Characterizing Genetic Regulatory Elements in Ovine Tissues

Abstract

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.

Differentially CTCF-Binding Sites in Cattle Rumen Tissue during Weaning

“…For example, more repressed sites were in promoter regions compared to the induced sites (+10%), while more induced sites were in exons (+7%), UTR (+2.7%), and downstream (+2.2%). However, most of the induced and repressed DCBS are in distal intergenic regions (~60%), in accordance with previous results in sheep [ 33 ] and humans [ 44 ], showing a potential role as insulators, indicating that these regions may also act as insulators by blocking enhancers and silencers present nearby these sequences [ 27 ]. In addition, a significant percentage of the sites are located within genes (~24% for the induced and 19% for the repressed sites).…”

“…Although large numbers of regulatory elements have been identified in cattle recently, many more regulatory non-coding elements are expected to be identified in additional tissues and conditions. A few studies have used CTCF ChIP-seq to identify functional regions in animals, such as mouse [ 29 , 30 , 31 ], sheep [ 32 , 33 ], and cattle [ 34 ]. A recent study in lactating dairy cows identified millions of functional regions in the cattle genome using ChIP-seq data from six tissues (heart, kidney, liver, lung, mammary, and spleen), including CTCF-binding sites and differential binding sites between tissues [ 18 , 34 ].…”

Examining the extent of environmental contributions toward DNA methylation and phenotypic variation

“…Both transcribed regions of genes and regulatory regions harbor important epigenetic modifications that vary by tissue, with regulatory regions being of particular interest. Davenport et al (2021) demonstrate the prevalence of diverse epigenetic modifications in regulatory regions in three tissues that have been used to characterize economically important traits in sheep. Chromatin immunoprecipitation with sequencing (ChIP-seq) and whole-genome bisulfite sequencing ( WGBS ) in the spleen, liver, and cerebellum assess CCCTC-binding factor ( CTCF ) binding, DNA methylation, and histone modifications relevant to chromatin state ( Davenport et al, 2021 ).…”

“… Davenport et al (2021) demonstrate the prevalence of diverse epigenetic modifications in regulatory regions in three tissues that have been used to characterize economically important traits in sheep. Chromatin immunoprecipitation with sequencing (ChIP-seq) and whole-genome bisulfite sequencing ( WGBS ) in the spleen, liver, and cerebellum assess CCCTC-binding factor ( CTCF ) binding, DNA methylation, and histone modifications relevant to chromatin state ( Davenport et al, 2021 ). Hypomethylation was mainly identified at active enhancers in all three tissues, whereas hypermethylation was discovered at CTCF-binding sites in the liver and poised enhancer H3K4me1 in the spleen and cerebellum.…”

“…Hypomethylation was mainly identified at active enhancers in all three tissues, whereas hypermethylation was discovered at CTCF-binding sites in the liver and poised enhancer H3K4me1 in the spleen and cerebellum. Methylation sites varied by tissue, but hypo- and hyper-methylation sites were mostly similar between the liver and cerebellum ( Davenport et al, 2021 ).…”