Role of DNA methylation in altered gene expression patterns in adult zebrafish (Danio rerio) exposed to 3, 3’, 4, 4’, 5-pentachlorobiphenyl (PCB 126)

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Environmental Epigenetics 4 (2018): dvy005, doi:10.1093/eep/dvy005.There is growing evidence that environmental toxicants can affect various physiological processes by altering DNA methylation patterns. However, very little is known about the impact of toxicant-induced DNA methylation changes on gene expression patterns. The objective of this study was to determine the genome-wide changes in DNA methylation concomitant with altered gene expression patterns in response to 3, 3’, 4, 4’, 5-pentachlorobiphenyl (PCB126) exposure. We used PCB126 as a model environmental chemical because the mechanism of action is well-characterized, involving activation of aryl hydrocarbon receptor, a ligand-activated transcription factor. Adult zebrafish were exposed to 10 nM PCB126 for 24 h (water-borne exposure) and brain and liver tissues were sampled at 7 days post-exposure in order to capture both primary and secondary changes in DNA methylation and gene expression. We used enhanced Reduced Representation Bisulfite Sequencing and RNAseq to quantify DNA methylation and gene expression, respectively. Enhanced reduced representation bisulfite sequencing analysis revealed 573 and 481 differentially methylated regions in the liver and brain, respectively. Most of the differentially methylated regions are located more than 10 kilobases upstream of transcriptional start sites of the nearest neighboring genes. Gene Ontology analysis of these genes showed that they belong to diverse physiological pathways including development, metabolic processes and regeneration. RNAseq results revealed differential expression of genes related to xenobiotic metabolism, oxidative stress and energy metabolism in response to polychlorinated biphenyl exposure. There was very little correlation between differentially methylated regions and differentially expressed genes suggesting that the relationship between methylation and gene expression is dynamic and complex, involving multiple layers of regulation.This work was supported by the National Institute of Health Outstanding New Environmental Scientist Award to NA (NIH R01ES024915) and Woods Hole Center for Oceans and Human Health [National Institutes of Health (NIH) grant P01ES021923 and National Science Foundation Grant OCE-1314642 to M. Hahn, J. Stegeman, NA and SK]

Exon-Mediated Activation of Transcription Starts

© 2019 Elsevier Inc. The processing of RNA transcripts from mammalian genes occurs in proximity to their transcription. Here, we describe a phenomenon affecting thousands of genes that we call exon-mediated activation of transcription starts (EMATS), in which the splicing of internal exons impacts promoter choice and the expression level of the gene. We observed that evolutionary gain of internal exons is associated with gain of new transcription start sites (TSSs) nearby and increased gene expression. Inhibiting exon splicing reduced transcription from nearby promoters, and creation of new spliced exons activated transcription from cryptic promoters. The strongest effects occurred for weak promoters located proximal and upstream of efficiently spliced exons. Together, our findings support a model in which splicing recruits transcription machinery locally to influence TSS choice and identify exon gain, loss, and regulatory change as major contributors to the evolution of alternative promoters and gene expression in mammals

Transcriptomic and Network Analyses Reveal Mechanistic-Based Biomarkers of Endocrine Disruption in the Marine Mussel, <i>Mytilus edulis</i>

Transcriptomics, high-throughput assays, and adverse outcome pathways (AOP) are promising approaches applied to toxicity monitoring in the 21st century, but development of these methods is challenging for nonmodel organisms and emerging contaminants. For example, Endocrine Disrupting Compounds (EDCs) may cause reproductive impairments and feminization of male bivalves; however, the mechanism linked to this adverse outcome is unknown. To develop mechanism-based biomarkers that may be linked through an AOP, we exposed <i>Mytilus edulis</i> to 17-alpha-ethinylestradiol (5 and 50 ng/L) and 4-nonylphenol (1 and 100 μg/L) for 32 and 39 days. When mussels were exposed to these EDCs, we found elevated female specific transcripts and significant female-skewed sex ratios using a RT-qPCR assay. We performed gene expression analysis on digestive gland tissue using an <i>M. edulis</i> microarray and through network and targeted analyses identified the nongenomic estrogen signaling pathway and steroidogenesis pathway as the likely mechanisms of action for a putative AOP. We also identified several homologues to genes within the vertebrate steroidogenesis pathway including the cholesterol side chain cleavage complex. From this AOP, we designed the Coastal Biosensor for Endocrine Disruption (C-BED) assay which was confirmed in the laboratory and tested in the field

Transcriptomic and Network Analyses Reveal Mechanistic-Based Biomarkers of Endocrine Disruption in the Marine Mussel, <i>Mytilus edulis</i>

Transcriptomics, high-throughput assays, and adverse outcome pathways (AOP) are promising approaches applied to toxicity monitoring in the 21st century, but development of these methods is challenging for nonmodel organisms and emerging contaminants. For example, Endocrine Disrupting Compounds (EDCs) may cause reproductive impairments and feminization of male bivalves; however, the mechanism linked to this adverse outcome is unknown. To develop mechanism-based biomarkers that may be linked through an AOP, we exposed <i>Mytilus edulis</i> to 17-alpha-ethinylestradiol (5 and 50 ng/L) and 4-nonylphenol (1 and 100 μg/L) for 32 and 39 days. When mussels were exposed to these EDCs, we found elevated female specific transcripts and significant female-skewed sex ratios using a RT-qPCR assay. We performed gene expression analysis on digestive gland tissue using an <i>M. edulis</i> microarray and through network and targeted analyses identified the nongenomic estrogen signaling pathway and steroidogenesis pathway as the likely mechanisms of action for a putative AOP. We also identified several homologues to genes within the vertebrate steroidogenesis pathway including the cholesterol side chain cleavage complex. From this AOP, we designed the Coastal Biosensor for Endocrine Disruption (C-BED) assay which was confirmed in the laboratory and tested in the field