Whole-Genome Mapping of Epigenetic Modification of 5-Formylcytosine at Single-Base Resolution by Chemical Labeling Enrichment and Deamination Sequencing.

DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays a critical role in a variety of biological and pathological processes in mammals. In active DNA demethylation, the 10-11 translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution is still highly desirable. Herein, we propose a chemical labeling enrichment and deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (APOBEC3A or A3A) and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mESCs. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution.

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA.

RNA molecules undergo various chemical modifications that play critical roles in a wide range of biological processes. N6,N6-Dimethyladenosine (m6,6A) is a conserved RNA modification and is essential for the processing of rRNA. To gain a deeper understanding of the functions of m6,6A, site-specific and accurate quantification of this modification in RNA is indispensable. In this study, we developed an AlkB-facilitated demethylation (AD-m6,6A) method for the site-specific detection and quantification of m6,6A in RNA. The N6,N6-dimethyl groups in m6,6A can cause reverse transcription to stall at the m6,6A site, resulting in truncated cDNA. However, we found that Escherichia coli AlkB demethylase can effectively demethylate m6,6A in RNA, generating full-length cDNA from AlkB-treated RNA. By quantifying the amount of full-length cDNA produced using quantitative real-time PCR, we were able to achieve site-specific detection and quantification of m6,6A in RNA. Using the AD-m6,6A method, we successfully detected and quantified m6,6A at position 1851 of 18S rRNA and position 937 of mitochondrial 12S rRNA in human cells. Additionally, we found that the level of m6,6A at position 1007 of mitochondrial 12S rRNA was significantly reduced in lung tissues from sleep-deprived mice compared with control mice. Overall, the AD-m6,6A method provides a valuable tool for easy, accurate, quantitative, and site-specific detection of m6,6A in RNA, which can aid in uncovering the functions of m6,6A in human diseases.

Single-Nucleotide Resolution Mapping of N6-Methyladenine in Genomic DNA.

N6-Methyladenine (6mA) is a naturally occurring DNA modification in both prokaryotes and eukaryotes. Herein, we developed a deaminase-mediated sequencing (DM-seq) method for genome-wide mapping of 6mA at single-nucleotide resolution. The method capitalizes on the selective deamination of adenine, but not 6mA, in DNA mediated by an evolved adenine deaminase, ABE8e. By employing this method, we achieved genome-wide mapping of 6mA in Escherichia coli and in mammalian mitochondrial DNA (mtDNA) at single-nucleotide resolution. We found that the 6mA sites are mainly located in the GATC motif in the E. coli genome. We also identified 17 6mA sites in mtDNA of HepG2 cells, where all of the 6mA sites are distributed in the heavy strand of mtDNA. We envision that DM-seq will be a valuable tool for uncovering new functions of 6mA in DNA and for exploring its potential roles in mitochondria-related human diseases.

Single-Base Resolution Detection of N6-Methyladenosine in RNA by Adenosine Deamination Sequencing.

N6-Methyladenosine (m6A) is one of the most abundant and prevalent natural modifications occurring in diverse RNA species. m6A plays a wide range of roles in physiological and pathological processes. Revealing the functions of m6A relies on the faithful detection of individual m6A sites in RNA. However, developing a simple method for the single-base resolution detection of m6A is still a challenging task. Herein, we report an adenosine deamination sequencing (AD-seq) technique for the facile detection of m6A in RNA at single-base resolution. The AD-seq approach capitalizes on the selective deamination of adenosine, but not m6A, by the evolved tRNA adenosine deaminase (TadA) variant of TadA8e or the dimer protein of TadA-TadA8e. In AD-seq, adenosine is deaminated by TadA8e or TadA-TadA8e to form inosine, which pairs with cytidine and is read as guanosine in sequencing. m6A resists deamination due to the interference of the methyl group at the N6 position of adenosine. Thus, the m6A base pairs with thymine and is still read as adenosine in sequencing. The differential readouts from A and m6A in sequencing can achieve the single-base resolution detection of m6A in RNA. Application of the proposed AD-seq successfully identified individual m6A sites in Escherichia coli 23S rRNA. Taken together, the proposed AD-seq allows simple and cost-effective detection of m6A at single-base resolution in RNA, which provides a valuable tool to decipher the functions of m6A in RNA.

Enzymatic Cleavage-Mediated Extension Stalling Enables Accurate Recognition and Quantification of Locus-Specific Uracil Modification in DNA.

Chemical modifications in DNA have profound influences on the structures and functions of DNA. Uracil, a naturally occurring DNA modification, can originate from the deamination of cytosine or arise from misincorporation of dUTP into DNA during DNA replication. Uracil in DNA will imperil genomic stability due to their potential in producing detrimental mutations. An in-depth understanding of the functions of uracil modification requires the accurate determination of its site as well as content in genomes. Herein, we characterized that a new member of the uracil-DNA glycosylase (UDG) family enzyme (UdgX-H109S) could selectively cleave both uracil-containing single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Based on this unique property of UdgX-H109S, we developed an enzymatic cleavage-mediated extension stalling (ECES) method for the locus-specific detection and quantification of uracil in genomic DNA. In the ECES method, UdgX-H109S specifically recognizes and cleaves the N-glycosidic bond of uracil from dsDNA and generates an apurinic/apyrimidinic (AP) site, which could be broken by APE1 to form a one-nucleotide gap. The specific cleavage by UdgX-H109S is then evaluated and quantified by qPCR. With the developed ECES approach, we demonstrated that the level of uracil at position Chr4:50566961 in genomic DNA of breast cancer tissues was significantly decreased. Collectively, the ECES method has been proved to be accurate and reproducible in the locus-specific quantification of uracil in genomic DNA from biological and clinical samples.

Quantitative and site-specific detection of inosine modification in RNA by acrylonitrile labeling-mediated elongation stalling.

RNA molecules contain diverse modifications that play crucial roles in a wide variety of biological processes. Inosine is one of the most prevalent modifications in RNA and dysregulation of inosine is correlated with many human diseases. Herein, we established an acrylonitrile labeling-mediated elongation stalling (ALES) method for quantitative and site-specific detection of inosine in RNA from biological samples. In ALES method, inosine is selectively cyanoethylated with acrylonitrile to form N1-cyanoethylinosine (ce1I) through a Michael addition reaction. The N1-cyanoethyl group of ce1I compromises the hydrogen bond between ce1I and other nucleobases, leading to the stalling of reverse transcription at original inosine site. This specific property of stalling at inosine site could be evaluated by subsequent real-time quantitative PCR (qPCR). With the proposed ALES method, we found the significantly increased level of inosine at position Chr1:63117284 of Ino80dos RNA of multiple tissues from sleep-deprived mice compared to the control mice. This is the first report on the investigation of inosine modification in sleep-deprived mice, which may open up new direction for deciphering insomnia from RNA modifications. In addition, we found the decreased level of inosine at GluA2 Q/R site (Chr4:157336723) in glioma tissues, indicating the decreased level of inosine at GluA2 Q/R site may serve as potential indicator for the diagnosis of glioma. Taken together, the proposed ALES method is capable of quantitative and site-specific detection of inosine in RNA, which provides a valuable tool to uncover the functions of inosine in human diseases.

Engineered APOBEC3C Sequencing Enables Bisulfite-Free and Direct Detection of DNA Methylation at a Single-Base Resolution.

DNA methylation (5-methylcytosine, 5mC) is the most important epigenetic modification in mammals. Deciphering the roles of 5mC relies on the quantitative detection of 5mC at the single-base resolution. Bisulfite sequencing (BS-seq) is the most often employed technique for mapping 5mC in DNA. However, bisulfite treatment may cause serious degradation of input DNA due to the harsh reaction conditions. Here, we engineered the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3C (A3C) protein to endow the engineered A3C (eA3C) protein with differential deamination activity toward cytosine and 5mC. By the virtue of the unique property of eA3C, we proposed an engineered A3C sequencing (EAC-seq) method for the bisulfite-free and quantitative mapping of 5mC in DNA at the single-base resolution. In EAC-seq, the eA3C protein can deaminate C but not 5mC, which is employed to differentiate C and 5mC in sequencing. Using the EAC-seq method, we quantitatively detected 5mC in genomic DNA of lung cancer tissue. In contrast to the harsh reaction conditions of BS-seq, which could lead to significant degradation of DNA, the whole procedure of EAC-seq is carried out under mild conditions, thereby preventing DNA damage. Taken together, the EAC-seq approach is bisulfite-free and straightforward, making it an invaluable tool for the quantitative detection of 5mC in limited DNA at the single-base resolution.

Comprehensive Profiling of Phosphomonoester Metabolites in Saccharomyces cerevisiae by the Chemical Isotope Labeling-LC-MS Method.

Phosphomonoesters are important biosynthetic and energy metabolism intermediates in microorganisms. A comprehensive analysis of phosphomonoester metabolites is of great significance for the understanding of their metabolic phosphorylation process and inner mechanism. In this study, we established a pair of isotope reagent d0/d5-2-diazomethyl-N-methyl-phenyl benzamide-labeling-based LC-MS method for the comprehensive analysis of phosphomonoester metabolites. By this method, the labeled phosphomonoester metabolites specifically produced characteristic isotope paired peaks with an m/z difference of 5.0314 in the MS1 spectra and a pair of diagnostic ions (m/z 320.0693/325.1077) in the MS2 spectra. Based on this, a diagnostic ion-based strategy was established for the rapid screening, identification, and relative quantification of phosphomonoester metabolites. Using this strategy, 42 phosphomonoester metabolites were highly accurately identified fromSaccharomyces cerevisiae (S. cerevisiae). Notably, two phosphomonoesters were first detected fromS. cerevisiae. The relative quantification results indicated that the contents of nine phosphomonoester metabolites including two intermediates (Ru5P and S7P) in the pentose phosphate pathway (PPP) were significantly different between lycopene-producible and wild-type S. cerevisiae. A further enzyme assay indicated that the activity of the PPP was closely related to the production of lycopene. Our findings provide new perspectives for the related mechanism study and valuable references for making informed microbial engineering decisions.

Whole-Genome Sequencing of 5-Hydroxymethylcytosine at Base Resolution by Bisulfite-Free Single-Step Deamination with Engineered Cytosine Deaminase.

The epigenetic modification 5-hydroxymethylcytosine (5hmC) plays a crucial role in the regulation of gene expression. Although some methods have been developed to detect 5hmC, direct genome-wide mapping of 5hmC at base resolution is still highly desirable. Herein, we proposed a single-step deamination sequencing (SSD-seq) method, designed to precisely map 5hmC across the genome at single-base resolution. SSD-seq takes advantage of a screened engineered human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A) protein, known as eA3A-v10, to selectively deaminate cytosine (C) and 5-methylcytosine (5mC) but not 5hmC. During sequencing, the deaminated C and 5mC are converted to uracil (U) and thymine (T), read as T in the sequencing data. However, 5hmC remains unaffected by eA3A-v10 and is read as C during sequencing. Consequently, the presence of C in the sequence reads indicates the original 5hmC. We applied SSD-seq to generate a base-resolution map of 5hmC in human lung tissue. Our findings revealed that 5hmC was predominantly localized to CpG dinucleotides. Furthermore, the base-resolution map of 5hmC generated by SSD-seq demonstrated a strong correlation with prior ACE-seq results. The advantages of SSD-seq are its single-step process, absence of bisulfite treatment or DNA glycosylation, cost effectiveness, and ability to detect and quantify 5hmC directly at single-base resolution.

Bisulfite-Free and Single-Base Resolution Detection of Epigenetic DNA Modification of 5-Methylcytosine by Methyltransferase-Directed Labeling with APOBEC3A Deamination Sequencing.

DNA methylation (5-methylcytosine, 5mC) is the most prevalent epigenetic modification that is predominantly found in CG dinucleotides in mammalian genomes. In-depth investigation of the functions of 5mC heavily relies on the quantitative measurement of 5mC at single-base resolution in genomes. Here, we proposed a methyltransferase-directed labeling with APOBEC3A (A3A) deamination sequencing (MLAD-seq) method for the single-base resolution and quantitative detection of 5mC in DNA. In MLAD-seq, a mutant of DNA methyltransferase, M.MpeI-N374K, is utilized to selectively transfer a carboxymethyl group to the 5 position of cytosine in the CG dinucleotide to form 5-carboxymethylcytosine (5camC) using carboxy-S-adenosyl-l-methionine (caSAM) as the cofactor. After A3A treatment, 5camC is resistant to the deamination and base pairs with guanine. Thus, the cytosines in CG sites are read as C in sequencing. On the contrary, the methyl group in 5mC inhibits its carboxymethylcytosine by M.MpeI-N374K and therefore is readily deaminated by A3A to produce thymine that pairs with adenine and is read as T in sequencing. The differential readouts from C and 5mC in the MLAD-seq enable the single-base resolution mapping of 5mC in CG sites in DNA. With the developed MLAD-seq method, we observed the hypermethylation in the promoter region of retinoic acid receptor ß (RARB) gene from human nonsmall cell lung tumor tissue. Compared to harsh reaction conditions in bisulfite sequencing that could lead to significant degradation of DNA, the whole procedure of MLAD-seq is carried out under mild conditions, which will avoid DNA damage. Thus, MLAD-seq is more suitable in the scenario where only limited input DNA is available. Taken together, the MLAD-seq offers a valuable tool for bisulfite-free, single-base resolution and quantitative detection of 5mC in limited DNA.

Genome-wide mapping of N 4-methylcytosine at single-base resolution by APOBEC3A-mediated deamination sequencing.

N 4-methylcytosine (4mC) is a natural DNA modification occurring in thermophiles and plays important roles in restriction-modification (R-M) systems in bacterial genomes. However, the precise location and sequence context of 4mC in the whole genome are limited. In this study, we developed an APOBEC3A-mediated deamination sequencing (4mC-AMD-seq) method for genome-wide mapping of 4mC at single-base resolution. In the 4mC-AMD-seq method, cytosine and 5-methylcytosine (5mC) are deaminated by APOBEC3A (A3A) protein to generate uracil and thymine, both of which are read as thymine in sequencing, while 4mC is resistant to deamination and therefore read as cytosine. Thus, the readouts of cytosines from sequencing could manifest the original 4mC sites in genomes. With the 4mC-AMD-seq method, we achieved the genome-wide mapping of 4mC in Deinococcus radiodurans (D. radiodurans). In addition, we confirmed that 4mC, but not 5mC, was the major modification in the D. radiodurans genome. We identified 1586 4mC sites in the genome of D. radiodurans, among which 564 sites were located in the CCGCGG motif. The average methylation levels in the CCGCGG motif and non-CCGCGG sequence were 70.0% and 22.8%, respectively. We envision that the 4mC-AMD-seq method will facilitate the investigation of 4mC functions, including the 4mC-involved R-M systems, in uncharacterized but potentially useful strains.

Formation and removal of 1,N6-dimethyladenosine in mammalian transfer RNA.

RNA molecules harbor diverse modifications that play important regulatory roles in a variety of biological processes. Over 150 modifications have been identified in RNA molecules. N6-methyladenosine (m6A) and 1-methyladenosine (m1A) are prevalent modifications occurring in various RNA species of mammals. Apart from the single methylation of adenosine (m6A and m1A), dual methylation modification occurring in the nucleobase of adenosine, such as N6,N6-dimethyladenosine (m6,6A), also has been reported to be present in RNA of mammals. Whether there are other forms of dual methylation modification occurring in the nucleobase of adenosine other than m6,6A remains elusive. Here, we reported the existence of a novel adenosine dual methylation modification, i.e. 1,N6-dimethyladenosine (m1,6A), in tRNAs of living organisms. We confirmed that m1,6A is located at position 58 of tRNAs and is prevalent in mammalian cells and tissues. The measured level of m1,6A ranged from 0.0049% to 0.047% in tRNAs. Furthermore, we demonstrated that TRMT6/61A could catalyze the formation of m1,6A in tRNAs and m1,6A could be demethylated by ALKBH3. Collectively, the discovery of m1,6A expands the diversity of RNA modifications and may elicit a new tRNA modification-mediated gene regulation pathway.

Bisulfite-free and single-nucleotide resolution sequencing of DNA epigenetic modification of 5-hydroxymethylcytosine using engineered deaminase.

The discovery of 5-hydroxymethylcytosine (5hmC) in mammalian genomes is a landmark in epigenomics study. Similar to 5-methylcytosine (5mC), 5hmC is viewed as a critical epigenetic modification. Deciphering the functions of 5hmC necessitates the location analysis of 5hmC in genomes. Here, we proposed an engineered deaminase-mediated sequencing (EDM-seq) method for the quantitative detection of 5hmC in DNA at single-nucleotide resolution. This method capitalizes on the engineered human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A) protein to produce differential deamination activity toward cytosine, 5mC, and 5hmC. In EDM-seq, the engineered A3A (eA3A) protein can deaminate C and 5mC but not 5hmC. The original C and 5mC in DNA are deaminated by eA3A to form U and T, both of which are read as T during sequencing, while 5hmC is resistant to deamination by eA3A and is still read as C during sequencing. Therefore, the remaining C in the sequence manifests the original 5hmC. By EDM-seq, we achieved the quantitative detection of 5hmC in genomic DNA of lung cancer tissue. The EDM-seq method is bisulfite-free and does not require DNA glycosylation or chemical treatment, which offers a valuable tool for the straightforward and quantitative detection of 5hmC in DNA at single-nucleotide resolution.

Single-Base Resolution Detection of Adenosine-to-Inosine RNA Editing by Endonuclease-Mediated Sequencing.

RNA molecules contain diverse modifications that play crucial roles in a wide variety of biological processes. Adenosine-to-inosine (A-to-Ino) RNA editing is one of the most prevalent modifications among all types of RNA. Abnormal A-to-InoRNA editing has been demonstrated to be associated with many human diseases. Identification of A-to-Ino editing sites is indispensable to deciphering their biological roles. Herein, by employing the unique property of human endonuclease V (hEndoV), we proposed a hEndoV-mediated sequencing (hEndoV-seq) method for the single-base resolution detection of A-to-InoRNA editing sites. In this approach, the terminal 3'OH of RNA is first blocked by 3'-deoxyadenosine (3'-deoxy-A). Specific cleavage of Ino sites by hEndoV protein produces new terminal 3'OH, which can be identified by sequencing analysis, and therefore offers the site-specific detection of Ino in RNA. The principle of hEndoV-seq is straightforward and the analytical procedure is simple. No chemical reaction is involved in the sequencing library preparation. The whole procedure in hEndoV-seq is carried out under mild conditions and RNA is not prone to degradation. Taken together, the proposed hEndoV-seq method is capable of site-specific identification of A-to-Ino editing in RNA, which provides a valuable tool for elucidating the functions of A-to-Ino editing in RNA.

Identification of Inosine and 2'-O-Methylinosine Modifications in Yeast Messenger RNA by Liquid Chromatography-Tandem Mass Spectrometry Analysis.

The discovery of reversible modifications in messenger RNA (mRNA) opens new research directions in RNA modification-mediated epigenetic regulation. Yeast is an extensively used model organism in molecular biology. Systematic investigation and profiling of modifications in yeast mRNA would promote our understanding of the physiological regulation mechanisms in yeast. However, due to the high abundance of ribosomal RNA (rRNA) and transfer RNA (tRNA) in total RNA, isolation of low abundance of mRNA frequently suffers from the contamination of rRNA and tRNA, which will lead to the false-positive determination and inaccurate quantification of modifications in mRNA. Therefore, obtaining high-purity mRNA is critical for precise determination and accurate quantification of modifications in mRNA, especially for studies that focus on discovering new ones. Herein, we proposed a successive orthogonal isolation method by combining polyT-based purification and agarose gel electrophoresis purification for extracting high-purity mRNA. With the extracted high-purity yeast mRNA, we systemically explored the modifications in yeast mRNA by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. The results showed that in addition to the previously reported eight kinds of modifications, two novel modifications of inosine (Ino) and 2'-O-methylinosine (Im) were identified to be prevalent in yeast mRNA. It is worth noting that Im was reported for the first time, to the best of our knowledge, to exist in living organisms in the three domains of life. Moreover, we observed that the levels of 10 kinds of modifications including Ino and Im in yeast mRNA exhibited dynamic change at different growth stages of yeast cells. Furthermore, Im in mRNA showed a significant decrease while in response to H2O2 treatment. These results indicated that the two newly identified modifications in yeast mRNA were involved in yeast cell growth and response to environmental stress. Taken together, we reported two new modifications of Ino and Im in yeast mRNA, which expends the diversity of RNA modifications in yeast and also suggests new regulators for modulating yeast physiological functions.

Ultrasensitive Determination of Sugar Phosphates in Trace Samples by Stable Isotope Chemical Labeling Combined with RPLC-MS.

Sugar phosphates are important metabolic intermediates in organisms and play a vital role in energy and central carbon metabolism. Profiling of sugar phosphates is of great significance but full of challenges due to their high structural similarity and low sensitivities in liquid chromatography (LC)-mass spectrometry (MS). In this study, we developed a novel stable isotope chemical labeling combined with the reversed-phase (RP)LC-MS method for ultrasensitive determination of sugar phosphates at the single-cell level. By chemical derivatization with 2-(diazo-methyl)-N-methyl-N-phenyl-benzamide (2-DMBA) and d5-2-DMBA, sugar phosphate isomers can obtain better separation and identification, and the detection sensitivities of sugar phosphates increased by 3.5-147 folds. The obtained limits of detection of sugar phosphates ranged from 5 to 16 pg/mL. Using this method, we achieved ultrasensitive and accurate quantification of 12 sugar phosphates in different trace biological samples. Benefiting from the improved separation and detection sensitivity, we successfully quantified five sugar phosphates (d-glucose 1-phosphate, d-mannose 6-phosphate, d-fructose 6-phosphate, d-glucose 6-phosphate, and seduheptulose 7-phosphate) in a single protoplast of Arabidopsis thaliana.

DNA-Protein Cross-Linking Sequencing for Genome-Wide Mapping of Thymidine Glycol.

Thymidine glycol (Tg) is the most prevalent form of oxidatively induced pyrimidine lesions in DNA. Tg can arise from direct oxidation of thymidine in DNA. In addition, 5-methyl-2'-deoxycytidine (5-mdC) can be oxidized to 5-mdC glycol, and its subsequent deamination also yields Tg. However, Tg's distribution in the human genome remains unknown. Here, we presented a DNA-protein cross-linking sequencing (DPC-Seq) method for genome-wide mapping of Tg in human cells. Our approach capitalizes on the specificity of a bifunctional DNA glycosylase, i.e., NTHL1, for the covalent labeling, as well as DPC pulldown, SDS-PAGE fractionation, and membrane transfer for highly efficient and selective enrichment of Tg-bearing DNA. By employing DPC-Seq, we detected thousands of Tg sites in the human genome, where dual ablation of NTHL1 and NEIL1, the major DNA glycosylases responsible for Tg repair, led to pronounced increases in the number of Tg peaks. In addition, Tg is depleted in genomic regions associated with active transcription but enriched at nucleosome-binding sites, especially at heterochromatin sites marked with H3K9me2. Collectively, we developed a DPC-Seq method for highly efficient enrichment of Tg-containing DNA and for genome-wide mapping of Tg in human cells. Our work offers a robust tool for future functional studies of Tg in DNA, and we envision that the method can also be adapted for mapping other modified nucleosides in genomic DNA in the future.

An enzyme-mediated bioorthogonal labeling method for genome-wide mapping of 5-hydroxymethyluracil.

DNA 5-hydroxymethyluracil (5hmU) is a thymine modification existing in the genomes of various organisms. The post-replicative formation of 5hmU occurs via hydroxylation of thymine by ten-eleven translocation (TET) dioxygenases in mammals and J-binding proteins (JBPs) in protozoans, respectively. In addition, 5hmU can also be generated through oxidation of thymine by reactive oxygen species or deamination of 5hmC by cytidine deaminase. While the biological roles of 5hmU have not yet been fully explored, determining its genomic location will highly assist in elucidating its functions. Herein, we report a novel enzyme-mediated bioorthogonal labeling method for selective enrichment of 5hmU in genomes. 5hmU DNA kinase (5hmUDK) was utilized to selectively install an azide (N3) group or alkynyl group into the hydroxyl moiety of 5hmU followed by incorporation of the biotin linker through click chemistry, which enabled the capture of 5hmU-containing DNA fragments via streptavidin pull-down. The enriched fragments were applied to deep sequencing to determine the genomic distribution of 5hmU. With this established enzyme-mediated bioorthogonal labeling strategy, we achieved the genome-wide mapping of 5hmU in Trypanosoma brucei. The method described here will allow for a better understanding of the functional roles and dynamics of 5hmU in genomes.

Direct decarboxylation of ten-eleven translocation-produced 5-carboxylcytosine in mammalian genomes forms a new mechanism for active DNA demethylation.

DNA cytosine methylation (5-methylcytosine, 5mC) is the most important epigenetic mark in higher eukaryotes. 5mC in genomes is dynamically controlled by writers and erasers. DNA (cytosine-5)-methyltransferases (DNMTs) are responsible for the generation and maintenance of 5mC in genomes. Active demethylation of 5-methylcytosine (5mC) is achieved by ten-eleven translocation (TET) dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are further processed by thymine DNA glycosylase (TDG)-initiated base excision repair (BER) to restore unmodified cytosines. The TET-TDG-BER pathway could cause the production of DNA strand breaks and therefore jeopardize the integrity of genomes. Here, we investigated the direct decarboxylation of 5caC in mammalian genomes by using metabolic labeling with 2'-fluorinated 5caC (F-5caC) and mass spectrometry analysis. Our results clearly demonstrated the decarboxylation of 5caC occurring in mammalian genomes, which unveiled that, in addition to the TET-TDG-BER pathway, the direct decarboxylation of TET-produced 5caC constituted a new pathway for active demethylation of 5mC in mammalian genomes.

Quantification and mapping of DNA modifications.

Apart from the four canonical nucleobases, DNA molecules carry a number of natural modifications. Substantial evidence shows that DNA modifications can regulate diverse biological processes. Dynamic and reversible modifications of DNA are critical for cell differentiation and development. Dysregulation of DNA modifications is closely related to many human diseases. The research of DNA modifications is a rapidly expanding area and has been significantly stimulated by the innovations of analytical methods. With the recent advances in methods and techniques, a series of new DNA modifications have been discovered in the genomes of prokaryotes and eukaryotes. Deciphering the biological roles of DNA modifications depends on the sensitive detection, accurate quantification, and genome-wide mapping of modifications in genomic DNA. This review provides an overview of the recent advances in analytical methods and techniques for both the quantification and genome-wide mapping of natural DNA modifications. We discuss the principles, advantages, and limitations of these developed methods. It is anticipated that new methods and techniques will resolve the current challenges in this burgeoning research field and expedite the elucidation of the functions of DNA modifications.