Table_1_Characterizing Genetic Regulatory Elements in Ovine Tissues.docx

The Ovine Functional Annotation of Animal Genomes (FAANG) project, part of the broader livestock species FAANG initiative, aims to identify and characterize gene regulatory elements in domestic sheep. Regulatory element annotation is essential for identifying genetic variants that affect health and production traits in this important agricultural species, as greater than 90% of variants underlying genetic effects are estimated to lie outside of transcribed regions. Histone modifications that distinguish active or repressed chromatin states, CTCF binding, and DNA methylation were used to characterize regulatory elements in liver, spleen, and cerebellum tissues from four yearling sheep. Chromatin immunoprecipitation with sequencing (ChIP-seq) was performed for H3K4me3, H3K27ac, H3K4me1, H3K27me3, and CTCF. Nine chromatin states including active promoters, active enhancers, poised enhancers, repressed enhancers, and insulators were characterized in each tissue using ChromHMM. Whole-genome bisulfite sequencing (WGBS) was performed to determine the complement of whole-genome DNA methylation with the ChIP-seq data. Hypermethylated and hypomethylated regions were identified across tissues, and these locations were compared with chromatin states to better distinguish and validate regulatory elements in these tissues. Interestingly, chromatin states with the poised enhancer mark H3K4me1 in the spleen and cerebellum and CTCF in the liver displayed the greatest number of hypermethylated sites. Not surprisingly, active enhancers in the liver and spleen, and promoters in the cerebellum, displayed the greatest number of hypomethylated sites. Overall, chromatin states defined by histone marks and CTCF occupied approximately 22% of the genome in all three tissues. Furthermore, the liver and spleen displayed in common the greatest percent of active promoter (65%) and active enhancer (81%) states, and the liver and cerebellum displayed in common the greatest percent of poised enhancer (53%), repressed enhancer (68%), hypermethylated sites (75%), and hypomethylated sites (73%). In addition, both known and de novo CTCF-binding motifs were identified in all three tissues, with the highest number of unique motifs identified in the cerebellum. In summary, this study has identified the regulatory regions of genes in three tissues that play key roles in defining health and economically important traits and has set the precedent for the characterization of regulatory elements in ovine tissues using the Rambouillet reference genome.

绵羊动物基因组功能注释（FAANG）项目是更大范围畜禽物种FAANG计划的组成部分，旨在鉴定并注释家养绵羊的基因调控元件。调控元件注释对于识别影响这一重要农业物种健康与生产性状的遗传变异至关重要，因为据估计，超过90%的遗传效应相关变异位于转录区域之外。研究采用区分活跃/抑制染色质状态的组蛋白修饰、CCCTC结合因子（CTCF）结合以及DNA甲基化，对4只周岁绵羊的肝脏、脾脏和小脑组织中的调控元件进行表征。针对H3K4me3、H3K27ac、H3K4me1、H3K27me3及CTCF开展了染色质免疫共沉淀测序（ChIP-seq）。利用ChromHMM软件在每个组织中鉴定出9类染色质状态，涵盖活跃启动子、活跃增强子、预备增强子、抑制型增强子及绝缘子等。全基因组亚硫酸氢盐测序（WGBS）被用于获取全基因组DNA甲基化数据，以与ChIP-seq数据形成互补。研究人员在不同组织中鉴定出高甲基化区域与低甲基化区域，并将这些区域与染色质状态进行比对，以更好地区分并验证上述组织中的调控元件。有趣的是，脾脏和小脑内带有预备增强子标记H3K4me1的染色质状态，以及肝脏内的CTCF结合位点，展现出最多的高甲基化位点。不出所料，肝脏与脾脏内的活跃增强子、小脑内的启动子区域，拥有最多的低甲基化位点。总体而言，由组蛋白修饰与CTCF定义的染色质状态在三类组织中均占据约22%的基因组区域。此外，肝脏与脾脏共享最高比例的活跃启动子（65%）与活跃增强子（81%）状态；而肝脏与小脑则共享最高比例的预备增强子（53%）、抑制型增强子（68%）、高甲基化位点（75%）与低甲基化位点（73%）。此外，在三类组织中均鉴定出已知及新发的CTCF结合基序，其中小脑内鉴定出的独特基序数量最多。综上，本研究鉴定出三类组织中的基因调控区域，这些区域在决定绵羊健康及经济重要性状中发挥关键作用；同时为利用兰布耶（Rambouillet）参考基因组开展绵羊组织调控元件注释工作树立了范例。