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• In addition to the four canonical nucleobases, many modified nucleobases have been discovered in nucleic acids across various organisms [1-6]. Among these, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) stands out as the most extensively studied epigenetic modification, exerting crucial functions in a diverse array of biological processes in mammals [7-15]. Apart from 5mC, N⁶-methyladenine (6mA) is also a naturally occurring DNA modification found in both prokaryotes and eukaryotes [16,17]. In prokaryotes, 6mA is known to be involved in the restrictive modification (R-M) system [18]. Additionally, 6mA plays important roles in DNA mismatch repair and gene regulation processes in E. coli and other bacterial species [19], as well as in transcriptional regulation in certain bacteria [20]. While initially believed to be absent in eukaryotes, recent advancements in deep sequencing have uncovered the presence of 6mA modification in a limited number of eukaryotic organisms such as Chlamydomonas rheinus [21], Caenorhabditis elegans [22], Drosophila melanogaster [23], fungi [24], mice [25], Arabidopsis thaliana [26], zebrafish and pig [27]. In plants and fungi, 6mA modification is abundant around transcription start sites and is often associated with active transcription [21,24,26]. Conversely, high level of 6mA in animals has been linked to the inhibition of downstream gene activity [25].

  6mA has been implicated in a wide range of human diseases, including various types of cancers [28]. Research has indicated that dysregulation of 6mA is associated with the pathogenesis of different cancer types, underscoring the importance of 6mA in oncology [29]. A previous study has shown decreased level of 6mA in human liver cancer tissues compared to adjacent non-tumor tissues [30]. Xiao et al. observed a reduced abundance of 6mA density in primary gastric and liver cancer tissues, and this downregulation of 6mA density is linked to tumorigenesis [31]. Knocking down the 6mA methylase of N6AMT1 significantly reduces 6mA in DNA, leading to increased colony formation and migration of breast cancer cells [31]. Silencing N6AMT1 decreases 6mA level and enhances the growth of breast cancer cells [31]. Conversely, glioblastoma cells exhibit an increased level of 6mA [31,32].

  The elucidation of 6mA functions relies on precise detection and mapping methods [33-37]. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a commonly employed technique for quantifying nucleic acid modifications and metabolites [38-44]. However, it lacks the capability to provide detailed location information of nucleic acid modifications. Direct sequencing of 6mA has posed challenges due to its resemblance to adenine in hydrogen bonding with thymine. Immunoprecipitation sequencing techniques, such as 6mA DIP-seq, have been proposed for mapping 6mA in mammalian cell genomes [32,45]; however, the resolution of 6mA mapping using 6mA DIP-seq is low [46]. Dpn I-assisted N⁶-methyladenine sequencing (DA-6mA-seq) uses Dpn I to cleave methylated adenine sites (5′-G6mATC-3′) in duplex DNA to map 6mA sites [47], but it is restricted to a specific subset of 6mA. 6mA cross-linking exonuclease sequencing (6mACE-seq) employs 6mA-specific antibodies for cross-linking to prevent 6mA-DNA fragments from exonuclease digestion [48]. The 6mA-CLIP-exo method integrates immunoprecipitation enrichment, photo-crosslinking, and exonuclease digestion to map 6mA sites [21]. However, the detection of 6mA is confined to sites recognized by these enzymes, potentially leading to the loss of site information if 6mA appears in other motifs [49]. We recently developed a deaminase-mediated sequencing (DM-seq) method to map 6mA in the mtDNA of HepG2 cells [50], which offers a straightforward approach for mapping 6mA in DNA at single-base resolution from low input DNA (~10 ng).

  It has been reported that mammalian mitochondrial DNA (mtDNA) is enriched with 6mA, which functions in regulating mitochondrial transcription, replication, and activity [51]. The level of 6mA in mtDNA is approximately 1300 times higher than that in genomic DNA [51]. However, it is currently unknown whether there is a difference in the levels of 6mA sites in mtDNA between cancer tissues and normal tissues. In this study, we utilized our recently developed DM-seq method to explore the alterations and physiological implications of mtDNA 6mA in hepatocellular carcinoma (HCC). This involved analyzing the levels of various 6mA sites at single-base resolution within mtDNA extracted from both HCC tissues and adjacent normal tissues.

  In the DM-seq method, the TadA8e protein effectively deaminates adenosine (A) in DNA to generate inosine (I), but it does not deaminate 6mA (Fig. 1a). I base pairs with cytidine (C), while 6mA base pairs with thymidine (T). During subsequent sequencing, I is interpreted as guanosine (G), whereas 6mA is interpreted as A (Fig. 1a). We first expressed and purified the TadA8e protein according to our previous protocol [50]. SDS-PAGE analysis confirmed the successful expression and purification of the TadA8e protein (Fig. S1 in Supporting information). Two synthesized DNA molecules, one containing a single A (A-DNA) and the other containing a single 6mA (6mA-DNA) (Table S1 in Supporting information), were utilized as substrates in the DM-seq experiment. The Sanger sequencing results demonstrated that the DM-seq method can accurately identify and map 6mA in DNA at single-base resolution (Fig. 1b).

  Figure 1

  

  Figure 1.  Principle of the DM-seq method. (a) Schematic illustration for mapping of 6mA in DNA by DM-seq method. (b) Sanger sequencing of A-DNA and 6mA-DNA with or without TadA8e treatment. (c) Schematic illustration for the preparation of library for high-throughput sequencing by DM-seq. The workflow includes the fragmentation of mtDNA, ligation of adapters at both ends of the DNA fragment, deamination through TadA8e treatment, and PCR amplification.

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  We next employed the DM-seq to map 6mA in mtDNA from HCC tissues and matched tumor-adjacent normal tissues (Table S2 in Supporting information), and an approval for this study was granted by the Ethics Committee of Wuhan University. The schematic illustration in Fig. 1c outlines the procedure for preparing the sequencing library to map 6mA by DM-seq. Initially, mtDNA was fragmented into 300 bp to 500 bp fragments. The mixture of fragmented DNA and spike-in DNA (Table S1) underwent end-repair and dA-tailing using the Hieff NGS Fast-Pace End Repair/dA-Tailing module. Subsequently, the DNA was ligated with an adapter using the Ultima DNA Ligation Module. The resulting DNA was denatured to generate single-stranded DNA, followed by deamination using TadA8e protein. The deaminated DNA was amplified by PCR, and the PCR products were purified using KAPA Pure Beads. A second round of PCR amplification was performed using P5 and P7 primers containing distinct index sequences (Table S3 in Supporting information). The resulting library was then subjected to high-throughput sequencing analysis.

  Four mtDNA samples from two paired HCC tissues were subjected to DM-seq analysis. The raw data from high-throughput sequencing were processed according to our previously established method [50]. Initially, nuclear DNA segments were filtered out to ensure that the reads exclusively mapped to mtDNA. Subsequently, 185,528, 196,256, 166,711, and 178,580 reads were obtained from these four samples, with an average sequencing depth of 134 (Table S4 in Supporting information). Our focus was on the 16 previously identified 6mA sites present in the mtDNA of HepG2 cells. The circus plot illustrates the 16 identified 6mA sites in the mtDNA of HCC tissues and their corresponding normal tissues in the current study (Fig. 2). In addition, the positions of 6mA in mtDNA are summarized in Table S5 (Supporting information).

  Figure 2

  

  Figure 2.  6mA sites identified in mtDNA of HCC tissues and their corresponding normal tissues using DM-seq. (a) Distribution of 6mA sites in mtDNA of sample 1. (b) Distribution of 6mA sites in mtDNA of sample 2. The outermost circle indicates the size of mtDNA; the colored circle represents the genes distributed in mtDNA; the bars in the inner circles illustrate the distribution of 6mA sites in mtDNA (red bars for HCC tissues and blue bars for corresponding normal tissues).

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  Subsequently, Sanger sequencing was utilized to validate the 6mA sites identified through high-throughput sequencing. Specifically, two 6mA sites at positions 6241 and 6293 were chosen for analysis. The mtDNA was denatured to generate single-stranded DNA, which underwent TadA8e treatment. Following PCR amplification, the resulting PCR products were subjected to Sanger sequencing. A comparison of the untreated and treated PCR products using Sanger sequencing revealed that, apart from positions 6241 and 6293 (Fig. 3), which remained as A after deamination, all other sites initially identified as A prior to deamination were converted to G after TadA8e treatment. This finding confirms that the two sites at positions 6241 and 6293 identified by high-throughput sequencing indeed represent authentic instance of 6mA modification.

  Figure 3

  

  Figure 3.  Validation of 6mA sites in mtDNA through Sanger sequencing. (a) Sanger sequencing result of the 6mA site at position 6241. (b) Sanger sequencing result of the 6mA site at position 6293. In Sanger sequencing, the 6mA sites identified in mtDNA by DM-seq were read as A after TadA8e treatment, whereas other A sites were read as G after TadA8e treatment. The arrows indicate the A sites.

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  Next, we conducted statistical analysis to assess the alteration in 6mA level between HCC and adjacent normal tissues. The analysis revealed an overall upregulation of 6mA level in both HCC tissues compared to the corresponding adjacent normal tissues (Fig. 4). Specifically, we observed an elevated level of 6mA in 11 out of the 16 sites in the HCC tissue compared to the adjacent normal tissue in sample 1 (Fig. S2 in Supporting information), and an increased level of 6mA in 15 out of the 16 sites in the HCC tissue compared to the adjacent normal tissue in sample 2 (Fig. S3 in Supporting information). In contrast, five 6mA sites and one 6mA site displayed a decreasing trend in HCC tissues compared to adjacent normal tissues in sample 1 and sample 2, respectively (Figs. S2 and S3).

  Figure 4

  

  Figure 4.  Statistical analysis of the 6mA levels in 16 sites in mtDNA of HCC tissues and the adjacent normal tissues obtained through DM-seq. (a) The 6mA levels in 16 sites in mtDNA from sample 1. (b) The 6mA levels in 16 sites in mtDNA from sample 2.

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  Among the 16 identified 6mA sites in mtDNA, one 6mA site was located within the D-loop region, while the remaining fifteen 6mA sites were located within exonic regions of the genes. Specifically, three 6mA sites were observed in both the NADH dehydrogenase 2 (ND2) and NADH dehydrogenase 4 (ND4) gene regions, and two 6mA sites were detected in both the NADH dehydrogenase 1 (ND1) and NADH dehydrogenase 5 (ND5) gene regions (Fig. 5). Notably, these four genes encode essential subunits of NADH dehydrogenase. Furthermore, three 6mA sites were identified within the cytochrome c oxidase Ⅰ (COX1) region and two within the cytochrome c oxidase Ⅱ (COX2) region, which encode distinct subunits of cytochrome c oxidase. Both enzymes play crucial roles in cellular respiration processes.

  Figure 5

  

  Figure 5.  The numbers of identified 6mA sites in different genes in mtDNA.

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  We next proceeded to assess the impact of alteration of 6mA level on mRNA expression. Taking the sample 2 as an example, the 6mA level in different gene regions of HCC tissues exhibited an increasing trend compared to adjacent normal tissues (Fig. 6a). Remarkably, a downregulation in mRNA levels encoded by six genes was observed (Fig. 6b). The qPCR primers are listed in Table S6 and the real-time amplification curves of mRNA were depicted in Fig. S4 (Supporting information). The results indicated that while there was an upregulation in 6mA level, a simultaneous downregulation occurred in the mRNA levels of these genes, suggesting that increased 6mA level impeded transcription processes associated with these genes. These findings are in line with a previous study that demonstrated a decrease in AlkB homolog 1 histone H2A dioxygenase (ALKBH1) expression resulting in an increased level of 6mA associated with mtDNA, which subsequently led to a decrease in the expression of the NADH dehydrogenase subunit encoded by mtDNA [52].

  Figure 6

  

  Figure 6.  The impact of 6mA sites on mRNA expression. (a) The 6mA levels in different gene regions of mtDNA. (b) mRNA expression levels of different genes in HCC tissue over adjacent normal tissue.

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  The overall increase in 6mA level in mtDNA could impact mitochondrial transcriptional activity. Previous study has shown that 6mA has the potential to inhibit DNA binding and bending by the mitochondrial transcription factor (TFAM), leading to a possible suppression of the transcription process [51]. The regulation of 6mA levels is governed by the methyltransferase of METTL4 [51]. In cases of HCC, targeting METTL4 could hold promise as a potential therapeutic approach. Looking ahead, manipulating the expression of METTL4 offers a potential avenue for modulating 6mA level in mtDNA in the future.

  In summary, utilizing DM-seq, we investigated the alterations in the level of 16 6mA sites in mtDNA obtained from HCC tissues and adjacent normal tissues. Our findings revealed that the overall 6mA level was elevated within mtDNA associated with HCC development. Furthermore, we noted an upregulation in 6mA content accompanied by a downregulation in their respective mRNA levels, suggesting that the increased 6mA content impeded transcription processes linked to these DNA fragments. These results offer compelling evidence supporting the inhibitory impact of heightened 6mA level on downstream transcriptional activity. This study sheds light on the intricate relationship between 6mA modification and transcriptional regulation in the context of HCC, providing valuable insights underlying HCC pathogenesis.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Wen-Xuan Shao: Writing – review & editing, Writing – original draft, Methodology, Formal analysis, Data curation, Conceptualization. Jianyuan Wu: Validation, Methodology, Formal analysis, Data curation. Gaojie Li: Software, Formal analysis. Yi-Hao Min: Data curation. Qiu-Shuang Hu: Data curation. Yu Liu: Supervision, Formal analysis. Weimin Ci: Software, Formal analysis. Bi-Feng Yuan: Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

  Acknowledgments

  The work is supported by the National Natural Science Foundation of China (No. 22277093), and the Key Research and Development Project of Hubei Province (No. 2023BCB094).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110747.

  
  
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  Figure 1  Principle of the DM-seq method. (a) Schematic illustration for mapping of 6mA in DNA by DM-seq method. (b) Sanger sequencing of A-DNA and 6mA-DNA with or without TadA8e treatment. (c) Schematic illustration for the preparation of library for high-throughput sequencing by DM-seq. The workflow includes the fragmentation of mtDNA, ligation of adapters at both ends of the DNA fragment, deamination through TadA8e treatment, and PCR amplification.

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  Figure 2  6mA sites identified in mtDNA of HCC tissues and their corresponding normal tissues using DM-seq. (a) Distribution of 6mA sites in mtDNA of sample 1. (b) Distribution of 6mA sites in mtDNA of sample 2. The outermost circle indicates the size of mtDNA; the colored circle represents the genes distributed in mtDNA; the bars in the inner circles illustrate the distribution of 6mA sites in mtDNA (red bars for HCC tissues and blue bars for corresponding normal tissues).

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  Figure 3  Validation of 6mA sites in mtDNA through Sanger sequencing. (a) Sanger sequencing result of the 6mA site at position 6241. (b) Sanger sequencing result of the 6mA site at position 6293. In Sanger sequencing, the 6mA sites identified in mtDNA by DM-seq were read as A after TadA8e treatment, whereas other A sites were read as G after TadA8e treatment. The arrows indicate the A sites.

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  Figure 4  Statistical analysis of the 6mA levels in 16 sites in mtDNA of HCC tissues and the adjacent normal tissues obtained through DM-seq. (a) The 6mA levels in 16 sites in mtDNA from sample 1. (b) The 6mA levels in 16 sites in mtDNA from sample 2.

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  Figure 5  The numbers of identified 6mA sites in different genes in mtDNA.

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  Figure 6  The impact of 6mA sites on mRNA expression. (a) The 6mA levels in different gene regions of mtDNA. (b) mRNA expression levels of different genes in HCC tissue over adjacent normal tissue.

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