Cloning of multiple ERα mRNA variants in killifish (Fundulus heteroclitus), and differential expression by tissue type, stage of reproduction, and estrogen exposure in fish from polluted and unpolluted environments

Abstract

To test the hypothesis that alternative splicing could be an adaptive mechanism for populations subject to multi-generational estrogenic exposures, we compared estrogen receptor alpha (ERα) splicing variants in two populations of killifish (Fundulus heteroclitus): one resident in an estrogenic polluted environment (New Bedford Harbor, NBH, MA, USA) and one from a relatively uncontaminated reference site (Scorton Creek, SC, MA, USA). In total we identified nineteen ERα variants, each with deletions of one or more coding exons. Four of the variants with potential functional relevance were analyzed by qPCR to test for population differences in expression by tissue type, site, sex, seasonal reproductive status and estrogen treatment. Significantly, a 5′-truncated short form variant (ERαS) was highly expressed in liver and ovary, and was associated with seasonal reproductive activity in SC but not NBH fish. Both ERαS and the full-length long variant (ERαL) were estrogen-inducible (ERαS > ERαL) but the induction response was lower in NBH than in SC fish. In contrast, NBH killifish were hyper-responsive to estrogen as measured by expression of two other estrogen responsive genes: vitellogenin (Vtg) and aromatase B (AroB). Most strikingly, two ERα deletion variants (Δ6 and Δ6–8), lacking ligand binding and activation function domains, were identified in a subset of NBH fish, where they were associated with reduced responsiveness to estrogen treatment. Together, these results support the hypothesis that alternative splicing of the esr1 gene of killifish could be an autoregulatory mechanism by which estrogen modulates the differential expression of ERα, and suggests a novel and adaptive mechanistic response to xenoestrogenic exposure.

Genetic basis for rapidly evolved tolerance in the wild: adaptation to toxic pollutants by an estuarine fish species

“…Genetic variation in this region might be consistent with other studies that have described differences between NB and reference killifish in form (splice variants) and function (estrogen responsiveness) of estrogen receptors (Cotter et al . ). The importance of marker Fhe_1205 ( hnf1ba , LG21) might also be inferred by this gene's position relative to AHR ‐related genes (hypoxia‐inducible factor alpha ( hif1a1 ) and zebrafish QTL1) in medaka Chr 13 and zebrafish Chr 15 which share synteny with killifish LG21 (Fig.…”

Annotation of the Nuclear Receptors in an Estuarine Fish species, Fundulus heteroclitus

“…Overall, there is a significant expansion of the 3A and 3B group in F. heteroclitus and other fish species relative to humans. Interestingly, Fundulus chronically exposed to estrogenic pollutants developed heritable splicing variants that show different transcriptional responses than wild-type ER potentially as an adaptive mechanism to the estrogens found in the polluted environment (11,18). …”

“…The abundance of F. heteroclitus in estuaries, their association with sediments due to hiding from predators or searching for food, and their non-migratory nature makes them an excellent bioindicator species. Fundulus species have been used as bioindicators at contaminated sites (1,2,5,13,14) and to follow the evolution of pollution resistance in a vertebrate (3,10,11,15–17). Thus, mummichogs are an excellent vertebrate bioindicator species for understanding rapid adaptation to environmental change in feral populations (5,8), including adaptations in transcription factors such as estrogen receptor alpha (ERα) splice variants (18), and evolutionary convergence in the aryl hydrocarbon receptor pathway (AHR1a, AHR2a, CYP1A) (5,8).…”

Novel splice isoforms of dairy goat DBC1 and their diverse mRNA expression profiles

“…Along with the rapid development of sequencing and bioinformatics, deep RNA sequencing (RNA-Seq) has become a powerful and useful tool for genome-wide analysis of AS (Shen et al, 2014;Sohail et al, 2014), uncovering novel AS data from animal transcriptome databases. AS is a common mode of gene regulation in diverse biological processes, such as cell differentiation (Kianianmomeni et al, 2014;Cotter et al, 2015), cell signaling (Wright et al, 2014;Li et al, 2015), and disease development (Wang et al, 2007;Cooper et al, 2009;Sohail et al, 2014).…”