DNA N6-methyldeoxyadenosine in mammals and human disease.

N6-methyladenine (6mA) is the most prevalent DNA modification in prokaryotes. However, its presence and significance in eukaryotes remain elusive. Recently, with methodology advances in detection and sequencing of 6mA in eukaryotes, 6mA is back in the spotlight. Although multiple studies have reported that 6mA is an important epigenetic mark in eukaryotes and plays a regulatory role in DNA transcription, transposon activation, stress response, and other bioprocesses, there are some discrepancies in the current literature. We review the recent advances in 6mA research in eukaryotes, especially in mammals. In particular, we describe the abundance/distribution of 6mA, its potential role in regulating gene expression, identified regulators, and pathological roles in human diseases, especially in cancer. The limitations faced by the field and future perspectives in 6mA research are also discussed.

A Mutation-Based Method for Pinpointing a DNA N6 -Methyladenine Methyltransferase Modification Site at Single Base Resolution.

DNA N6 -methyladenine (6mA) has recently received notable attention due to an increased finding of its functional roles in higher eukaryotes. Here we report an enzyme-assisted chemical labeling method to pinpoint the DNA 6mA methyltransferase (MTase) substrate modification site at single base resolution. A designed allyl-substituted MTase cofactor was applied in the catalytic transfer reaction, and the allyl group was installed to the N6 -position of adenine within a specific DNA sequence to form N6 -allyladenine (6aA). The iodination of 6aA allyl group induced the formation of 1, N6 -cyclized adenine which caused mutations during DNA replication by a polymerase. Thus the modification site could be precisely detected by a mutation signal. We synthesized 6aA deoxynucleoside and deoxynucleotide model compounds and a 6aA-containing DNA probe, and screened nine DNA polymerases to define an optimal system capable of detecting the substrate modification site of a DNA 6mA MTase at single-base resolution.

Interplay of DNA and RNA N 6 -methyladenine with R-loops in regulating gene transcription in Arabidopsis.

R-loops and covalent modifications of N 6 -methyladenine on DNA (D-6 mA) or RNA (R-m6A) have been documented to function in various cellular processes in eukaryotes. However, the relationships between R-loops and both covalent modifications are still elusive in plants. Here, we integrated existing ssDRIP-seq with D-6 mA and R-m6A data from Arabidopsis thaliana. We found that the presence of either of both modifications facilitates R-loop formation and transcription of overlapping genes. Interestingly, our study suggests that the presence of R-m6A is key to affect R-loop intensity and positively regulate gene transcription. Moreover, the presence of D-6 mA plays an additive role to facilitate the effect of R-m6A on R-loop intensity, however, D-6 mA may negatively regulate gene transcription when coexisted with R-m6A. Our analyses indicate that D-6 mA, R-m6A, or histone marks may act individually and cooperatively with R-loops in regulating gene transcription. Our study is the first to link R-loops with D-6 mA and R-m6A in plants, thereby providing new insights into interactions between R-loops with D-6 mA, R-m6A, and histone marks for regulating gene transcription. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01010-5.

i6mA-Fuse: improved and robust prediction of DNA 6 mA sites in the Rosaceae genome by fusing multiple feature representation.

DNA N6-methyladenine (6 mA) is one of the most vital epigenetic modifications and involved in controlling the various gene expression levels. With the avalanche of DNA sequences generated in numerous databases, the accurate identification of 6 mA plays an essential role for understanding molecular mechanisms. Because the experimental approaches are time-consuming and costly, it is desirable to develop a computation model for rapidly and accurately identifying 6 mA. To the best of our knowledge, we first proposed a computational model named i6mA-Fuse to predict 6 mA sites from the Rosaceae genomes, especially in Rosa chinensis and Fragaria vesca. We implemented the five encoding schemes, i.e., mononucleotide binary, dinucleotide binary, k-space spectral nucleotide, k-mer, and electron-ion interaction pseudo potential compositions, to build the five, single-encoding random forest (RF) models. The i6mA-Fuse uses a linear regression model to combine the predicted probability scores of the five, single encoding-based RF models. The resultant species-specific i6mA-Fuse achieved remarkably high performances with AUCs of 0.982 and 0.978 and with MCCs of 0.869 and 0.858 on the independent datasets of Rosa chinensis and Fragaria vesca, respectively. In the F. vesca-specific i6mA-Fuse, the MBE and EIIP contributed to 75% and 25% of the total prediction; in the R. chinensis-specific i6mA-Fuse, Kmer, MBE, and EIIP contribute to 15%, 65%, and 20% of the total prediction. To assist high-throughput prediction for DNA 6 mA identification, the i6mA-Fuse is publicly accessible at https://kurata14.bio.kyutech.ac.jp/i6mA-Fuse/.

DNA 6mA demethylase ALKBH1 regulates DDX18 expression to promote proliferation of human head and neck squamous cell carcinoma.

PURPOSE: Human head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Currently, surgical resection plus a combination of chemotherapy and radiotherapy is the standard treatment for HNSCC, and the 5-year survival rate of patients with HNSCC remains very low because of the higher incidence of metastasis with consequent recurrence. Here, we aimed to investigate the potential role of DNA N6-methyladenine (6mA) demethylase ALKBH1 in tumor cell proliferation in HNSCC. METHODS: The expression of ALKBH1 in 10 pairs of HNSCC/normal tissues and 3 HNSCC cell lines were measured by qRT‒PCR and western blotting. Colony formation, flow cytometry, patient-derived HNSCC organoid assays were used to assess the role of ALKBH1 in HNSCC cell proliferation in cell lines and human HNSCC patients. MeDIP-seq, RNA sequencing, Dot blotting and western blotting were used to evaluate the regulatory effect of ALKBH1 on the expression of DEAD-box RNA helicase DDX18. A dual-luciferase reporter assay was used to assess the putative effect of DNA 6mA levels on DDX18 transcription. RESULTS: ALKBH1 was highly expressed in HNSCC cells and patient tissues. Functional experiments revealed that ALKBH1 knockdown in SCC9, SCC25, and CAL27 cells inhibited their proliferation in vitro. Using patient-derived HNSCC organoid assay, we found that knockdown of ALKBH1 inhibited the proliferation and colony formation of HNSCC patients-derived organoids. Moreover, we found that ALKBH1 can enhance DDX18 expression by erasing DNA 6mA level and regulating its promoter activity. ALKBH1 deficiency blocked tumor cell proliferation by inhibiting DDX18 expression. Exogenous overexpression of DDX18 rescued the cell proliferation arrest caused by ALKBH1 knockdown. CONCLUSION: Our data reveal the important role of ALKBH1 in regulating proliferation of HNSCC.

SNN6mA: Improved DNA N6-methyladenine site prediction using Siamese network-based feature embedding.

DNA N6-methyladenine (6mA) is one of the most common and abundant modifications, which plays essential roles in various biological processes and cellular functions. Therefore, the accurate identification of DNA 6mA sites is of great importance for a better understanding of its regulatory mechanisms and biological functions. Although significant progress has been made, there still has room for further improvement in 6mA site prediction in DNA sequences. In this study, we report a smart but accurate 6mA predictor, termed as SNN6mA, using Siamese network. To be specific, DNA segments are firstly encoded into feature vectors using the one-hot encoding scheme; then, these original feature vectors are mapped to a low-dimensional embedding space derived from Siamese network to capture more discriminative features; finally, the obtained low-dimensional features are fed to a fully connected neural network to perform final prediction. Stringent benchmarking tests on the datasets of two species demonstrated that the proposed SNN6mA is superior to the state-of-the-art 6mA predictors. Detailed data analyses show that the major advantage of SNN6mA lies in the utilization of Siamese network, which can map the original features into a low-dimensional embedding space with more discriminative capability. In summary, the proposed SNN6mA is the first attempt to use Siamese network for 6mA site prediction and could be easily extended to predict other types of modifications. The codes and datasets used in the study are freely available at https://github.com/YuXuan-Glasgow/SNN6mA for academic use.

DNA 6mA Demethylase ALKBH1 Orchestrates Fatty Acid Metabolism and Suppresses Diet-Induced Hepatic Steatosis.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is a major cause of liver-related morbidity and mortality whereas the pathogenic mechanism remains largely elusive. DNA N6-methyladenosine (6mA) modification is a recently identified epigenetic mark indicative of transcription in eukaryotic genomes. Here, we aimed to investigate the role and mechanism of DNA 6mA modification in NAFLD progression. METHODS: Dot blot and immunohistochemistry were used to detect DNA 6mA levels. Liver-specific AlkB homolog 1 (ALKBH1)-knockout mice and mice with ALKBH1 overexpression in liver were subjected to a high-fat diet or methionine choline-deficient diet to evaluate the critical role of ALKBH1-demethylated DNA 6mA modification in the pathogenesis of hepatic steatosis during NAFLD. RNA sequencing and chromatin immunoprecipitation sequencing were performed to investigate molecular mechanisms underlying this process. RESULTS: The DNA 6mA level was increased significantly with hepatic steatosis, while ALKBH1 expression was down-regulated markedly in both mouse and human fatty liver. Deletion of ALKBH1 in hepatocytes increased genomic 6mA levels and accelerated diet-induced hepatic steatosis and metabolic dysfunction. Comprehensive analyses of transcriptome and chromatin immunoprecipitation sequencing data indicated that ALKBH1 directly bound to and exclusively demethylated 6mA levels of genes involved in fatty acid uptake and lipogenesis, leading to reduced hepatic lipid accumulation. Importantly, ALKBH1 overexpression was sufficient to suppress lipid uptake and synthesis, and alleviated diet-induced hepatic steatosis and insulin resistance. CONCLUSIONS: Our findings show an indispensable role of ALKBH1 as an epigenetic suppressor of DNA 6mA in hepatic fatty acid metabolism and offer a potential therapeutic target for NAFLD treatment.

Distribution Pattern of N6-Methyladenine DNA Modification in the Seashore Paspalum (Paspalum vaginatum) Genome.

N6-methyladenine (6mA) DNA modification has been detected in several eukaryotic organisms, in some of them, it plays important role in the regulation process of stress-resistance response. However, the genome-wide distribution patterns and potential functions of 6mA DNA modification in halophyte Seashore paspalum (Paspalum vaginatum) remain largely unknown. Here, we examined the 6mA landscape in the P. vaginatum genome by adopting single molecule real-time sequencing technology and found that 6mA modification sites were broadly distributed across the P. vaginatum genome. We demonstrated distinct 6mA methylation levels and 6mA distribution patterns in different types of transcription genes, which hinted at different epigenetic rules. Furthermore, the moderate 6mA density genes in P. vaginatum functionally correlated with stress resistance, which also maintained a higher transcriptional level. On the other hand, a specific 6mA distribution pattern in the gene body and near TSS was observed in gene groups with higher RNA expression, which maybe implied some kind of regularity between 6mA site distribution and the protein coding genes transcription was possible. Our study provides new insights into the association between 6mA methylation and gene expression, which may also contribute to key agronomic traits in P. vaginatum.

N 6-Methyladenine DNA Modification in the Woodland Strawberry (Fragaria vesca) Genome Reveals a Positive Relationship With Gene Transcription.

N 6-methyladenine (6mA) DNA modification has been detected in several eukaryotic organisms, where it plays important roles in gene regulation and epigenetic memory maintenance. However, the genome-wide distribution patterns and potential functions of 6mA DNA modification in woodland strawberry (Fragaria vesca) remain largely unknown. Here, we examined the 6mA landscape in the F. vesca genome by adopting single-molecule real-time sequencing technology and found that 6mA modification sites were broadly distributed across the woodland strawberry genome. The pattern of 6mA distribution in the long non-coding RNA was significantly different from that in protein-coding genes. The 6mA modification influenced the gene transcription and was positively associated with gene expression, which was validated by computational and experimental analyses. Our study provides new insights into the DNA methylation in F. vesca.