Karchner
Sibel I.
Karchner
Sibel I.
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ArticleRole of DNA methylation in altered gene expression patterns in adult zebrafish (Danio rerio) exposed to 3, 3’, 4, 4’, 5-pentachlorobiphenyl (PCB 126)(Oxford University Press, 2018-04-13) Aluru, Neelakanteswar ; Karchner, Sibel I. ; Krick, Keegan S. ; Zhu, Wei ; Liu, JiangThere 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.
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ArticleThe landscape of extreme genomic variation in the highly adaptable Atlantic killifish(Oxford University Press, 2017-03-01) Reid, Noah M. ; Jackson, Craig E. ; Gilbert, Don ; Minx, Patrick ; Montague, Michael J. ; Hampton, Thomas H. ; Helfrich, Lily W. ; King, Benjamin L. ; Nacci, Diane E. ; Aluru, Neelakanteswar ; Karchner, Sibel I. ; Colbourne, John K. ; Hahn, Mark E. ; Shaw, Joseph R. ; Oleksiak, Marjorie F. ; Crawford, Douglas L. ; Warren, Wesley C. ; Whitehead, AndrewUnderstanding and predicting the fate of populations in changing environments require knowledge about the mechanisms that support phenotypic plasticity and the adaptive value and evolutionary fate of genetic variation within populations. Atlantic killifish (Fundulus heteroclitus) exhibit extensive phenotypic plasticity that supports large population sizes in highly fluctuating estuarine environments. Populations have also evolved diverse local adaptations. To yield insights into the genomic variation that supports their adaptability, we sequenced a reference genome and 48 additional whole genomes from a wild population. Evolution of genes associated with cell cycle regulation and apoptosis is accelerated along the killifish lineage, which is likely tied to adaptations for life in highly variable estuarine environments. Genome-wide standing genetic variation, including nucleotide diversity and copy number variation, is extremely high. The highest diversity genes are those associated with immune function and olfaction, whereas genes under greatest evolutionary constraint are those associated with neurological, developmental, and cytoskeletal functions. Reduced genetic variation is detected for tight junction proteins, which in killifish regulate paracellular permeability that supports their extreme physiological flexibility. Low-diversity genes engage in more regulatory interactions than high-diversity genes, consistent with the influence of pleiotropic constraint on molecular evolution. High genetic variation is crucial for continued persistence of species given the pace of contemporary environmental change. Killifish populations harbor among the highest levels of nucleotide diversity yet reported for a vertebrate species, and thus may serve as a useful model system for studying evolutionary potential in variable and changing environments.
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ArticleMolecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family(National Academy of Sciences, 1997-12) Hahn, Mark E. ; Karchner, Sibel I. ; Shapiro, Miriam A. ; Perera, Samanthi A.The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene expression and toxicity. The AHR belongs to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, we sequenced the PAS homology domain of AHRs from several species of early vertebrates and performed phylogenetic analyses of these AHR amino acid sequences in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences were identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, were found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that we propose have arisen by gene duplication early in vertebrate evolution. Overall, our analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function.
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ArticleTranscriptomic assessment of resistance to effects of an aryl hydrocarbon receptor (AHR) agonist in embryos of Atlantic killifish (Fundulus heteroclitus) from a marine Superfund site(BioMed Central, 2011-05-24) Oleksiak, Marjorie F. ; Karchner, Sibel I. ; Jenny, Matthew J. ; Franks, Diana G. ; Mark Welch, David B. ; Hahn, Mark E.Populations of Atlantic killifish (Fundulus heteroclitus) have evolved resistance to the embryotoxic effects of polychlorinated biphenyls (PCBs) and other halogenated and nonhalogenated aromatic hydrocarbons that act through an aryl hydrocarbon receptor (AHR)-dependent signaling pathway. The resistance is accompanied by reduced sensitivity to induction of cytochrome P450 1A (CYP1A), a widely used biomarker of aromatic hydrocarbon exposure and effect, but whether the reduced sensitivity is specific to CYP1A or reflects a genome-wide reduction in responsiveness to all AHR-mediated changes in gene expression is unknown. We compared gene expression profiles and the response to 3,3',4,4',5-pentachlorobiphenyl (PCB-126) exposure in embryos (5 and 10 dpf) and larvae (15 dpf) from F. heteroclitus populations inhabiting the New Bedford Harbor, Massachusetts (NBH) Superfund site (PCB-resistant) and a reference site, Scorton Creek, Massachusetts (SC; PCB-sensitive). Analysis using a 7,000-gene cDNA array revealed striking differences in responsiveness to PCB-126 between the populations; the differences occur at all three stages examined. There was a sizeable set of PCB-responsive genes in the sensitive SC population, a much smaller set of PCB-responsive genes in NBH fish, and few similarities in PCB-responsive genes between the two populations. Most of the array results were confirmed, and additional PCB-regulated genes identified, by RNA-Seq (deep pyrosequencing). The results suggest that NBH fish possess a gene regulatory defect that is not specific to one target gene such as CYP1A but rather lies in a regulatory pathway that controls the transcriptional response of multiple genes to PCB exposure. The results are consistent with genome-wide disruption of AHR-dependent signaling in NBH fish.
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ArticleThe molecular basis for differential dioxin sensitivity in birds : role of the aryl hydrocarbon receptor(National Academy of Sciences, 2006-04-10) Karchner, Sibel I. ; Franks, Diana G. ; Kennedy, Sean W. ; Hahn, Mark E.2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatic hydrocarbons (HAHs) are highly toxic to most vertebrate animals, but there are dramatic differences in sensitivity among species and strains. Aquatic birds including the common tern (Sterna hirundo) are highly exposed to HAHs in the environment, but are up to 250-fold less sensitive to these compounds than the typical avian model, the domestic chicken (Gallus gallus). The mechanism of HAH toxicity involves altered gene expression subsequent to activation of the aryl hydrocarbon receptor (AHR), a basic helix–loop–helix-PAS transcription factor. AHR polymorphisms underlie mouse strain differences in sensitivity to HAHs and polynuclear aromatic hydrocarbons, but the role of the AHR in species differences in HAH sensitivity is not well understood. Here, we show that although chicken and tern AHRs both exhibit specific binding of [3H]TCDD, the tern AHR has a lower binding affinity and exhibits a reduced ability to support TCDD-dependent transactivation as compared to AHRs from chicken or mouse. We further show through use of chimeric AHR proteins and site-directed mutagenesis that the difference between the chicken and tern AHRs resides in the ligand-binding domain and that two amino acids (Val-325 and Ala-381) are responsible for the reduced activity of the tern AHR. Other avian species with reduced sensitivity to HAHs also possess these residues. These studies provide a molecular understanding of species differences in sensitivity to dioxin-like compounds and suggest an approach to using the AHR as a marker of dioxin susceptibility in wildlife.
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ArticleThe African coelacanth genome provides insights into tetrapod evolution(Nature Publishing Group, 2013-04-17) Amemiya, Chris T. ; Alfoldi, Jessica ; Lee, Alison P. ; Fan, Shaohua ; Philippe, Herve ; MacCallum, Iain ; Braasch, Ingo ; Manousaki, Tereza ; Schneider, Igor ; Rohner, Nicolas ; Organ, Chris ; Chalopin, Domitille ; Smith, Jeramiah J. ; Robinson, Mark ; Dorrington, Rosemary A. ; Gerdol, Marco ; Aken, Bronwen ; Assunta Biscotti, Maria ; Barucca, Marco ; Baurain, Denis ; Berlin, Aaron M. ; Blatch, Gregory L. ; Buonocore, Francesco ; Burmester, Thorsten ; Campbell, Michael S. ; Canapa, Adriana ; Cannon, John P. ; Christoffels, Alan ; De Moro, Gianluca ; Edkins, Adrienne L. ; Fan, Lin ; Fausto, Anna Maria ; Feiner, Nathalie ; Forconi, Mariko ; Gamieldien, Junaid ; Gnerre, Sante ; Gnirke, Andreas ; Goldstone, Jared V. ; Haerty, Wilfried ; Hahn, Mark E. ; Hesse, Uljana ; Hoffmann, Steve ; Johnson, Jeremy ; Karchner, Sibel I. ; Kuraku, Shigehiro ; Lara, Marcia ; Levin, Joshua Z. ; Litman, Gary W. ; Mauceli, Evan ; Miyake, Tsutomu ; Mueller, M. Gail ; Nelson, David R. ; Nitsche, Anne ; Olmo, Ettore ; Ota, Tatsuya ; Pallavicini, Alberto ; Panji, Sumir ; Picone, Barbara ; Ponting, Chris P. ; Prohaska, Sonja J. ; Przybylski, Dariusz ; Ratan Saha, Nil ; Ravi, Vydianathan ; Ribeiro, Filipe J. ; Sauka-Spengler, Tatjana ; Scapigliati, Giuseppe ; Searle, Stephen M. J. ; Sharpe, Ted ; Simakov, Oleg ; Stadler, Peter F. ; Stegeman, John J. ; Sumiyama, Kenta ; Tabbaa, Diana ; Tafer, Hakim ; Turner-Maier, Jason ; van Heusden, Peter ; White, Simon ; Williams, Louise ; Yandell, Mark ; Brinkmann, Henner ; Volff, Jean-Nicolas ; Tabin, Clifford J. ; Shubin, Neil ; Schartl, Manfred ; Jaffe, David B. ; Postlethwait, John H. ; Venkatesh, Byrappa ; Di Palma, Federica ; Lander, Eric S. ; Meyer, Axel ; Lindblad-Toh, KerstinThe discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.
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ArticlePCB126 exposure revealed alterations in m6A RNA modifications in transcripts associated with AHR activation(Oxford University Press, 2020-10-16) Aluru, Neelakanteswar ; Karchner, Sibel I.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.
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ArticleGenetic variation at aryl hydrocarbon receptor (AHR) loci in populations of Atlantic killifish (Fundulus heteroclitus) inhabiting polluted and reference habitats(BioMed Central, 2014-01-14) Reitzel, Adam M. ; Karchner, Sibel I. ; Franks, Diana G. ; Evans, Brad R. ; Nacci, Diane E. ; Champlin, Denise ; Vieira, Veronica M. ; Hahn, Mark E.The non-migratory killifish Fundulus heteroclitus inhabits clean and polluted environments interspersed throughout its range along the Atlantic coast of North America. Several populations of this species have successfully adapted to environments contaminated with toxic aromatic hydrocarbon pollutants such as polychlorinated biphenyls (PCBs). Previous studies suggest that the mechanism of resistance to these and other “dioxin-like compounds” (DLCs) may involve reduced signaling through the aryl hydrocarbon receptor (AHR) pathway. Here we investigated gene diversity and evidence for positive selection at three AHR-related loci (AHR1, AHR2, AHRR) in F. heteroclitus by comparing alleles from seven locations ranging over 600 km along the northeastern US, including extremely polluted and reference estuaries, with a focus on New Bedford Harbor (MA, USA), a PCB Superfund site, and nearby reference sites. We identified 98 single nucleotide polymorphisms within three AHR-related loci among all populations, including synonymous and nonsynonymous substitutions. Haplotype distributions were spatially segregated and F-statistics suggested strong population genetic structure at these loci, consistent with previous studies showing strong population genetic structure at other F. heteroclitus loci. Genetic diversity at these three loci was not significantly different in contaminated sites as compared to reference sites. However, for AHR2 the New Bedford Harbor population had significant FST values in comparison to the nearest reference populations. Tests for positive selection revealed ten nonsynonymous polymorphisms in AHR1 and four in AHR2. Four nonsynonymous SNPs in AHR1 and three in AHR2 showed large differences in base frequency between New Bedford Harbor and its reference site. Tests for isolation-by-distance revealed evidence for non-neutral change at the AHR2 locus. Together, these data suggest that F. heteroclitus populations in reference and polluted sites have similar genetic diversity, providing no evidence for strong genetic bottlenecks for populations in polluted locations. However, the data provide evidence for genetic differentiation among sites, selection at specific nucleotides in AHR1 and AHR2, and specific AHR2 SNPs and haplotypes that are associated with the PCB-resistant phenotype in the New Bedford Harbor population. The results suggest that AHRs, and especially AHR2, may be important, recurring targets for selection in local adaptation to dioxin-like aromatic hydrocarbon contaminants.
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ArticleThe transcriptional response to oxidative stress during vertebrate development : effects of tert-butylhydroquinone and 2,3,7,8-tetrachlorodibenzo-p-dioxin(Public Library of Science, 2014-11-17) Hahn, Mark E. ; McArthur, Andrew G. ; Karchner, Sibel I. ; Franks, Diana G. ; Jenny, Matthew J. ; Timme-Laragy, Alicia R. ; Stegeman, John J. ; Woodin, Bruce R. ; Cipriano, Michael J. ; Linney, Elwood A.Oxidative stress is an important mechanism of chemical toxicity, contributing to teratogenesis and to cardiovascular and neurodegenerative diseases. Developing animals may be especially sensitive to chemicals causing oxidative stress. The developmental expression and inducibility of anti-oxidant defenses through activation of NF-E2-related factor 2 (NRF2) affect susceptibility to oxidants, but the embryonic response to oxidants is not well understood. To assess the response to chemically mediated oxidative stress and how it may vary during development, zebrafish embryos, eleutheroembryos, or larvae at 1, 2, 3, 4, 5, and 6 days post fertilization (dpf) were exposed to DMSO (0.1%), tert-butylhydroquinone (tBHQ; 10 µM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 2 nM) for 6 hr. Transcript abundance was assessed by real-time qRT-PCR and microarray. qRT-PCR showed strong (4- to 5-fold) induction of gstp1 by tBHQ as early as 1 dpf. tBHQ also induced gclc (2 dpf), but not sod1, nqo1, or cyp1a. TCDD induced cyp1a but none of the other genes. Microarray analysis showed that 1477 probes were significantly different among the DMSO-, tBHQ-, and TCDD-treated eleutheroembryos at 4 dpf. There was substantial overlap between genes induced in developing zebrafish and a set of marker genes induced by oxidative stress in mammals. Genes induced by tBHQ in 4-dpf zebrafish included those involved in glutathione synthesis and utilization, signal transduction, and DNA damage/stress response. The strong induction of hsp70 determined by microarray was confirmed by qRT-PCR and by use of transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under control of the hsp70 promoter. Genes strongly down-regulated by tBHQ included mitfa, providing a molecular explanation for the loss of pigmentation in tBHQ-exposed embryos. These data show that zebrafish embryos are responsive to oxidative stress as early as 1 dpf, that responsiveness varies with development in a gene-specific manner, and that the oxidative stress response is substantially conserved in vertebrate animals.