PCB126 (3,3',4,4',5-Pentachlorobiphenyl) induced alterations in m6A RNA modifications in zebrafish embryos
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. Overall design: AB strain of zebrafish embryos were exposed to 10 nM PCB126 (3,3',4,4',5-pentachlorobiphenyl, 99.2% purity; UltraScientific, RI, USA) or DMSO (solvent control; 0.01%) starting at 72 hours post-fertilization (hpf) for 6 hours. This duration of exposure was chosen based on previous studies that demonstrated differential gene expression after 6 hour exposure. In addition, short term exposure allows us to characterize primary changes in the m6A patterns. Each treatment consisted of four biological replicates with 48 embryos per replicate. Embryos were maintained in glass petri dishes at 28±0.5oC at a density of 1 embryo per mL in 0.3X Danieau's solution (pH 7.2). At the end of the exposure, embryos were thoroughly rinsed with 0.3X Danieau's solution and sampled for MeRIP sequencing.  Four biological replicates per treatment condition.