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Abstract N 1-methyladenosine (m1A) RNA methylation is critical for regulating mRNA translation; however, its role in the development, progression, and immunotherapy response of head and neck squamous cell carcinoma (HNSCC) remains largely unknown. Using Tgfbr1 and Pten conditional knockout (2cKO) mice, we found the neoplastic transformation of oral mucosa was accompanied by increased m1A modification levels. Analysis of m1A-associated genes identified TRMT61A as a key m1A writer linked to cancer progression and poor prognosis. Mechanistically, TRMT61A-mediated tRNA-m1A modification promotes MYC protein synthesis, upregulating programmed death-ligand 1 (PD-L1) expression. Moreover, m1A modification levels were also elevated in tumors treated with oncolytic herpes simplex virus (oHSV), contributing to reactive PD-L1 upregulation. Therapeutic m1A inhibition sustained oHSV-induced antitumor immunity and reduced tumor growth, representing a promising strategy to alleviate resistance. These findings indicate that m1A inhibition can prevent immune escape after oHSV therapy by reducing PD-L1 expression, providing a mutually reinforcing combination immunotherapy approach.

Lipid reprogramming metabolism is crucial for supporting tumor growth in breast cancer and investigating potential tumor biomarkers. Fatty acid esters of hydroxy fatty acids (FAHFAs) are a class of endogenous lipid metabolites with anti-diabetic and anti-inflammatory properties that have been discovered in recent years. Our previous targeted analysis of sera from breast cancer patients revealed a significant down-regulation of several FAHFAs. In this study, we aimed to further explore the relationship between FAHFAs and breast cancer by employing chemical isotope labeling combined with liquid chromatography−mass spectrometry (CIL-LC-MS) for profiling of FAHFAs in tumors and adjacent normal tissues from breast cancer patients. Statistical analysis identified 13 altered isomers in breast cancer. These isomers showed the potential to distinguish breast cancer tissues with an area under the curve (AUC) value above 0.9 in a multivariate receiver operating curve model. Furthermore, the observation of up-regulated 9-oleic acid ester of hydroxy stearic acid (9-OAHSA) and down-regulated 9-hydroxystearic acid (9-HSA) in tumors suggests that breast cancer shares similarities with colorectal cancer, and their potential mechanism is to attenuate the effects of pro-apoptotic 9-HSA by enhancing the synthesis of FAHFAs, thereby promoting tumor survival and progression through this buffering system.

Abstract Background Both testis and ovary can be produced sequentially in an individual with the same genome when sex reversal occurs in the teleost Monopterus albus, and epigenetic modification is supposed to be involved in gonadal differentiation. However, DNA methylation regulation mechanism underlying the gonadal differentiation remains unclear. Results Here, we used liquid chromatography-electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) to simultaneously determine endogenous levels of both 5-methyl-2′-deoxycytidine (m5dC) and 5-hydroxymethyl-2′-deoxycytidine (hm5dC) during gonadal differentiation. Overall DNA methylation level was upregulated from ovary to testis via ovotestis. As a de novo methylase, dnmt3aa expression was also upregulated in the process. Notably, we determined transcription factor Foxa1 for dnmt3aa gene expression. Site-specific mutations and chromatin immunoprecipitation showed that Foxa1 can bind to and activate the dnmt3aa promoter. Furthermore, DNA methylation levels of key genes foxl2 (forkhead box L2) and cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a) in regulation of female hormone synthesis were consistently upregulated during gonadal differentiation. Conclusions These data suggested that dynamic change of DNA methylation modification is associated with gonadal differentiation.

TET mediated RNA-hydroxymethylation (5hmC) has been detected in mammals, but its physiological role remains unclear. Here the authors map 5hmC during embryonic stem cell (ESC) differentiation and find that Tet-mediated RNA hydroxymethylation reduces the stability of crucial pluripotency related transcripts.

Abstract Background 5-Hydroxymethylcytosine (5hmC) is an oxidation product of 5-methylcytosine (5mC), and adjacent CpG sites in mammalian genome can be co-methylated and co-hydroxymethylated due to the processivity of DNMT and TET enzymes. Results We applied TAB-seq and oxBS-seq to selectively detect 5hmC and 5mC at base resolution in the mouse cortex, olfactory bulb and cerebellum tissues. We found that majority of the called 5hmC CpG sites frequently have 5mC modification simultaneously and are enriched in gene body regions of neuron development-related genes in brain tissues. Strikingly, by a systematic search of regions that show highly coordinated methylation and hydroxymethylation (MHBs and hMHBs), we found that MHBs significantly overlapped with hMHBs in gene body regions. Moreover, using a metric called methylation haplotype load, we defined a subset of 1361 tissue-specific MHBs and 3818 shared MHBs. Shared MHBs with low MHL correspond with developmental enhancers, and tissue-specific MHBs resemble the regulatory elements for tissue identity. Conclusions Our results provide new insights into the role of coordinately oxidized 5mC to 5hmC as distal regulatory elements may involve in regulating tissue identity.

Abstract Background Alterations in DNA methylation are demonstrated in atherosclerosis pathogenesis. However, changing rules of global DNA methylation and hydroxymethylation in peripheral blood leukocytes (PBLs) and different blood cell subtypes of coronary artery disease (CAD) patients are still inconclusive, and much less is known about mechanisms underlying. Results We recruited 265 CAD patients and 270 healthy controls with genomic DNA from PBLs, of which 50 patients and 50 controls were randomly chosen with DNA from isolated neutrophils, lymphocytes and monocytes, and RNA from PBLs. Genomic 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) contents were quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assay. Genomic 5-mC contents were negatively associated with the serum total cholesterol (TC) level (P = 0.010), age (P = 0.016), and PBL classifications (P = 0.023), explaining 6.8% individual variation in controls. Furthermore, genomic 5-mC contents were inversely associated with an increased risk of CAD (odds ratio (OR) = 0.325, 95% confidence interval (CI) = 0.237~0.445, P = 2.62 × 10− 12), independent of PBL counts and classifications, age, sex, histories of hyperlipidemia, hypertension, and diabetes. Within-individual analysis showed a general 5-mC decrease in PBL subtypes, but significant difference was found in monocytes only (P = 0.001), accompanied by increased 5-hmC (P = 3.212 × 10− 4). In addition, coincident to the reduced DNMT1 expression in patients’ PBLs, the expression level of DNMT1 was significantly lower (P = 0.022) in oxidized low-density lipoprotein (ox-LDL) stimulated THP-1-derived foam cells compared to THP-1 monocytes, with decreased genomic 5-mdC content (P = 0.038). Conclusions Global hypomethylation of blood cells defined dominantly by the monocyte DNA hypomethylation is independently associated with the risk of CAD in Chinese Han population. The importance of monocytes in atherosclerosis pathophysiology may demonstrate via an epigenetic pathway, but prospective studies are still needed to test the causality.

To counteract the deleterious effects of DNA damage, cells are equipped with specialized polymerases to bypass DNA lesions. Previous biochemical studies revealed that DinB family DNA polymerases, including Escherichia coli DNA polyrnerase IV and human DNA polymerase κ, efficiently incorporate the correct nucleotide opposite some N2-modified 2′-deoxyguanosine derivatives. Herein, we used shuttle vector technology and demonstrated that deficiency in Polkor Poli in mouse embryonic fibroblast (MEF) cells resulted in elevated frequencies of G→T and G→A mutations at N2-(1-carboxyethyl)-2′-deoxyguanosine (N2-CEdG) and N2-carboxymethyl-2′-deoxyguanosine (N2-CMdG) sites. Steady-state kinetic measurements revealed that human DNA polymerase ɩ preferentially inserts the correct nucleotide, dCMP, opposite N2-CEdG lesions. In contrast, no mutation was found after the N2-CEdG- and N2-CMdG-bearing plasmids were replicated in POLH-deficient human cells or Rev3-deficient MEF cells. Together, our results revealed that, in mammalian cells, both polymerases κ and ɩ are necessary for the error-free bypass of N2-CEdG and N2-CMdG. However, in the absence of polymerase κ or ɩ, other translesion synthesis polymerase(s) could incorporate nucleotide(s) opposite these lesions but would do so inaccurately. [ABSTRACT FROM AUTHOR]

The purpose of our study was to develop suitable methods to quantify oxidative DNA lesions in the setting of transition metal-related diseases. Transition metal-driven Fenton reactions constitute an important endogenous source of reactive oxygen species (ROS). In genetic diseases with accumulation of transition metal ions, excessive ROS production causes pathophysiological changes, including DNA damage. Wilson′s disease is an autosomal recessive disorder with copper toxicosis due to deficiency of ATP7B protein needed for excreting copper into bile. The Long-Evans Cinnamon (LEC) rat bears a deletion in Atp7b gene and serves as an excellent model for hepatic Wilson′s disease. We used a sensitive capillary liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS/MS) method in conjunction with the stable isotope-dilution technique to quantify several types of oxidative DNA lesions in the liver and brain of LEC rats. These lesions included 5-formyl-2′-deoxyuridine, 5-hydroxymethyl-2′-deoxyuridine, and the 5′R and 5′S diastereomers of 8,5′-cyclo-2′-deoxyguanosine and 8,5′-cyclo-2′-deoxyadenosine. Moreover, the levels of these DNA lesions in the liver and brain increased with age and correlated with age-dependent regulation of the expression of DNA repair genes in LEC rats. These results provide significant new knowledge for better understanding the implications of oxidative DNA lesions in transition metal-induced diseases, such as Wilson′s disease, as well as in aging and aging-related pathological conditions. [ABSTRACT FROM AUTHOR]

Reactive oxygen species can induce the formation of not only single-nucleobase lesions, which have been extensively studied, but also tandem lesions. Herein, we report a high frequency of formation of a type of tandem lesion, where two commonly observed oxidatively induced single-nucleobase lesions, that is, thymidine glycol (Tg) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), are vicinal to each other in calf thymus DNA upon exposure to Cu(II)/ascorbate along with H2O2or γ-rays. We further explored how the tandem lesions perturb the efficiency and fidelity of DNA replication by assessing the replication products formed from the propagation, in Escherichia colicells, of the single-stranded pYMV1 shuttle vectors containing two tandem lesions [5′-(8-oxodG)-Tg-3′ and 5′-Tg-(8-oxodG)-3′] or an isolated Tg or 8-oxodG. The bypass efficiencies for the two tandem lesions were approximately one-half of those for the two isolated single-nucleobase lesions. The presence of an adjacent Tg could lead to significant increases in G→T transversion at the 8-oxodG site as compared to that of a single 8-oxodG lesion; the frequencies of G→T mutation were approximately 18, 32, and 28% for 8-oxodG that is isolated, in 5′-(8-oxodG)-Tg-3′ and in 5′-Tg-(8-oxodG)-3′, respectively. Moreover, both pol IV and pol V are involved, in part, in bypassing the Tg, either present alone or as part of the tandem lesions, in E. colicells. Together, our results support that complex lesions could exert greater cytotoxic and mutagenic effects than when the composing individual lesions are present alone. [ABSTRACT FROM AUTHOR]

Thiopurine drugs, including 6-thioguanine (SG), 6-mercaptopurine, and azathioprine, are widely employed anticancer agents and immunosuppressants. The formation of SG nucleotides from the thiopurine prodrugs and their subsequent incorporation into nucleic acids are important for the drugs to exert their cytotoxic effects. SG in DNA can be methylated by S-adenosyl-L-methionine to give SG-methylthioguanine (S6mG) and oxidized by UVA light to render guanine-S6-sulfonic acid (SO3HG) Here, we constructed single-stranded M13 shuttle vectors carrying a SG, S6mG, or SO3HG at a unique site and allowed the vectors to propagate in wild-type and bypass polymerase-deficient Escherichia coli cells, Analysis of the replication products by using the competitive replication and adduct bypass and a slightly modified restriction enzyme digestion and post-labeling assays revealed that, although none of the three thionucleosides considerably blocked DNA replication in all transfected E. coli cells, both S6mG and SO3HG were highly mutagenic, which resulted in G→A mutation at frequencies of 94 and 77%, respectively, in wild-type E. coli cells. Deficiency in bypass polymerases does not result in alteration of mutation frequencies of these two lesions. In contrast to what was found from previous steady-state kinetic analysis, our data demonstrated that 6-thioguanine is mutagenic, with G→A transition occurring at a frequency of -10%. The mutagenic properties of 6-thioguanine and its derivatives revealed in the present study offered important knowledge about the biological implications of these thionucleosides. [ABSTRACT FROM AUTHOR]

DinB, a Y-family DNA polymerase, is conserved among all domains of life; however, its endogenous substrates have not been identified. DinB is known to synthesize accurately across a number of N2-dG lesions. Methylglyoxal (MG) is a common byproduct of the ubiquitous glycolysis pathway and induces the formation of N2- (1-carboxyethyl)-2′-deoxyguanosine (N2-CEdG) as the major stable DNA adduct. Here, we found that N2-CEdG could be detected at a frequency of one lesion per 107 nucleosides in WM-266-4 human melanoma cells, and treatment of these cells with MG or glucose led to a dose-responsive increase in N2-CEdG formation. We further constructed single-stranded M13 shuttle vectors harboring individual diastereomers of N2-CEdG at a specific site and assessed the cytotoxic and mutagenic properties of the lesion in wild-type and bypass polymerase-deficient Escherichia coli cells. Our results revealed that N2-CEdG is weakly mutagenic, and DinB (i.e., polymerase IV) is the major DNA polymerase responsible for bypassing the lesion in vivo. Moreover, steady-state kinetic measurements showed that nucleotide insertion, catalyzed by E. coli pol IV or its human counterpart (i.e., polymerase κ), opposite the N2-CEdG is both accurate and efficient. Taken together, our data support that N2-CEdG, a minor-groove DNA adduct arising from MG, is an important endogenous substrate for DinB DNA polymerase. [ABSTRACT FROM AUTHOR]

Aberrant cytosine methylation is known to be associated with cancer development Here, we assessed how common cancer chemotherapeutic agents perturb cytosine methylation in Jurkat-T acute lymphoblastic leukemia cells. We tested six antitumor agents and found that cyclophosphamide induced the most pronounced increase in global DNA cytosine methylation after a 24-h treatment Long-term treatment with cyclophosphamide led to a time-dependent increase in cytosine methylation level with up to 4 days of treatment, and the 1 extent of cytosine methylation returned to normal level after 8 days. The trend of 5 change in DNA methylation level paralleled that of the expression level of DNMT1 protein, whereas no significant increase in DNMT1 mRNA level was observed. Previous studies showed that the stability of endogenous DNMT1 protein is regulated by lysine methylation through histone lysine memyltransferase Set7 and lysine-specific demethylase 1 (LSD 1), with the methylated DNMT1 being the target for proteasomal degradation. We observed that the elevated expression of DNMT1 protein at 4 days of treatment was correlated with die increased expression of LSD1 protein and with the decreased frequency of K142 methylation in DNMT1. Taken together, our results showed that cyclophosphamide perturbed temporarily global cytosine methylation in Jurkat-T cells via regulation of the lysine mediation level in DNMT1. [ABSTRACT FROM AUTHOR]

Thiopurine drugs are extensively used as chemotherapeutic agents in clinical practice, even though there is concern about the risk of therapy-related cancers. It has been previously suggested that the cytotoxicity of thiopurine drugs involves their metabolic activation, the resultant generation of 6-thioguanine (SG) and S6-methylthioguanine (S6mG) in DNA, and the futile mismatch repair triggered by replication-induced SG:T and S6mG:T mispairs. Disruption of transcription is known to be one of the major consequences of DNA damage induced by many antiviral and antitumor agents; however, it remains undefined how SG and S6mG compromise the efficiency and fidelity of transcription. Using our recently developed competitive transcription and adduct bypass assay, herein we examined the impact of SG and S6mG on transcription in vitro and in human cells. Our results revealed that, when situated on the transcribed strand, S6mG exhibited both inhibitory and mutagenic effects during transcription mediated by single-subunit T7 RNA polymerase or multisubunit human RNA polymerase II in vitro and in human cells. Moreover, we found that the impact of S6mG on transcriptional efficiency and fidelity is modulated by the transcription-coupled nucleotide excision repair capacity. In contrast, SG did not considerably compromise the efficiency or fidelity of transcription, and it was a poor substrate for NER.We propose that S6mG might contribute, at least in part, to thiopurine- mediated cytotoxicity through inhibition of transcription and to potential therapy-related carcinogenesis via transcriptional mutagenesis. [ABSTRACT FROM AUTHOR]

Reactive oxygen species can give rise to a battery ofDNAdamage products including the 8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG) tandem lesions. The 8,5′-cyclopurine-2′-deoxynucleosides are quite stable lesions and are valid and reliable markers of oxidative DNA damage. However, it remains unclear how these lesions compromise DNAreplication in mammalian cells. Previous in vitro biochemical assays have suggested a role forhumanpolymerase (Pol) ƞ in the insertion step of translesion synthesis (TLS) across the (5′S) diastereomers of cdA and cdG. Using in vitro steady-state kinetic assay, herein we showed that human Pol Ɩ and a two-subunit yeast Pol ζ complex (REV3/REV7) could function efficiently in the insertion and extension steps, respectively, of TLS across S-cdA and S-cdG; human Pol κ and Pol η could also extend past these lesions, albeit much less efficiently. Results from a quantitative TLS assay showed that, in human cells, S-cdA and S-cdG inhibited strongly DNA replication and induced substantial frequencies of mutations at the lesion sites. Additionally, Polη, PolƖ, and Polζ, but not Polκ, had important roles in promoting replication through S-cdA and S-cdG in human cells. Based on these results, we propose a model for TLS across S-cdA and S-cdG in human cells, where Pol η and/or Pol Ɩ carries out nucleotide insertion opposite the lesion, whereas Pol ζ executes the extension step. [ABSTRACT FROM AUTHOR]