this, several fundamental details about the precise role and interpretation of DNA methylation patterns remain under investigation. Here, we review the field of DNA methylation from its beginning to present day, with an emphasis on findings in mammalian systems, and point the reader to select experiments that form the foundation of this field.

Translational adaptation to heat stress is mediated by RNA 5-methylcytosine in Caenorhabditis elegans.

Methylation of carbon-5 of cytosines (m5 C) is a post-transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual m5 C-methyltransferases have been studied, the impact of the global cytosine-5 methylome on development, homeostasis and stress remains unknown. Here, using Caenorhabditis elegans, we generated the first organism devoid of m5 C in RNA, demonstrating that this modification is non-essential. Using this genetic tool, we determine the localisation and enzymatic specificity of m5 C sites in the RNome in vivo. We find that NSUN-4 acts as a dual rRNA and tRNA methyltransferase in C. elegans mitochondria. In agreement with leucine and proline being the most frequently methylated tRNA isoacceptors, loss of m5 C impacts the decoding of some triplets of these two amino acids, leading to reduced translation efficiency. Upon heat stress, m5 C loss leads to ribosome stalling at UUG triplets, the only codon translated by an m5 C34-modified tRNA. This leads to reduced translation efficiency of UUG-rich transcripts and impaired fertility, suggesting a role of m5 C tRNA wobble methylation in the adaptation to higher temperatures.

Germline MBD4 deficiency causes a multi-tumor predisposition syndrome.

We report an autosomal recessive, multi-organ tumor predisposition syndrome, caused by bi-allelic loss-of-function germline variants in the base excision repair (BER) gene MBD4. We identified five individuals with bi-allelic MBD4 variants within four families and these individuals had a personal and/or family history of adenomatous colorectal polyposis, acute myeloid leukemia, and uveal melanoma. MBD4 encodes a glycosylase involved in repair of G:T mismatches resulting from deamination of 5'-methylcytosine. The colorectal adenomas from MBD4-deficient individuals showed a mutator phenotype attributable to mutational signature SBS1, consistent with the function of MBD4. MBD4-deficient polyps harbored somatic mutations in similar driver genes to sporadic colorectal tumors, although AMER1 mutations were more common and KRAS mutations less frequent. Our findings expand the role of BER deficiencies in tumor predisposition. Inclusion of MBD4 in genetic testing for polyposis and multi-tumor phenotypes is warranted to improve disease management.

No evidence for epitranscriptomic m5C modification of SARS-CoV-2, HIV and MLV viral RNA.

The addition of chemical groups to cellular RNA to modulate RNA fate and/or function is summarized under the term epitranscriptomic modification. More than 170 different modifications have been identified on cellular RNA, such as tRNA, rRNA and, to a lesser extent, on other RNA types. Recently, epitranscriptomic modification of viral RNA has received considerable attention as a possible additional mechanism regulating virus infection and replication. N6-methyladenosine (m6A) and C5-methylcytosine (m5C) have been most broadly studied in different RNA viruses. Various studies, however, reported varying results with regard to number and extent of the modification. Here we investigated the m5C methylome of SARS-CoV-2, and we reexamined reported m5C sites in HIV and MLV. Using a rigorous bisulfite-sequencing protocol and stringent data analysis, we found no evidence for the presence of m5C in these viruses. The data emphasize the necessity for optimizing experimental conditions and bioinformatic data analysis.

MLm5C: A high-precision human RNA 5-methylcytosine sites predictor based on a combination of hybrid machine learning models.

RNA modification serves as a pivotal component in numerous biological processes. Among the prevalent modifications, 5-methylcytosine (m5C) significantly influences mRNA export, translation efficiency and cell differentiation and are also associated with human diseases, including Alzheimer's disease, autoimmune disease, cancer, and cardiovascular diseases. Identification of m5C is critically responsible for understanding the RNA modification mechanisms and the epigenetic regulation of associated diseases. However, the large-scale experimental identification of m5C present significant challenges due to labor intensity and time requirements. Several computational tools, using machine learning, have been developed to supplement experimental methods, but identifying these sites lack accuracy and efficiency. In this study, we introduce a new predictor, MLm5C, for precise prediction of m5C sites using sequence data. Briefly, we evaluated eleven RNA sequence-derived features with four basic machine learning algorithms to generate baseline models. From these 44 models, we ranked them based on their performance and subsequently stacked the Top 20 baseline models as the best model, named MLm5C. The MLm5C outperformed the-state-of-the-art predictors. Notably, the optimization of the sequence length surrounding the modification sites significantly improved the prediction performance. MLm5C is an invaluable tool in accelerating the detection of m5C sites within the human genome, thereby facilitating in the characterization of their roles in post-transcriptional regulation.

Tethered Oligonucleotide-Primed Sequencing, TOP-Seq: A High-Resolution Economical Approach for DNA Epigenome Profiling.

Modification of CG dinucleotides in DNA is part of epigenetic regulation of gene function in vertebrates and is associated with complex human disease. Bisulfite sequencing permits high-resolution analysis of cytosine modification in mammalian genomes; however, its utility is often limited due to substantial cost. Here, we describe an alternative epigenome profiling approach, named TOP-seq, which is based on covalent tagging of individual unmodified CG sites followed by non-homologous priming of the DNA polymerase action at these sites to directly produce adjoining regions for their sequencing and precise genomic mapping. Pilot TOP-seq analyses of bacterial and human genomes showed a better agreement of TOP-seq with published bisulfite sequencing maps as compared to widely used MBD-seq and MRE-seq and permitted identification of long-range and gene-level differential methylation among human tissues and neuroblastoma cell types. Altogether, we propose an affordable single CG-resolution technique well suited for large-scale epigenome studies.

Epigenetic regulation of DNA repair gene program by Hippo/YAP1-TET1 axis mediates sorafenib resistance in HCC.

Hepatocellular carcinoma (HCC) is a malignancy that occurs worldwide and is generally associated with poor prognosis. The development of resistance to targeted therapies such as sorafenib is a major challenge in clinical cancer treatment. In the present study, Ten-eleven translocation protein 1 (TET1) was found to be highly expressed in sorafenib-resistant HCC cells and knockdown of TET1 can substantially improve the therapeutic effect of sorafenib on HCC, indicating the potential important roles of TET1 in sorafenib resistance in HCC. Mechanistic studies determined that TET1 and Yes-associated protein 1 (YAP1) synergistically regulate the promoter methylation and gene expression of DNA repair-related genes in sorafenib-resistant HCC cells. RNA sequencing indicated the activation of DNA damage repair signaling was extensively suppressed by the TET1 inhibitor Bobcat339. We also identified TET1 as a direct transcriptional target of YAP1 by promoter analysis and chromatin-immunoprecipitation assays in sorafenib-resistant HCC cells. Furthermore, we showed that Bobcat339 can overcome sorafenib resistance and synergized with sorafenib to induce tumor eradication in HCC cells and mouse models. Finally, immunostaining showed a positive correlation between TET1 and YAP1 in clinical samples. Our findings have identified a previously unrecognized molecular pathway underlying HCC sorafenib resistance, thus revealing a promising strategy for cancer therapy.

5-methylcytosine (m5C) RNA modification controls the innate immune response to virus infection by regulating type I interferons.

5-methylcytosine (m5C) is one of the most prevalent modifications of RNA, playing important roles in RNA metabolism, nuclear export, and translation. However, the potential role of RNA m5C methylation in innate immunity remains elusive. Here, we show that depletion of NSUN2, an m5C methyltransferase, significantly inhibits the replication and gene expression of a wide range of RNA and DNA viruses. Notably, we found that this antiviral effect is largely driven by an enhanced type I interferon (IFN) response. The antiviral signaling pathway is dependent on the cytosolic RNA sensor RIG-I but not MDA5. Transcriptome-wide mapping of m5C following NSUN2 depletion in human A549 cells revealed a marked reduction in the m5C methylation of several abundant noncoding RNAs (ncRNAs). However, m5C methylation of viral RNA was not noticeably altered by NSUN2 depletion. In NSUN2-depleted cells, the host RNA polymerase (Pol) III transcribed ncRNAs, in particular RPPH1 and 7SL RNAs, were substantially up-regulated, leading to an increase of unshielded 7SL RNA in cytoplasm, which served as a direct ligand for the RIG-I-mediated IFN response. In NSUN2-depleted cells, inhibition of Pol III transcription or silencing of RPPH1 and 7SL RNA dampened IFN signaling, partially rescuing viral replication and gene expression. Finally, depletion of NSUN2 in an ex vivo human lung model and a mouse model inhibits viral replication and reduces pathogenesis, which is accompanied by enhanced type I IFN responses. Collectively, our data demonstrate that RNA m5C methylation controls antiviral innate immunity through modulating the m5C methylome of ncRNAs and their expression.

NSUN2-Mediated m5C Methylation Impairs Endometrial Receptivity.

Impaired endometrial decidualization is the primary cause of recurrent implantation failure (RIF). RNA methylation modification, especially NSUN family mediated m5C, is crucial for various physiological events, such as maternal-to-zygotic transition, gametogenesis, embryonic development, organismal lifespan, and cell cycle. However, the regulatory mechanisms between NSUN family mediated m5C modification and RIF remain unknown. We acquired NSUN2 expression data of 15 human endometrium samples at proliferative and secretory stages from reproductive cell atlas. The overall pattern of m5C sites and genes was elucidated through m5C-BS-seq, whereas the overall m5C levels in different groups were revealed by dot blot assay. BrdU and western blotting assays were carried out to evaluate the role of NSUN2 in proliferation and autophagy. The effects of NSUN2-mediated m5C modification on embryo attachment were evaluated by an in vitro model of a confluent monolayer of Ishikawa cells cocultured with BeWo spheroids, and its downstream targets were evaluated by real-time reverse-transcription PCR and western blotting in Ishikawa cells. The molecular mechanism for NSUN2 regulating its downstream targets' expression was determined by Cut&Tag and coimmunoprecipitation assays. NSUN2 was increased in SOX9+ cells and widespread in epithelial cell type at the proliferative stage by previous single-cell RNA sequencing data. NSUN2 overexpression (NSUN2OE) in the Ishikawa cell line elevated m5C levels and promoted cell proliferation and autophagy. NSUN2OE reduced attachment efficiency of BeWo cell spheres. Overexpressed NSUN2 was found to increase STAT1 and MMP14 mRNA expressions by inducing exon skipping. NSUN2 interacted with CLDN4 through m5C modification, and NSUN2OE or NSUN2 knockdown resulted in a similar variation tendency of CLDN4. Overexpression of NSUN2 increased CLDN4 H3K9ac modification by downregulating SIRT4 expression at the protein level, leading to the upregulation of CLDN4 mRNA expression. Our results uncovered a novel intricate regulatory mechanism between NSUN2-mediated m5C and RIF and suggested a potential new therapeutic strategy for RIF.

Pathological remodeling of reactive astrocytes: Involvement of DNA methylation and downregulation of homeostatic genes.

Astrocytes provide metabolic support to neurons, maintain ionic and water homeostasis, and uptake and recycle neurotransmitters. After exposure to the prototypical PAMP lipopolysaccharide (LPS), reactive astrocytes increase the expression of pro-inflammatory genes, facilitating neurodegeneration. In this study, we analyzed the expression of homeostatic genes in astrocytes exposed to LPS and identified the epigenetic factors contributing to the suppression of homeostatic genes in reactive astrocytes. Primary astrocytic cultures were acutely exposed to LPS and allowed to recover for 24, 72 h, and 7 days. As expected, LPS exposure induced reactive astrogliosis and increased the expression of pro-inflammatory IL-1B and IL-6. Interestingly, the acute exposure resulted in persistent hypermethylation of astroglial DNA. Similar hypermethylation was observed in highly reactive astrocytes from the traumatic brain injury (TBI) penumbra in vivo. Hypermethylation was accompanied by decreased expression of homeostatic genes including LDHA and Scl16a1 (MCT1) both involved in the lactate shuttle to neurons; glutamine synthase (GS) responsible for glutamate processing; Kcnj10 (Kir4.1) important for K+ homeostasis, and the water channel aquaporin-4 (Aqp4). Furthermore, the master regulator of DNA methylation, MAFG-1, as well as DNA methyl transferases DNMT1 and DNMT3a were overexpressed. The downregulation of homeostatic genes correlated with increased methylation of CpG islands in their promoters, as assessed by methylation-sensitive PCR and increased DNMT3a binding to the GS promoter. Treatment with decitabine, a DNMT inhibitor, prevented the LPS- and the HMGB-1-induced downregulation of homeostatic genes. Decitabine treatment also prevented the neurotoxic effects of these astrocytes in primary cortical cultures. In summary, our findings reveal that the pathological remodeling of reactive astrocytes encompasses not only the pro-inflammatory response but, significantly, also entails a long-term suppression of homeostatic gene expression with methylation of crucial CpG islands within their promoters.

Epigenetic and Molecular Alterations in Obesity: Linking CRP and DNA Methylation to Systemic Inflammation.

Obesity is marked by excessive fat accumulation in the adipose tissue, which disrupts metabolic processes and causes chronic systemic inflammation. Commonly, body mass index (BMI) is used to assess obesity-related risks, predicting potential metabolic disorders. However, for a better clustering of obese patients, we must consider molecular and epigenetic changes which may be responsible for inflammation and metabolic changes. Our study involved two groups of patients, obese and healthy donors, on which routine analysis were performed, focused on BMI, leukocytes count, and C-reactive protein (CRP) and completed with global DNA methylation and gene expression analysis for genes involved in inflammation and adipogenesis. Our results indicate that obese patients exhibited elevated leukocytes levels, along with increased BMI and CRP. The obese group revealed a global hypomethylation and upregulation of proinflammatory genes, with adipogenesis genes following the same trend of being overexpressed. The study confirms that obesity is linked to systematic inflammation and metabolic dysfunction through epigenetic and molecular alterations. The CRP was correlated with the hypomethylation status in obese patients, and this fact may contribute to a better understanding of the roles of specific genes in adipogenesis and inflammation, leading to a better personalized therapy.

The Emerging Roles of Cytosine-5 Methylation in mRNAs.

Recent studies have unequivocally confirmed the presence of 5-methylcytosine (m5C) in mammalian mRNAs while indicating significant functional roles for this internal base modification type. Here, a brief history of m5C epitranscriptome research and a discussion of the important ways in which the field may now progress is presented.