热应激对奶牛GNAS启动子区DNA甲基化水平的影响

Effect of Heat Stress on DNA Methylation of GNAS Promoter Region in Dairy Cows

【目的】热应激严重影响奶牛的生产和健康,是制约奶业持续健康发展的重要因素。DNA甲基化作为一种重要的表观遗传调控机制参与动物的热应激反应,但其在奶牛热应激过程中的作用和分子机制研究较少。研究通过检测奶牛热应激相关的DNA甲基化变化,筛选和验证DNA甲基化相关的关键基因,为奶牛热应激的表观遗传机制研究积累数据。【方法】以北京市三元绿荷金银岛牧场的24头中国荷斯坦牛(泌乳阶段及胎次相同)为研究对象,分别于春季(非应激期,2017年4月份)和夏季(热应激期,2017年7月份)采集试验个体的血液,提取DNA,共获得48份DNA样本。首先,随机选择其中15头奶牛,并随机分为3组,每组5份DNA样本混合,通过全基因组重亚硫酸盐测序法(Whole-genome bisulfite sequencing,WGBS)检测奶牛基因组DNA的甲基化状态,筛选差异甲基化区域(Differential methylation region,DMR;1000 bp windows,500 bp overlap,P<0.05)及相关基因,利用PROMO和Methprimer软件预测基因启动子的转录因子结合位点和CpG岛区。然后,39℃热处理牛乳腺上皮细胞(Bovine mammary gland epithelial cells,Mac-T)不同时间(24 h,48 h,72 h),MTT法检测细胞活力。最后,利用亚硫酸盐测序法(Bisulfite sequencing PCR,BSP)分别对春夏季的24头奶牛及39℃热处理不同时间的Mac-T细胞中目的基因启动子区的甲基化状态进行分析。【结果】通过WGBS共得到49861个DMRs,其中一个DMR注释到基因GNAS复合体基因座(GNAS complex locus,GNAS)的启动子区,其整体甲基化水平在夏季热应激期极显著上调(P<0.001),且该区域预测到一个352 bp的CpG岛,包含Sp1、C/EBP等重要转录因子的结合区域。24头奶牛个体中GNAS启动子区31个CG位点的整体甲基化水平在热应激期显著上调(P<0.05),与WGBS结果一致,其中,21号(-113 bp,Chr13:57532733)和27号CG(-63 bp,Chr13:57532683)位点甲基化水平显著上调(P<0.05)。Mac-T细胞热处理48和72 h后,细胞活力极显著下降(P<0.01),GNAS启动子的CG位点整体甲基化水平显著上调(P<0.05),21号和27号CG均为显著上调的差异甲基化位点,与个体水平结果一致。【结论】热应激会引起奶牛GNAS启动子甲基化水平增加,GNAS是奶牛热应激DNA甲基化调控的潜在靶基因。

【Objective】Heat stress has seriously impaired the production and health of dairy cows,causing the subsequent limitation in sustainable development of dairy industry.DNA methylation is an important epigenetic regulatory mechanism involved in an animal’s heat stress response,but the potential functions and molecular mechanisms of which are not clear.The current study was conducted to detect the DNA methylation related to heat stress in dairy cows and to identify target genes related to DNA methylation,so as to provide a better insight into the epigenetics mechanism of heat stress in dairy cows.【Method】In the study,24 Chinese Holstein lactation cows(same lactation stage and same parity)in Sanyuan dairy farm were used for the blood samples collection in heat stress period(July in the summer of 2017)and non-heat stress period(April in spring 2017),respectively,followed by DNA extraction.To explore the DNA methylation differences in dairy cows from different heat stress period,15 of 24 animals were randomly assigned to 3 groups(N=5 animals/group),5 DNA samples in one group were mixed together to get a single pooled DNA sample,thus 6 pooled DNA samples including 3 from spring and 3 from summer were used for the DNA methylation detection by the whole-genome bisulfite sequencing(WGBS),then the differential methylation region(DMR;1000 bp windows,500 bp overlap,P<0.05)and key gene were identified.PROMO and Methprimer software were used to predict transcription factor binding sites and CpG islands,respectively.Then,the bovine mammary gland epithelial cells(Mac-T)were treated at 39℃for 24 h,48 h,and 72 h,and the cell viability were detected by MTT method.Finally,using the bisulfite sequencing PCR(BSP),the methylation levels of target gene promoter in 24 dairy cows in spring and summer and Mac-T cells treated in 39℃were examined,respectively.【Result】Based on the DNA methylation analysis of WGBS data,49861 differential methylation regions(DMRs)associated with heat stress were identified.One of DMRs was attributed to the promoter area of GNAS complex locus(GNAS),whose methylation level significantly increased in heat-stressed animals(P<0.001).Also,there was a 352 bp CpG island in the promoter of GNAS containing potential binding sites for Sp1,C/EBP and other important transcription factors.Further the methylation status of the GNAS gene promoter region in heat stressed dairy cows were verified by BSP,and the average methylation level in all cytosine of 31 CpG sites was higher in heat stress cows than that in control groups(P<0.05),which corresponding to the above WGBS results.Moreover,the 21(-113 bp,Chr13:57532733)and 27(-63 bp,Chr13:57532683)CpG sites showed significant differences between the spring and summer groups(P<0.05).In Mac-T cells,after 48 h and 72 h heat treatment,the cell viability decreased significantly(P<0.01),but the overall CG methylation level of 31 CpG sites in the GNAS gene promoter region increased significantly(P<0.05),and also the similar significant methylation changes appeared in the site 21 and 27 CpG in cell.【Conclusion】Heat stress increased the methylation levels of the promoter region of the GNAS in dairy cows as well as in cells,which indicated that GNAS was a potential target gene regulated DNA methylation in heat stress response of dairy cows.