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Environmental Science & Technology Oct 2023Contaminants of emerging concern (CECs) in the environment undergo various transformations, leading to the formation of transformation products (TPs) with a modified...
Contaminants of emerging concern (CECs) in the environment undergo various transformations, leading to the formation of transformation products (TPs) with a modified ecological risk potential. Although the environmental significance of TPs is increasingly recognized, there has been relatively little research to understand the influences of such transformations on subsequent ecotoxicological safety. In this study, we used four pairs of CECs and their methylated or demethylated derivatives as examples to characterize changes in bioaccumulation and acute toxicity in , as a result of methylation or demethylation. The experimental results were further compared to quantitative structure-activity relationship (QSAR) predictions. The methylated counterpart in each pair generally showed greater acute toxicity in , which was attributed to their increased hydrophobicity. For example, the LC values of methylparaben (34.4 ± 4.3 mg L) and its demethylated product (225.6 ± 17.3 mg L) differed about eightfold in . The methylated derivative generally exhibited greater bioaccumulation than the demethylated counterpart. For instance, the bioaccumulation of methylated acetaminophen was about 33-fold greater than that of acetaminophen. predictions via QSARs aligned well with the experimental results and suggested an increased persistence of the methylated forms. The study findings underline the consequences of simple changes in chemical structures induced by transformations such as methylation and demethylation and highlight the need to consider TPs to achieve a more holistic understanding of the environmental fate and risks of CECs.
PubMed: 37769124
DOI: 10.1021/acs.est.3c03242 -
Scientific Reports Mar 2024As the most prevalent epitranscriptomic modification, N-methyladenosine (mA) shows important roles in a variety of diseases through regulating the processing, stability...
METTL3 and METTL14-mediated N-methyladenosine modification of SREBF2-AS1 facilitates hepatocellular carcinoma progression and sorafenib resistance through DNA demethylation of SREBF2.
As the most prevalent epitranscriptomic modification, N-methyladenosine (mA) shows important roles in a variety of diseases through regulating the processing, stability and translation of target RNAs. However, the potential contributions of mA to RNA functions are unclear. Here, we identified a functional and prognosis-related mA-modified RNA SREBF2-AS1 in hepatocellular carcinoma (HCC). The expression of SREBF2-AS1 and SREBF2 in HCC tissues and cells was measured by RT-qPCR. mA modification level of SREBF2-AS1 was measured by methylated RNA immunoprecipitation assay. The roles of SREBF2-AS1 in HCC progression and sorafenib resistance were investigated by proliferation, apoptosis, migration, and cell viability assays. The regulatory mechanisms of SREBF2-AS1 on SREBF2 were investigated by Chromatin isolation by RNA purification, RNA immunoprecipitation, CUT&RUN, and bisulfite DNA sequencing assays. Our findings showed that the expression of SREBF2-AS1 was increased in HCC tissues and cells, and positively correlated with poor survival of HCC patients. mA modification level of SREBF2-AS1 was also increased in HCC and positively correlated with poor prognosis of HCC patients. METTL3 and METTL14-induced mA modification upregulated SREBF2-AS1 expression through increasing SREBF2-AS1 transcript stability. Functional assays showed that only mA-modified, but not non-modified SREBF2-AS1 promoted HCC progression and sorafenib resistance. Mechanistic investigations revealed that mA-modified SREBF2-AS1 bound and recruited mA reader FXR1 and DNA 5-methylcytosine dioxygenase TET1 to SREBF2 promoter, leading to DNA demethylation at SREBF2 promoter and the upregulation of SREBF2 transcription. Functional rescue assays showed that SREBF2 was the critical mediator of the oncogenic roles of SREBF2-AS1 in HCC. Together, this study showed that mA-modified SREBF2-AS1 exerted oncogenic roles in HCC through inducing DNA demethylation and transcriptional activation of SREBF2, and suggested mA-modified SREBF2-AS1 as a prognostic biomarker and therapeutic target for HCC.
Topics: Humans; Carcinoma, Hepatocellular; Sorafenib; Liver Neoplasms; DNA Demethylation; Cell Line, Tumor; MicroRNAs; RNA-Binding Proteins; Mixed Function Oxygenases; Proto-Oncogene Proteins; Methyltransferases; Adenosine; Sterol Regulatory Element Binding Protein 2
PubMed: 38486042
DOI: 10.1038/s41598-024-55932-7 -
Oncogene Mar 2022Non-small cell lung cancer (NSCLC) is a fatal disease, and its metastatic process is poorly understood. Although aberrant methylation is involved in tumor progression,...
Non-small cell lung cancer (NSCLC) is a fatal disease, and its metastatic process is poorly understood. Although aberrant methylation is involved in tumor progression, the mechanisms underlying dynamic DNA methylation remain to be elucidated. It is significant to study the molecular mechanism of NSCLC metastasis and identify new biomarkers for NSCLC early diagnosis. Here, we performed MeDIP-seq and hMeDIP-seq analyses to detect the genes regulated by dynamic DNA methylation. Comparison of the 5mC and 5hmC sites revealed that the CD147 gene underwent active demethylation in NSCLC tissues compared with normal tissues, and this demethylation upregulated CD147 expression. Significantly high levels of CD147 expression and low levels of promoter methylation were observed in NSCLC tissues. Then, we identified the CD147 promoter as a target of KLF6, MeCP2, and DNMT3A. Treatment of cells with TGF-β triggered active demethylation involving loss of KLF6/MeCP2/DNMT3A and recruitment of Sp1, Tet1, TDG, and SMAD2/3 transcription complexes. A dCas9-SunTag-DNMAT3A-sgCD147-targeted methylation system was constructed to reverse CD147 expression. The targeted methylation system downregulated CD147 expression and inhibited NSCLC proliferation and metastasis in vitro and in vivo. Accordingly, we used cfDNA to detect the levels of CD147 methylation in NSCLC tissues and found that the CD147 methylation levels exhibited an inverse relationship with tumor size, lymphatic metastasis, and TNM stage. In conclusion, this study clarified the mechanism of active demethylation of CD147 and suggested that the targeted methylation of CD147 could inhibit NSCLC invasion and metastasis, providing a highly promising therapeutic target for NSCLC.
Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; DNA Methylation; Demethylation; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mixed Function Oxygenases; Proto-Oncogene Proteins
PubMed: 35132181
DOI: 10.1038/s41388-022-02213-0 -
The Journal of Biological Chemistry Feb 2024Increased expression of angiotensin II AT receptor (encoded by Agtr1a) and Na-K-Cl cotransporter-1 (NKCC1, encoded by Slc12a2) in the hypothalamic paraventricular...
Increased expression of angiotensin II AT receptor (encoded by Agtr1a) and Na-K-Cl cotransporter-1 (NKCC1, encoded by Slc12a2) in the hypothalamic paraventricular nucleus (PVN) contributes to hypertension development. However, little is known about their transcriptional control in the PVN in hypertension. DNA methylation is a critical epigenetic mechanism that regulates gene expression. Here, we determined whether transcriptional activation of Agtr1a and Slc12a2 results from altered DNA methylation in spontaneously hypertensive rats (SHR). Methylated DNA immunoprecipitation and bisulfite sequencing-PCR showed that CpG methylation at Agtr1a and Slc12a2 promoters in the PVN was progressively diminished in SHR compared with normotensive Wistar-Kyoto rats (WKY). Chromatin immunoprecipitation-quantitative PCR revealed that enrichment of DNA methyltransferases (DNMT1 and DNMT3A) and methyl-CpG binding protein 2, a DNA methylation reader protein, at Agtr1a and Slc12a2 promoters in the PVN was profoundly reduced in SHR compared with WKY. By contrast, the abundance of ten-eleven translocation enzymes (TET1-3) at Agtr1a and Slc12a2 promoters in the PVN was much greater in SHR than in WKY. Furthermore, microinjecting of RG108, a selective DNMT inhibitor, into the PVN of WKY increased arterial blood pressure and correspondingly potentiated Agtr1a and Slc12a2 mRNA levels in the PVN. Conversely, microinjection of C35, a specific TET inhibitor, into the PVN of SHR markedly reduced arterial blood pressure, accompanied by a decrease in Agtr1a and Slc12a2 mRNA levels in the PVN. Collectively, our findings suggest that DNA hypomethylation resulting from the DNMT/TET switch at gene promoters in the PVN promotes transcription of Agtr1a and Slc12a2 and hypertension development.
Topics: Animals; Rats; Blood Pressure; DNA; DNA Demethylation; Hypertension; Hypothalamus; Paraventricular Hypothalamic Nucleus; Rats, Inbred SHR; Rats, Inbred WKY; Receptor, Angiotensin, Type 1; RNA, Messenger; Sympathetic Nervous System; Solute Carrier Family 12, Member 2
PubMed: 38160798
DOI: 10.1016/j.jbc.2023.105597 -
Vavilovskii Zhurnal Genetiki I Selektsii Jul 2022One of the main mechanisms of epigenetic regulation in higher eukaryotes is based on the methylation of cytosine at the C5 position with the formation of...
One of the main mechanisms of epigenetic regulation in higher eukaryotes is based on the methylation of cytosine at the C5 position with the formation of 5-methylcytosine (mC), which is further recognized by regulatory proteins. In mammals, methylation mainly occurs in CG dinucleotides, while in plants it targets CG, CHG, and CHH sequences (H is any base but G). Correct maintenance of the DNA methylation status is based on the balance of methylation, passive demethylation, and active demethylation. While in mammals active demethylation is based on targeted regulated damage to mC in DNA followed by the action of repair enzymes, demethylation in plants is performed by specialized DNA glycosylases that hydrolyze the N-glycosidic bond of mC nucleotides. The genome of the model plant Arabidopsis thaliana encodes four paralogous proteins, two of which, DEMETER (DME) and REPRESSOR OF SILENCING 1 (ROS1), possess 5-methylcytosine-DNA glycosylase activity and are necessary for the regulation of development, response to infections and abiotic stress and silencing of transgenes and mobile elements. Homologues of DME and ROS1 are present in all plant groups; however, outside A. thaliana, they are poorly studied. Here we report the properties of a recombinant fragment of the ROS1 protein from Nicotiana tabacum (NtROS1), which contains all main structural domains required for catalytic activity. Using homologous modeling, we have constructed a structural model of NtROS1, which revealed folding characteristic of DNA glycosylases of the helix- hairpin-helix structural superfamily. The recombinant NtROS1 protein was able to remove mC bases from DNA, and the enzyme activity was barely affected by the methylation status of CG dinucleotides in the opposite strand. The enzyme removed 5-hydroxymethylcytosine (hmC) from DNA with a lower eff iciency, showing minimal activity in the presence of mC in the opposite strand. Expression of the NtROS1 gene in cultured human cells resulted in a global decrease in the level of genomic DNA methylation. In general, it can be said that the NtROS1 protein and other homologues of DME and ROS1 represent a promising scaffold for engineering enzymes to analyze the status of epigenetic methylation and to control gene activity.
PubMed: 35860677
DOI: 10.18699/VJGB-22-41 -
Molecular Oncology Feb 2023Colorectal cancer (CRC) is one of the most common malignancies, and the main cause of death from CRC is tumor metastasis. m A RNA modification plays critical role in...
Colorectal cancer (CRC) is one of the most common malignancies, and the main cause of death from CRC is tumor metastasis. m A RNA modification plays critical role in many biological processes. However, the role of m A modification in CRC remains unclear. Here, we find that the m A demethylase alkB homolog 1, histone H2A dioxygenase (ALKBH1) is overexpressed in CRC and is associated with metastasis and poor prognosis. Upregulation of ALKBH1 expression promotes CRC metastasis in vitro and in vivo. Mechanistically, knockdown of ALKBH1 results in a decrease in methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit (METTL3) expression, probably due to m A modification of METTL3 mRNA, followed by m A demethylation of SMAD family member 7 (SMAD7) mRNA. In addition, downregulation of SMAD7 establishes an aggressive phenotype. More importantly, the cell migration and invasion defects caused by ALKBH1 depletion or METTL3 depletion are significantly reversed by SMAD7 silencing. Considering these results collectively, we propose that ALKBH1 promotes CRC metastasis by destabilizing SMAD7 through METTL3.
Topics: Humans; RNA, Messenger; Methyltransferases; Up-Regulation; Demethylation; Colorectal Neoplasms; AlkB Homolog 1, Histone H2a Dioxygenase; Smad7 Protein
PubMed: 36550779
DOI: 10.1002/1878-0261.13366 -
The Plant Cell Mar 2022Cytosine methylation is a reversible epigenetic modification of DNA. In plants, removal of cytosine methylation is accomplished by the four members of the DEMETER (DME)...
Cytosine methylation is a reversible epigenetic modification of DNA. In plants, removal of cytosine methylation is accomplished by the four members of the DEMETER (DME) family of 5-methylcytosine DNA glycosylases, named DME, DEMETER-LIKE2 (DML2), DML3, and REPRESSOR OF SILENCING1 (ROS1) in Arabidopsis thaliana. Demethylation by DME is critical for seed development, preventing experiments to determine the function of the entire gene family in somatic tissues by mutant analysis. Here, we bypassed the reproductive defects of dme mutants to create somatic quadruple homozygous mutants of the entire DME family. dme; ros1; dml2; and dml3 (drdd) leaves exhibit hypermethylated regions compared with wild-type leaves and rdd triple mutants, indicating functional redundancy among all four demethylases. Targets of demethylation include regions co-targeted by RNA-directed DNA methylation and, surprisingly, CG gene body methylation, indicating dynamic methylation at these less-understood sites. Additionally, many tissue-specific methylation differences are absent in drdd, suggesting a role for active demethylation in generating divergent epigenetic states across wild-type tissues. Furthermore, drdd plants display an early flowering phenotype, which involves 5'-hypermethylation and transcriptional down-regulation of FLOWERING LOCUS C. Active DNA demethylation is therefore required for proper methylation across somatic tissues and defines the epigenetic landscape of intergenic and coding regions.
Topics: Arabidopsis Proteins; DNA Demethylation; DNA Methylation; Gene Expression Regulation, Plant; Protein-Tyrosine Kinases; Proto-Oncogene Proteins
PubMed: 34954804
DOI: 10.1093/plcell/koab319 -
Nature Communications May 2024Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. The DDR is required during germ cell...
Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. The DDR is required during germ cell development and meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1 or Pcna) or extension (Rev7 ) result in a > 150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both activation of the germ cell transcriptional program and in DNA demethylation, a critical step in germline epigenetic reprogramming. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for progression through the earliest stages of germ cell development in mammals.
Topics: Animals; Humans; DNA Demethylation; Mice; DNA Repair; Germ Cells; DNA-Directed DNA Polymerase; Male; Nucleotidyltransferases; Female; DNA Damage; Mice, Knockout; Meiosis; DNA Replication; Proliferating Cell Nuclear Antigen; Epigenesis, Genetic; Translesion DNA Synthesis
PubMed: 38702312
DOI: 10.1038/s41467-024-47219-2 -
Molecular Therapy Oncolytics Sep 2020miR-21 is an oncogenic microRNA (miRNA) that is upregulated in many solid tumors. However, the effect of hypomethylation on miR-21 expression in tumors and the...
miR-21 is an oncogenic microRNA (miRNA) that is upregulated in many solid tumors. However, the effect of hypomethylation on miR-21 expression in tumors and the mechanism of miR-21 DNA demethylation remain unclear. In this study, we confirmed that the expression of miR-21 was significantly increased in multiple tumors. We analyzed eight types of cancer, including breast cancer (BRCA), lung adenocarcinoma (LUAD), renal and renal clear cell carcinoma (KIRC), bladder urothelial carcinoma (BLCA), hepatocellular carcinoma (LIHC), lung squamous cell cancer (LUSC), renal papillary cell carcinoma (KIRP), and pancreatic adenocarcinoma (PAAD). DNA methylation levels were elevated in these cancers. CpG loci located approximately 200 bp upstream of the transcription initiation site strongly affect MIR21 expression. We also confirmed hypomethylation by pyrosequencing of fresh clear cell renal cell carcinoma (ccRCC) samples. Demethylating agent was proved to increase hsa-miR-21-5p level in HEK293T cells, while knockdown of DNA demethylases TET3 and TDG decreased expression. In addition, we showed that the cg02515217 CpG locus in promoter was a conserved binding site of transcription factors CEBPB, MEIS3, and TEAD4, which were co-expressed with miR-21 in tumors. These observations identified that gene hypomethylation regulated the expression of in tumors.
PubMed: 32637580
DOI: 10.1016/j.omto.2020.05.011 -
Nature Communications Jan 2024The intracellular ATP-ribosyltransferases PARP1 and PARP2, contribute to DNA base excision repair (BER) and DNA demethylation and have been implicated in epigenetic...
The intracellular ATP-ribosyltransferases PARP1 and PARP2, contribute to DNA base excision repair (BER) and DNA demethylation and have been implicated in epigenetic programming in early mammalian development. Recently, proteomic analyses identified BER proteins to be covalently poly-ADP-ribosylated by PARPs. The role of this posttranslational modification in the BER process is unknown. Here, we show that PARP1 senses AP-sites and SSBs generated during TET-TDG mediated active DNA demethylation and covalently attaches PAR to each BER protein engaged. Covalent PARylation dissociates BER proteins from DNA, which accelerates the completion of the repair process. Consistently, inhibition of PARylation in mESC resulted both in reduced locus-specific TET-TDG-targeted DNA demethylation, and in reduced general repair of random DNA damage. Our findings establish a critical function of covalent protein PARylation in coordinating molecular processes associated with dynamic DNA methylation.
Topics: Animals; DNA Repair; Excision Repair; Poly ADP Ribosylation; DNA Demethylation; Proteomics; Poly (ADP-Ribose) Polymerase-1; DNA Damage; DNA; Mammals
PubMed: 38167803
DOI: 10.1038/s41467-023-44209-8