Influence of tissue, age, and environmental quality on DNA methylation in Alligator mississippiensis

Abstract

Epigenetic modifications are key mediators of the interactions between the environment and an organism's genome. DNA methylation represents the best-studied epigenetic modification to date and is known to play key roles in regulating transcriptional activity and promoting chromosome stability. Our laboratory has previously demonstrated the utility of the American alligator (Alligator mississippiensis) as a sentinel species to investigate the persistent effects of environmental contaminant exposure on reproductive health. Here, we incorporate a liquid chromatography-tandem mass spectrometry method to directly measure the total (global) proportion of 5-methyl-2 0 -deoxycytidine (5mdC) in ovarian and whole blood DNA from alligators. Global DNA methylation in ovaries was significantly elevated in comparison with that of whole blood. However, DNA methylation appeared similar in juvenile alligators reared under controlled laboratory conditions but originating from three sites with dissimilar environmental qualities, indicating an absence of detectable site-of-origin effects on persistent levels of global 5mdC content. Analyses of tissues across individuals revealed a surprising lack of correlation between global methylation levels in blood and ovary. In addition, global DNA methylation in blood samples from juvenile alligators was elevated compared with those from adults, suggesting that age, as observed in mammals, may negatively influence global DNA methylation levels in alligators. To our knowledge, this is the first study examining global levels of DNA methylation in the American alligator and provides a reference point for future studies examining the interplay of epigenetics and environmental factors in a long-lived sentinel species.

Global DNA methylation loss associated with mercury contamination and aging in the American alligator (Alligator mississippiensis)

“…The percent global methylation (% 5mdC, or 5mdC/dG) was calculated using a calibration curve constructed by relating the calibration solution peak area ratios to the gravimetrically determined weight ratios of 5mdC and dG, as previously described, with some modification (contribution of deoxyribonucleoside adducts was negligible and were not factored into this analysis) (Parrott et al, 2014; Quinlivan and Gregory, 2008b). To normalize differences in ionization efficiency between 5mdC and dG, the dG peak area was multiplied by a response factor (0.8971) before the peak area ratio was calculated, as previously described using an equimolar solution (Parrott et al, 2014). The QC and calibration solutions were run throughout the sample queue and the resultant calibration line is shown in Figure A3 (r 2 = 0.9994).…”

“…Although some studies have begun to examine epigenome-by-environment interactions in human populations, the aforementioned impediments have restricted the majority of wildlife studies in terms of both the species and the number of individuals (Alvarado et al, 2014; Calvanese et al, 2009; Christensen et al, 2009). Over the past decade, direct measurement of changes in global DNA methylation using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become a viable approach, and the inherent advantage of this approach (cost, throughput, simplicity) make it suitable for both environmental applications and studies at the population level (Kok et al, 2007; Le et al, 2011; Liu et al, 2009; Ma et al, 2009; Parrott et al, 2014; Quinlivan and Gregory, 2008b; Song et al, 2005). In this study, LC-MS/MS was used to characterize changes in global DNA methylation in a long-lived environmental sentinel to investigate the relationship between a fundamental epigenetic modification and whole blood mercury concentrations.…”

“…DNA methylation, consisting of the covalent addition of a methyl-group to the 5′ carbon of cytosine, is an epigenetic modification functioning in roles that include regulating gene expression, promoting chromosome stability, and silencing the transcription of transposons (Di Giacomo et al, 2013; Parrott et al, 2014; Rodriguez et al, 2006). Alterations to the methylome can occur at specific loci or more broadly across the genome.…”

“…While methods of resolving the methylome at base pair resolution allow for the identification of loci-specific alterations, analyses of the comprehensive methylome at base pair are not currently feasible at the population level. However, measures of global DNA methylation can be made across a large number of samples and DNA methylation at the global level is intimately linked to one carbon metabolism as this process provides key methyl donors for both maintenance and de novo DNA methylation (Anderson et al, 2012; Parrott et al, 2014). Alterations to one-carbon metabolism, either through environmental or genetic factors, have been shown to influence measures of global DNA methylation (Drake et al, 2015; Kruman and Fowler, 2014).…”

Identification and Characterization of the Androgen Receptor From the American Alligator,Alligator mississippiensis

“…Notably, vinclozolin induces decreased spermatogenic capacity, and such abnormalities can be transferred to subsequent generations, accompanied by altered DNA methylation patterns (59). DNA methylation is implicated as a key regulatory mechanism of sexual development in the alligator (60,61). However, the contribution of the AR to such epigenetic modification during development has not been studied.…”