Genome-Wide Assessment of DNA Methylation in Chicken Cardiac Tissue Exposed to Different Incubation Temperatures and CO2 Levels.

Ryan J Corbett,Catherine W Ernst,Ole Madsen,Richard P M A Crooijmans,Marinus F W Te Pas,Henry Van Den Brand,Martien A M Groenen

doi:10.3389/fgene.2020.558189

Ryan J Corbett, Catherine W Ernst + Show 5 more

Open Access

https://doi.org/10.3389/fgene.2020.558189

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Abstract

Temperature and CO2 concentration during incubation have profound effects on broiler chick development, and numerous studies have identified significant effects on hatch heart weight (HW) as a result of differences in these parameters. Early life environment has also been shown to affect broiler performance later in life; it has thus been suggested that epigenetic mechanisms may mediate long-term physiological changes induced by environmental stimuli. DNA methylation is an epigenetic modification that can confer heritable changes in gene expression. Using reduced-representation bisulfite sequencing (RRBS), we assessed DNA methylation patterns in cardiac tissue of 84 broiler hatchlings incubated at two egg shell temperatures (EST; 37.8°C and 38.9°C) and three CO2 concentrations (0.1%, 0.4%, and 0.8%) from day 8 of incubation onward. We assessed differential methylation between EST treatments and identified 2,175 differentially methylated (DM) CpGs (1,121 hypermethylated, 1,054 hypomethylated at 38.9° vs. 37.8°) in 269 gene promoters and 949 intragenic regions. DM genes (DMGs) were associated with heart developmental processes, including cardiomyocyte proliferation and differentiation. We identified enriched binding motifs among DM loci, including those for transcription factors associated with cell proliferation and heart development among hypomethylated CpGs that suggest increased binding ability at higher EST. We identified 9,823 DM CpGs between at least two CO2 treatments, with the greatest difference observed between 0.8 and 0.1% CO2 that disproportionately impacted genes involved in cardiac muscle development and response to low oxygen levels. Using HW measurements from the same chicks, we performed an epigenome-wide association study (EWAS) for HW, and identified 23 significantly associated CpGs, nine of which were also DM between ESTs. We found corresponding differences in transcript abundance between ESTs in three DMGs (ABLIM2, PITX2, and THRSP). Hypomethylation of an exonic CpG in PITX2 at 38.9°C was associated with increased expression, and suggests increased cell proliferation in broiler hatchlings incubated at higher temperatures. Overall, these results identified numerous epigenetic associations between chick incubation factors and heart development that may manifest in long-term differences in animal performance.

Highlights

Life environmental parameters have profound effects on broiler chick development and post-hatch performance
Enriched terms among TFs of hypermethylated motifs were not directly related to heart development or function; TFs of hypomethylated motifs were uniquely enriched for the GO terms “cardiovascular system development” and “positive regulation of cell population proliferation” (Table 3). Genes associated with these terms included several with known roles in positively regulating cardiomyocyte proliferation, including retinoic acid receptor alpha (RARA) (Xavier-Neto et al, 2015), T-box transcription factor 20 (TBX20) (Lu et al, 2017), lymphoid enhancer binding factor 1 (LEF1) (Ye et al, 2019) and PITX2, which was found to be hypomethylated at 38.9◦C. These results reveal the potential for decreased binding ability of TFs involved in negatively regulating cell proliferation, and increased binding ability of TFs involved in positively regulating cardiomyocyte proliferation at 38.9 vs. 37.8◦C
To the best of our knowledge, this study represents the largest epigenome-wide analysis in livestock species, the first epigenome-wide association studies (EWAS) in broiler chickens, and the first study assessing genomewide DNA methylation between chickens incubated in different environments

Summary

Introduction

Life environmental parameters have profound effects on broiler chick development and post-hatch performance. In studies assessing the effects of incubation egg shell temperature (EST) on organ growth, heart weight (HW) is consistently observed to be negatively correlated with EST (Leksrisompong et al, 2007; Molenaar et al, 2010, 2011; Maatjens et al, 2014, 2016). Chicks incubated at a higher EST from embryonic day 7 (E7) to hatch were found to have a higher incidence of ascites mortality at 6 weeks of age (Molenaar et al, 2011; Sözcü and Ipek, 2015). Broilers exposed to increased temperature early in life exhibited decreased mortality when exposed to heat stress again at 6 weeks of age relative to unexposed animals, suggesting that early heat stress confers thermotolerance that can improve performance upon subsequent exposures (Yahav and Hurwitz, 1996)

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