Embryonic heat conditioning in chicks induces transgenerational heat/immunological resilience via methylation on regulatory elements. Embryonic heat conditioning in chicks induces transgenerational heat/immunological resilience via methylation on regulatory elements

We used in ovo embryonic heat conditioning (EHC) of first-generation chicks and assessed the effect on their untreated offspring by measuring genome-wide alterations in DNA-methylation patterns. DNA was extracted from the APH and reduced representation bisulfite sequencing (RRBS) was performed Overall design: DNA was isolated by TRI Reagent (Molecular Research Center). RRBS libraries were prepared using Zymo-Seq RRBS Library Kit (Zymo) and prepared from 70–140 ng of DNA. Sequencing was done on a NovaSeq 6000 instrument (Illumina) using an SP 100 cycle kit (paired end sequencing). Trimming of Illumina adapters, as well as quality trimming, were performed with Trim Galore, using the following options: --nextseq --non_directional –rrbs. Reads were mapped to the chick genome (galGal5.0) using Bismark v. 0-22-3, bowtie mode. Methylation calls were extracted with Bismark, methylation extractor mode. DMS was extracted and analyzed by edgeR (39), with the recommended cutoff of at least 8 reads in every sample and FDR < 0.05. DMRs were extracted and analyzed by metilene v. 0.2-8. Motif enrichment analysis and DMS/DMR annotation were performed by HOMER tools. Segregating Hyper/Hypo-TADs according to TAD methylation was achieved considering only TADs with more than three DMSs. ChromHMM was used to establish a chromatin state model from an independent study that utilized bulk hypothalamic tissue, and to determine fold enrichment of DMS/DMR over state model. APH mRNA profile of 10 day old noncoditioned F1 chicks, nonchallenged (t0)/6 hours into heat challenge/6 hours into LPS challenge, by 3'-mRNA- sequencing (RNA-seq).

本研究对初代雏鸡实施胚胎内热应激预处理（in ovo embryonic heat conditioning, EHC），并通过检测全基因组DNA甲基化模式的全局改变，评估该处理对其未处理后代的影响。研究样本取自雏鸡下丘脑前室旁区（anterior periventricular hypothalamus, APH），从该区域提取DNA并开展简化代表性亚硫酸氢盐测序（reduced representation bisulfite sequencing, RRBS）。实验整体设计如下：采用TRI Reagent（Molecular Research Center）提取DNA；使用Zymo-Seq RRBS文库制备试剂盒（Zymo），以70~140 ng的DNA为起始量构建RRBS文库；采用Illumina NovaSeq 6000测序平台，搭配SP 100循环试剂盒完成双端测序。使用Trim Galore软件进行Illumina接头修剪及质量修剪，具体参数为：--nextseq --non_directional --rrbs。使用Bismark v0.22.3的Bowtie比对模式，将测序reads比对至鸡参考基因组galGal5.0；通过Bismark的甲基化提取器模式提取甲基化位点信息。差异甲基化位点（differentially methylated sites, DMS）的提取与分析采用edgeR软件（39），筛选标准遵循官方推荐：每个样本中至少覆盖8条reads，且错误发现率（false discovery rate, FDR）<0.05。差异甲基化区域（differentially methylated regions, DMRs）的提取与分析采用metilene v0.2-8软件完成。使用HOMER工具完成基序富集分析以及DMS/DMR的注释工作。根据拓扑关联结构域（topologically associating domains, TADs）的甲基化水平区分高甲基化/低甲基化TADs时，仅纳入包含至少3个DMS的TADs。使用ChromHMM软件，基于一项利用大块下丘脑组织完成的独立研究构建染色质状态模型，并以此计算DMS/DMR在各染色质状态下的富集倍数。此外，本研究采用3'端mRNA测序（RNA-seq）检测10日龄未接受预处理的F1雏鸡下丘脑前室旁区（APH）的mRNA表达谱，处理分组包括未受刺激对照组（t0）、热应激处理6小时组以及脂多糖（lipopolysaccharide, LPS）刺激6小时组。