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PCB126 Exposure Revealed Alterations in m6A RNA Modifications in Transcripts Associated With AHR Activation.

ABSTRACT: Chemical modifications of proteins, DNA, and RNA moieties play critical roles in regulating gene expression. Emerging evidence suggests the RNA modifications (epitranscriptomics) have substantive roles in basic biological processes. One of the most common modifications in mRNA and noncoding RNAs is N6-methyladenosine (m6A). In a subset of mRNAs, m6A sites are preferentially enriched near stop codons, in 3' UTRs, and within exons, suggesting an important role in the regulation of mRNA processing and function including alternative splicing and gene expression. Very little is known about the effect of environmental chemical exposure on m6A modifications. As many of the commonly occurring environmental contaminants alter gene expression profiles and have detrimental effects on physiological processes, it is important to understand the effects of exposure on this important layer of gene regulation. Hence, the objective of this study was to characterize the acute effects of developmental exposure to PCB126, an environmentally relevant dioxin-like PCB, on m6A methylation patterns. We exposed zebrafish embryos to PCB126 for 6 h starting from 72 h post fertilization and profiled m6A RNA using methylated RNA immunoprecipitation followed by sequencing (MeRIP-seq). Our analysis revealed 117 and 217 m6A peaks in the DMSO and PCB126 samples (false discovery rate 5%), respectively. The majority of the peaks were preferentially located around the 3' UTR and stop codons. Statistical analysis revealed 15 m6A marked transcripts to be differentially methylated by PCB126 exposure. These include transcripts that are known to be activated by AHR agonists (eg, ahrra, tiparp, nfe2l2b) as well as others that are important for normal development (vgf, cebpd, sned1). These results suggest that environmental chemicals such as dioxin-like PCBs could affect developmental gene expression patterns by altering m6A levels. Further studies are necessary to understand the functional consequences of exposure-associated alterations in m6A levels.

SUBMITTER: Aluru N

PROVIDER: S-EPMC8453794 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Chemical modifications of proteins, DNA and RNA moieties play critical roles in regulating gene expression. Emerging evidence suggests these RNA modifications (epitranscriptomics) have substantive roles in basic biological processes. One of the most common modifications in mRNA and noncoding RNAs is N6-methyladenosine (m6A). In a subset of mammalian mRNAs, m6A sites are preferentially enriched near stop codons, in 3′ UTRs, and within exons, suggesting an important role in the regulation of mRNA processing and function including alternative splicing and gene expression. Very little is known about the effect of environmental chemical exposure on m6A modifications. As many of the commonly occurring environmental contaminants alter gene expression profiles and have detrimental effects on physiological processes, it is important to understand the effects of exposure on this important layer of gene regulation. Hence, the objective of this study was to characterize the acute effects of developmental exposure to PCB126, an environmentally relevant dioxin-like PCB, on m6A methylation patterns. We exposed zebrafish embryos to PCB126 for 6 hours starting from 72 hours post-fertilization and profiled m6A RNA using methylated RNA immunoprecipitation followed by sequencing (MeRIP-seq), as well as assessing changes in mRNA splicing. We observed several hundreds of peaks that are differentially expressed in response to PCB126 exposure (FDR 5%). The majority of the peaks are preferentially located around the 3’UTR and stop codons. Pathway analysis of m6A marked transcripts induced by PCB126 exposure revealed that these transcripts are associated with important developmental pathways (MAPK, Hedgehog, Notch and Wnt). These results suggest that PCB126 could affect developmental gene expression patterns by altering m6A levels. Gene expression analysis revealed upregulation of classical aryl hydrocarbon receptor (AHR) target genes such as cytochrome P450s in response to PCB126 exposure. Interestingly, none of the AHR target genes overlapped with the m6A altered transcripts, suggesting that xenobiotic metabolism may not be under m6A regulation. Further studies are necessary to understand the functional consequences of exposure-associated alterations in m6A levels. This work is supported by NIEHS ES024915.

Project description:Two year cancer bioassays conducted by the National Toxicology Program have shown chronic exposure to dioxin-like compounds (DLCs) to lead to the development of both neoplastic and non-neoplastic lesions in the hepatic tissue of female Sprague Dawley rats. Most, if not all, of the hepatotoxic effects induced by DLC's are believed to involve the binding and activation of the transcription factor, the aryl hydrocarbon receptor (AhR). Toxicogenomics was implemented to identify genomic responses that may be contributing to the development of hepatotoxicity in rats.Through comparative analysis of time-course microarray data, unique hepatic gene expression signatures were identified for the DLCs, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (100 ng/kg/day) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) (1000 ng/kg/day) and the non-DLC 2,2',4,4',5,5',-hexachlorobiphenyl (PCB153) (1000 ?g/kg/day). A common time independent signature of 41 AhR genomic biomarkers was identified which exhibited at least a 2-fold change in expression following subchronic (13-wk) and chronic (52-wk) p.o. exposure to TCDD and PCB126, but not the non DLC, PCB153. Real time qPCR analysis validated that 30 of these genes also exhibited at least a 2-fold change in hepatic expression at 24 hr following a single exposure to TCDD (5 ?g/kg, po). Phenotypic anchoring was conducted which identified forty-six genes that were differently expressed both following chronic p.o. exposure to DLCs and in previously reported studies of cholangiocarcinoma or hepatocellular adenoma.Together these analyses provide a comprehensive description of the genomic responses which occur in rat hepatic tissue with exposure to AhR ligands and will help to isolate those genomic responses which are contributing to the hepatotoxicity observed with exposure to DLCs. In addition, the time independent gene expression signature of the AhR ligands may assist in identifying other agents with the potential to elicit dioxin-like hepatotoxic responses.

Project description:The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in the regulation of biological responses to more planar aromatic hydrocarbons, like TCDD. We previously described the sequence of events following exposure of male rats to a dioxin-like polychlorinated biphenyl (PCB) congener, 3,3',4,4',5-pentachlorobiphenyl (PCB126), that binds avidly to the AhR and causes various types of toxicity including metabolic syndrome, fatty liver, and disruption of energy homeostasis. The purpose of this study was, to investigate the role of AhR to mediate those toxic manifestations following sub-acute exposure to PCB126 and to examine possible sex differences in effects. For this goal, we created an AhR knockout (AhR-KO) model using CRISPR/Cas9. Comparison was made to the wild type (WT) male and female Holtzman Sprague Dawley rats. Rats were injected with a single IP dose of corn oil vehicle or 5 μmol/kg PCB126 in corn oil and necropsied after 28 days. PCB126 caused significant weight loss, reduced relative thymus weights, and increased relative liver weights in WT male and female rats, but not in AhR-KO rats. Similarly, significant pathologic changes were visible which included necrosis and regeneration in female rats, micro- and macro-vesicular hepatocellular vacuolation in males, and a paucity of glycogen in livers of both sexes in WT rats only. Hypoglycemia and lower IGF1, and reduced serum non-esterified fatty acids (NEFAs) were found in serum of both sexes of WT rats, low serum cholesterol levels only in the females, and no changes in AhR-KO rats. The expression of genes encoding enzymes related to xenobiotic metabolism (e.g. CYP1A1), gluconeogenesis, glycogenolysis, and fatty acid oxidation were unaffected in the AhR-KO rats following PCB126 exposure as opposed to WT rats where expression was significantly upregulated (PPARα, females only) or downregulated suggesting a disrupted energy homeostasis. Interestingly, Acox2, Hmgcs, G6Pase and Pc were affected in both sexes, the gluconeogenesis and glucose transporter genes Pck1, Glut2, Sds, and Crem only in male WT-PCB rats. These results show the essential role of the AhR in glycogenolysis, gluconeogenesis, and fatty acid oxidation, i.e. in the regulation of energy production and homeostasis, but also demonstrate a significant difference in the effects of PCB126 in males verses females, suggesting higher vulnerability of glucose homeostasis in males and more changes in fatty acid/lipid homeostasis in females. These differences in effects, which may apply to more/all AhR agonists, should be further analyzed to identify health risks to specific groups of highly exposed human populations.

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PCB126 Exposure Revealed Alterations in m6A RNA Modifications in Transcripts Associated With AHR Activation.

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