Global DNA Methylation Changes in Nile Tilapia Gonads during High Temperature-Induced Masculinization

RESEARCH ARTICLE Global DNA Methylation Changes in Nile Tilapia Gonads during High TemperatureInduced Masculinization Li-Xue Sun1☯, Yi-Ya Wang1☯, Yan Zhao1☯, Hui Wang1, Ning Li2, Xiang Shan Ji1* 1 College of Animal Science and Technology, Shandong Agricultural University, Taian, 271018, China, 2 College of Life Sciences, Shandong Agricultural University, Taian, 271018, China a11111 ☯ These authors contributed equally to this work. * Abstract OPEN ACCESS Citation: Sun L-X, Wang Y-Y, Zhao Y, Wang H, Li N, Ji XS (2016) Global DNA Methylation Changes in Nile Tilapia Gonads during High Temperature-Induced Masculinization. PLoS ONE 11(8): e0158483. doi:10.1371/journal.pone.0158483 Editor: Hanping Wang, The Ohio State University, UNITED STATES Received: January 25, 2016 Accepted: June 16, 2016 Published: August 3, 2016 Copyright: © 2016 Sun et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files and all MeDIP-seq data files are available from the GEO database (accession number(s) GSE72386). Funding: This study was supported by the National Natural Science Foundation of China (http://www. nsfc.gov.cn/) (31472270 to XSJ), the Research Award Fund for outstanding young scientists of Shandong Province (http://jihlx.sdstc.gov.cn/ STDPMS/BS/Default.aspx) (2014BSB01303 to YZ), and the earmarked fund for Modern Agro-industry Technology Research System in Shandong Province (SDAIT-13 and SDAIT-15-011 to XSJ). The funders In some fish species, high or low temperature can switch the sex determination mechanisms and induce fish sex reversal when the gonads are undifferentiated. During this high or low temperature-induced sex reversal, the expressions of many genes are altered. However, genome-wide DNA methylation changes in fish gonads after high or low temperature treatment are unclear. Herein, we compared the global DNA methylation changes in the gonads from control females (CF), control males (CM), high temperature-treated females (TF), and high temperature-induced males (IM) from the F8 family of Nile tilapia (Oreochromis niloticus) using methylated DNA immunoprecipitation sequencing. The DNA methylation level in CF was higher than that in CM for various chromosomes. Both females and males showed an increase in methylation levels on various chromosomes after high-temperature induction. We identified 64,438 (CF/CM), 63,437 (TF/IM), 98,675 (TF/CF), 235,270 (IM/CM) and 119,958 (IM/CF) differentially methylated regions (DMRs) in Nile tilapia gonads, representing approximately 0.70% (CF/CM), 0.69% (TF/IM), 1.07% (TF/CF), 2.56% (IM/CM), and 1.30% (IM/CF)of the length of the genome. A total of 89 and 65 genes that exhibited DMRs in their gene bodies and promoters were mapped to the Nile tilapia genome. Furthermore, more than half of the genes with DMRs in the gene body in CF/CM were also included in the IM/CM, TF/CF, TF/IM, and IM/CF groups. Additionally, many important pathways, including neuroactive ligand-receptor interaction, extracellular matrixreceptor interaction, and biosynthesis of unsaturated fatty acids were identified. This study provided an important foundation to investigate the molecular mechanism of high temperature-induced sex reversal in fish species. Introduction Sex-determining mechanisms in gonochoristic fish can broadly be classified as genotypic (GSD), temperature-dependent (TSD), or genotypic plus temperature effects (GSD+TE) [1,2]. For fish species with TSD, they should have a sex ratio response to temperature within the PLOS ONE | DOI:10.1371/journal.pone.0158483 August 3, 2016 1 / 16 DNA Methylation and Induced Masculinization had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. range of temperature during development in the wild and should not have sex chromosomes [1]. However, artificially high or low temperatures during critical thermosensitive periods, which is ecologically irrelevant, also results in sex ratio changes in many fish, which are defined as the GSD+TE type. For GSD + TE, a high or low temperature can override the influence of genetics and switch the sex determination mechanisms when the gonads are undifferentiated [3]. However, long-term extreme temperature treatment even resulted in the sterility in female Nile tilapia [4]. In 2011, Navarro-Martín et al. first described DNA methylation of cyp19a (cytochrome P450, family 19, subfamily a), which regulated the cyp19a mRNA expression, and temperature effects on sex ratios in European sea bass [5]. During high or low temperature-induced sex reversal in fish species, the expressions of many genes are altered, such as cyp19a1a, foxl2 (forkhead box L2), sox9 (sex-determining region Y box 9), dmrt1 (doublesex and mab-3 related transcription factor 1), ERα, and ERβ [6–11]. Thus, a high or low temperature could cause a global change of DNA methylation and gene expression. To the best of our knowledge, studies focusing on genome-wide DNA methylation changes in the gonads after high or low temperature treatment in Nile tilapia (Oreochromis niloticus) have not been performed. Nile tilapia is the third most important aquaculture fish after carp and salmon, and the males grow significantly faster than females. A considerable amount of previous research has made the sex determination mechanism Nile tilapia relatively clear [12]. In Nile tilapia, a GSD + TE sex determination fish, high temperature treatment applied after hatching (around 10 days post-fertilization) and lasting from 10 to 28 days, significantly increased the male ratio [3,13–18]. Therefore, the thermal control of sex in Nile tilapia is an economic and environmentally friendly method [19]. In 2012, the whole genome of Nile tilapia was sequenced. Thus, Nile tilapia is an appealing model to study the molecular mechanisms underlying GSD + TE. Methylated DNA immunoprecipitation sequencing (MeDIP) is a recently devised method to determine the distribution of DNA methylation within functional regions (e.g., promoters) or in the entire genome, robustly and cost efficiently [20]. In this study, we used Nile tilapia as a model to perform a genome-wide survey of DNA methylation differences in female and male gonads between control and high temperature-induced groups using MeDIP. The objectives of present study were: 1) to identify the genomic DNA methylation changes after high temperature treatment in Nile tilapia; 2) to identify the high temperature induction-related differentially methylated regions (DMRs); and 3) to identify critical pathways associated with high temperature-induced masculinization. Results Sex ident (...truncated)