DNA recognition by transcription activator-like effector (TALE) proteins is mediated by tandem repeats that specify nucleotides through repeat-variable diresidues. These repeat-variable diresidues form direct and sequence-specific contacts to DNA bases; hence, TALE-DNA interaction is sensitive to DNA chemical modifications. Here we conduct a thorough investigation, covering all theoretical repeat-variable diresidue combinations, for their recognition capabilities for 5-methylcytosine and 5-hydroxymethylcytosine, two important epigenetic markers in higher eukaryotes. We identify both specific and degenerate repeat-variable diresidues for 5-methylcytosine and 5-hydroxymethylcytosine. Utilizing these novel repeat-variable diresidues, we achieve methylation-dependent gene activation and genome editing in vivo; we also report base-resolution detection of 5hmC in an in vitro assay. Our work deciphers repeat-variable diresidues for 5-methylcytosine and 5-hydroxymethylcytosine, and provides tools for TALE-dependent epigenome recognition.Transcription activator-like effector proteins recognise specific DNA sequences via tandem repeats. Here the authors demonstrate TALEs can recognise the methylated bases 5mC and 5hmC, enabling them to detect epigenetic modifications.

5-Methylcytosine is found in all domains of life, but the bacterial cytosine deaminase from Escherichia coli (CodA) will not accept 5-methylcytosine as a substrate. Since significant amounts of 5-methylcytosine are produced in both prokaryotes and eukaryotes, this compound must eventually be catabolized and the fragments recycled by enzymes that have yet to be identified. We therefore initiated a comprehensive phylogenetic screen for enzymes that may be capable of deaminating 5-methylcytosine to thymine. From a systematic analysis of sequence homologues of CodA from thousands of bacterial species, we identified putative cytosine deaminases where a "discriminating" residue in the active site, corresponding to Asp-314 in CodA from E. coli, was no longer conserved. Representative examples from Klebsiella pneumoniae (locus tag: Kpn00632), Rhodobacter sphaeroides (locus tag: Rsp0341), and Corynebacterium glutamicum (locus tag: NCgl0075) were demonstrated to efficiently deaminate 5-methylcytosine to thymine with values of kcat/Km of 1.4 × 10(5), 2.9 × 10(4), and 1.1 × 10(3) M(-1) s(-1), respectively. These three enzymes also catalyze the deamination of 5-fluorocytosine to 5-fluorouracil with values of kcat/Km of 1.2 × 10(5), 6.8 × 10(4), and 2.0 × 10(2) M(-1) s(-1), respectively. The three-dimensional structure of Kpn00632 was determined by X-ray diffraction methods with 5-methylcytosine (PDB id: 4R85 ), 5-fluorocytosine (PDB id: 4R88 ), and phosphonocytosine (PDB id: 4R7W ) bound in the active site. When thymine auxotrophs of E. coli express these enzymes, they are capable of growth in media lacking thymine when supplemented with 5-methylcytosine. Expression of these enzymes in E. coli is toxic in the presence of 5-fluorocytosine, due to the efficient transformation to 5-fluorouracil.

5-Methylcytosine (m5C) is a well-characterized DNA modification, and is also predominantly reported in abundant non-coding RNAs in both prokaryotes and eukaryotes. However, the distribution and biological functions of m5C in plant mRNAs remain largely unknown. Here, we report transcriptome-wide profiling of RNA m5C in Arabidopsis thaliana by applying m5C RNA immunoprecipitation followed by a deep-sequencing approach (m5C-RIP-seq). LC-MS/MS and dot blot analyses reveal a dynamic pattern of m5C mRNA modification in various tissues and at different developmental stages. m5C-RIP-seq analysis identified 6045 m5C peaks in 4465 expressed genes in young seedlings. We found that m5C is enriched in coding sequences with two peaks located immediately after start codons and before stop codons, and is associated with mRNAs with low translation activity. We further demonstrated that an RNA (cytosine-5)-methyltransferase, tRNA-specific methyltransferase 4B (TRM4B), exhibits m5C RNA methyltransferase activity. Mutations in TRM4B display defects in root development and decreased m5C peaks. TRM4B affects the transcript levels of the genes involved in root development, which is positively correlated with their mRNA stability and m5C levels. Our results suggest that m5C in mRNA is a new epitranscriptome marker inArabidopsis, and that regulation of this modification is an integral part of gene regulatory networks underlying plant development.

Previous studies have revealed that mouse primordial germ cells (PGCs) undergo genome-wide DNA methylation reprogramming to reset the epigenome for totipotency. However, the precise 5-methylcytosine (5mC) dynamics and its relationship with the generation of 5-hydroxymethylcytosine (5hmC) are not clear. Here we analyzed the dynamics of 5mC and 5hmC during PGC reprograming and germ cell development. Unexpectedly, we found a specific period (E8.5-9.5) during which both 5mC and 5hmC levels are low. Subsequently, 5hmC levels increase reaching its peak at E11.5 and gradually decrease until E13.5 likely by replication-dependent dilution. Interestingly, 5hmC is enriched in chromocenters during this period. While this germ cell-specific 5hmC subnuclear localization pattern is maintained in female germ cells even in mature oocytes, such pattern is gradually lost in male germ cells as mitotic proliferation resumes during the neonatal stage. Pericentric 5hmC plays an important role in silencing major satellite repeat, especially in female PGCs. Global transcriptome analysis by RNA-seq revealed that the great majority of differentially expressed genes from E9.5 to 13.5 are upregulated in both male and female PGCs. Although only female PGCs enter meiosis during the prenatal stage, meiosis-related and a subset of imprinted genes are significantly upregulated in both male and female PGCs at E13.5. Thus, our study not only reveals the dynamics of 5mC and 5hmC during PGC reprogramming and germ cell development, but also their potential role in epigenetic reprogramming and transcriptional regulation of meiotic and imprinted genes.

Altered placental function as a consequence of aberrant imprinted gene expression may be one mechanism mediating the association between low birth weight and increased cardiometabolic disease risk. Imprinted gene expression is regulated by epigenetic mechanisms, particularly DNA methylation (5mC) at differentially methylated regions (DMRs). While 5-hydroxymethylcytosine (5hmC) is also present at DMRs, many techniques do not distinguish between 5mC and 5hmC. Using human placental samples, we show that the expression of the imprinted gene CDKN1C associates with birth weight. Using specific techniques to map 5mC and 5hmC at DMRs controlling the expression of CDKN1C and the imprinted gene IGF2, we show that 5mC enrichment at KvDMR and DMR0, and 5hmC enrichment within the H19 gene body, associate positively with birth weight. Importantly, the presence of 5hmC at imprinted DMRs may complicate the interpretation of DNA methylation studies in placenta; future studies should consider using techniques that distinguish between, and permit quantification of, both modifications.

Whilst 5-methylcytosine (5mC) is a major epigenetic mark in the nuclear DNA in mammals, whether or not mitochondrial DNA (mtDNA) receives 5mC modification remains controversial. Herein, we exhaustively analysed mouse mtDNA using three methods that are based upon different principles for detecting 5mC. Next-generation bisulfite sequencing did not give any significant signatures of methylation in mtDNAs of liver, brain and embryonic stem cells (ESCs). Also, treatment with methylated cytosine-sensitive endonuclease McrBC resulted in no substantial decrease of mtDNA band intensities in Southern hybridisation. Furthermore, mass spectrometric nucleoside analyses of highly purified liver mtDNA preparations did not detect 5-methyldeoxycytidine at the levels found in the nuclear DNA but at a range of only 0.3-0.5% of deoxycytidine. Taken together, we propose that 5mC is not present at any specific region(s) of mtDNA and that levels of the methylated cytosine are fairly low, provided the modification occurs. It is thus unlikely that 5mC plays a universal role in mtDNA gene expression or mitochondrial metabolism.

Oxidation of 5-methylcytosine (5mC) in DNA by the ten-eleven translocation (TET) family of enzymes is indispensable for gene regulation in mammals. More recently, evidence has emerged to support a biological function for TET-mediated m5C oxidation in messenger RNA. Here, we describe a previously uncharacterized role of TET-mediated m5C oxidation in transfer RNA (tRNA). We found that the TET-mediated oxidation product 5-hydroxylmethylcytosine (hm5C) is specifically enriched in tRNA inside cells and that the oxidation activity of TET2 on m5C in tRNAs can be readily observed in vitro. We further observed that hm5C levels in tRNA were significantly decreased in Tet2 KO mouse embryonic stem cells (mESCs) in comparison with wild-type mESCs. Reciprocally, induced expression of the catalytic domain of TET2 led to an obvious increase in hm5C and a decrease in m5C in tRNAs relative to uninduced cells. Strikingly, we also show that TET2-mediated m5C oxidation in tRNA promotes translation in vitro. These results suggest TET2 may influence translation through impacting tRNA methylation and reveal an unexpected role for TET enzymes in regulating multiple nodes of the central dogma.

Methylation of cytosine to 5-methylcytosine (mC) is a prevalent reversible epigenetic mark in vertebrates established by DNA methyltransferases (MTases); the methylation mark can be actively erased via a multi-step demethylation mechanism involving oxidation by Ten-eleven translocation (TET) enzyme family dioxygenases, excision of the latter oxidation products by thymine DNA (TDG) or Nei-like 1 (NEIL1) glycosylases followed by base excision repair to restore the unmodified state. Here we probed the activity of the mouse TET1 (mTET1) and Naegleria gruberi TET (nTET) oxygenases with DNA substrates containing extended derivatives of the 5-methylcytosine carrying linear carbon chains and adjacent unsaturated CC bonds. We found that the nTET and mTET1 enzymes were active on modified mC residues in single-stranded and double-stranded DNA in vitro, while the extent of the reactions diminished with the size of the extended group. Iterative rounds of nTET hydroxylations of ssDNA proceeded with high stereo specificity and included not only the natural alpha position but also the adjoining carbon atom in the extended side chain. The regioselectivity of hydroxylation was broken when the reactive carbon was adjoined with an sp1 or sp2 system. We also found that NEIL1 but not TDG was active with bulky TET-oxidation products. These findings provide important insights into the mechanism of these biologically important enzymatic reactions.

Scrapie is a neurodegenerative disorder belonging to the group of transmissible spongiform encephalopathies or prion diseases, which are caused by an infectious isoform of the innocuous cellular prion protein (PrPC) known as PrPSc. DNA methylation, one of the most studied epigenetic mechanisms, is essential for the proper functioning of the central nervous system. Recent findings point to possible involvement of DNA methylation in the pathogenesis of prion diseases, but there is still a lack of knowledge about the behavior of this epigenetic mechanism in such neurodegenerative disorders. Here, we evaluated by immunohistochemistry the 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels in sheep and mouse brain tissues infected with scrapie. Expression analysis of different gene coding for epigenetic regulatory enzymes (DNMT1, DNMT3A, DNMT3B, HDAC1, HDAC2, TET1, and TET2) was also carried out. A decrease in 5mC levels was observed in scrapie-affected sheep and mice compared to healthy animals, whereas 5hmC displayed opposite patterns between the two models, demonstrating a decrease in 5hmC in scrapie-infected sheep and an increase in preclinical mice. 5mC correlated with prion-related lesions in mice and sheep, but 5hmC was associated with prion lesions only in sheep. Differences in the expression changes of epigenetic regulatory genes were found between both disease models, being differentially expressed Dnmt3b, Hdac1, and Tet1 in mice and HDAC2 in sheep. Our results support the evidence that DNA methylation in both forms, 5mC and 5hmC, and its associated epigenetic enzymes, take part in the neurodegenerative course of prion diseases.

Epigenetic modifications of DNA, such as 5-methylcytosine and 5-hydroxymethycytosine, play important roles in development and disease. Here, we present a cost-effective and versatile methodology for the analysis of DNA methylation in targeted genomic regions, which comprises multiplexed, PCR-based preparation of bisulfite DNA libraries followed by customized MiSeq sequencing.

Tet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC), and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We applied quantitative mass-spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC), and adult mouse brain tissue. Readers for these modifications are only partially overlapping, and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation.

N.ϕGamma is a strand-specific and site-specific DNA nicking enzyme (YCG↓GT or AC↑CGR). Here we describe the isolation of single and double mutants of N.ϕGamma with attenuated activity. The nicking domains (NDs) of E59A and 11 double mutants were fused to the 5mCG-binding domain of MBD2 and generated fusion enzymes that preferentially nick 5mCG-modified DNA. The CG dinucleotide can be modified by C5 methyltransferases (MTases) such as M.SssI, M.HhaI or M.HpaII to create composite sites AC↑YGG N(8-15) 5mCG. We also constructed a fusion enzyme 2xMBD2-ND(N.BceSVIII) targeting more frequent composite sites AS↑YS N(5-12) 5mCG in Mn2+ buffer. 5mCG-dependent nicking requires special digestion conditions in high salt (0.3 M KCl) or in Ni2+ buffer. The fusion enzyme can be used to nick and label 5mCG-modified plasmid and genomic DNAs with fluorescently labeled Cy3-dUTP and potentially be useful for diagnostic applications, DNA sequencing and optical mapping of epigenetic markers. The importance of the predicted catalytic residues D89, H90, N106 and H115 in N.ϕGamma was confirmed by mutagenesis. We found that the wild-type enzyme N.ϕGamma prefers to nick 5mCG-modified DNA in Ni2+ buffer even though the nicking activity is sub-optimal compared to the activity in Mg2+ buffer.

No abstract available

Tools for actively targeted DNA demethylation are required to increase our knowledge about regulation and specific functions of this important epigenetic modification. DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5-meC), which may promote its replication-dependent dilution and/or active removal through base excision repair (BER). However, it is still unclear whether oxidized derivatives of 5-meC are simply DNA demethylation intermediates or rather epigenetic marks on their own. Unlike animals, plants have evolved enzymes that directly excise 5-meC without previous modification. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5-meC DNA glycosylase to a CRISPR-associated null-nuclease (dCas9) and analyzed its capacity for targeted reactivation of methylation-silenced genes, in comparison to other dCas9-effectors. We found that dCas9-ROS1, but not dCas9-TET1, is able to reactivate methylation-silenced genes and induce partial demethylation in a replication-independent manner. We also found that reactivation induced by dCas9-ROS1, as well as that achieved by two different CRISPR-based chromatin effectors (dCas9-VP160 and dCas9-p300), generally decreases with methylation density. Our results suggest that plant 5-meC DNA glycosylases are a valuable addition to the CRISPR-based toolbox for epigenetic editing.

Methylation of cytosine to 5-methylcytosine (5mC) is important for gene expression, gene imprinting, X-chromosome inactivation, and transposon silencing. Active demethylation in animals is believed to proceed by DNA glycosylase removal of deaminated or oxidized 5mC. In plants, 5mC is removed from the genome directly by the DEMETER (DME) family of DNA glycosylases. Arabidopsis thaliana DME excises 5mC to activate expression of maternally imprinted genes. Although the related Repressor of Silencing 1 (ROS1) enzyme has been characterized, the molecular basis for 5mC recognition by DME has not been investigated. Here, we present a structure-function analysis of DME and the related DME-like 3 (DML3) glycosylases for 5mC and its oxidized derivatives. Relative to 5mC, DME and DML3 exhibited robust activity toward 5-hydroxymethylcytosine, limited activity for 5-carboxylcytosine, and no activity for 5-formylcytosine. We used homology modeling and mutational analysis of base excision and DNA binding to identify residues important for recognition of 5mC within the context of DNA and inside the enzyme active site. Our results indicate that the 5mC binding pocket is composed of residues from discrete domains and is responsible for discrimination against 5mC derivatives, and suggest that DME, ROS1, and DML3 utilize subtly different mechanisms to probe the DNA duplex for cytosine modifications.

The rye (Secale L.) genome is large, and it contains many classes of repetitive sequences. Secale species differ in terms of genome size, heterochromatin content, and global methylation level; however, the organization of individual types of sequences in chromosomes is relatively similar. The content of the abundant subtelomeric heterochromatin fraction in rye do not correlate with the global level of cytosine methylation, hence immunofluorescence detection of 5-methylcytosine (5-mC) distribution in metaphase chromosomes was performed. The distribution patterns of 5-methylcytosine in the chromosomes of Secale species/subspecies were generally similar. 5-methylcytosine signals were dispersed along the entire length of the chromosome arms of all chromosomes, indicating high levels of methylation, especially at retrotransposon sequences. 5-mC signals were absent in the centromeric and telomeric regions, as well as in subtelomeric blocks of constitutive heterochromatin, in each of the taxa studied. Pericentromeric domains were methylated, however, there was a certain level of polymorphism in these areas, as was the case with the nucleolus organizer region. Sequence methylation within the region of the heterochromatin intercalary bands were also demonstrated to be heterogenous. Unexpectedly, there was a lack of methylation in rye subtelomeres, indicating that heterochromatin is a very diverse fraction of chromatin, and its epigenetic regulation or potential influence on adjacent regions can be more complex than has conventionally been thought. Like telomeres and centromeres, subtelomeric heterochromatin can has a specific role, and the absence of 5-mC is required to maintain the heterochromatin state.

5-Methylcytosine (m5C) is a prevalent base modification in tRNA and rRNA but it also occurs more broadly in the transcriptome, including in mRNA, where it serves incompletely understood molecular functions. In pursuit of potential links of m5C with mRNA translation, we performed polysome profiling of human HeLa cell lysates and subjected RNA from resultant fractions to efficient bisulfite conversion followed by RNA sequencing (bsRNA-seq). Bioinformatic filters for rigorous site calling were devised to reduce technical noise.

Exposure of airborne particulate matter (PM) with an aerodynamic diameter less than 2.5 μm (PM2.5) is epidemiologically associated with lung dysfunction and respiratory symptoms, including pulmonary fibrosis. However, whether epigenetic mechanisms are involved in PM2.5-induced pulmonary fibrosis is currently poorly understood. Herein, using a PM2.5-induced pulmonary fibrosis mouse model, we found that PM2.5 exposure leads to aberrant mRNA 5-methylcytosine (m5C) gain and loss in fibrotic lung tissues. Moreover, we showed the m5C-mediated regulatory map of gene functions in pulmonary fibrosis after PM2.5 exposure. Several genes act as m5C gain-upregulated factors, probably critical for the development of PM2.5-induced fibrosis in mouse lungs. These genes, including Lcn2, Mmp9, Chi3l1, Adipoq, Atp5j2, Atp5l, Atpif1, Ndufb6, Fgr, Slc11a1, and Tyrobp, are highly related to oxidative stress response, inflammatory responses, and immune system processes. Our study illustrates the first epitranscriptomic RNA m5C profile in PM2.5-induced pulmonary fibrosis and will be valuable in identifying biomarkers for PM2.5 exposure-related lung pathogenesis with translational potential.

5-methylcytosine DNA methylation regulates gene expression and developmental programming in a broad range of eukaryotes. However, its presence and potential roles in ciliates, complex single-celled eukaryotes with germline-somatic genome specialization via nuclear dimorphism, are largely uncharted. While canonical cytosine methyltransferases have not been discovered in published ciliate genomes, recent studies performed in the stichotrichous ciliate Oxytricha trifallax suggest de novo cytosine methylation during macronuclear development. In this study, we applied bisulfite genome sequencing, DNA mass spectrometry and antibody-based fluorescence detection to investigate the presence of DNA methylation in Paramecium tetraurelia. While the antibody-based methods suggest cytosine methylation, DNA mass spectrometry and bisulfite sequencing reveal that levels are actually below the limit of detection. Our results suggest that Paramecium does not utilize 5-methylcytosine DNA methylation as an integral part of its epigenetic arsenal.

TET2 is a close relative of TET1, an enzyme that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. The gene encoding TET2 resides at chromosome 4q24, in a region showing recurrent microdeletions and copy-neutral loss of heterozygosity (CN-LOH) in patients with diverse myeloid malignancies. Somatic TET2 mutations are frequently observed in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes including chronic myelomonocytic leukaemia (CMML), acute myeloid leukaemias (AML) and secondary AML (sAML). We show here that TET2 mutations associated with myeloid malignancies compromise catalytic activity. Bone marrow samples from patients with TET2 mutations displayed uniformly low levels of 5hmC in genomic DNA compared to bone marrow samples from healthy controls. Moreover, small hairpin RNA (shRNA)-mediated depletion of Tet2 in mouse haematopoietic precursors skewed their differentiation towards monocyte/macrophage lineages in culture. There was no significant difference in DNA methylation between bone marrow samples from patients with high 5hmC versus healthy controls, but samples from patients with low 5hmC showed hypomethylation relative to controls at the majority of differentially methylated CpG sites. Our results demonstrate that Tet2 is important for normal myelopoiesis, and suggest that disruption of TET2 enzymatic activity favours myeloid tumorigenesis. Measurement of 5hmC levels in myeloid malignancies may prove valuable as a diagnostic and prognostic tool, to tailor therapies and assess responses to anticancer drugs.