Genetic improvement of domestic animal species has been crucial in reducing the environmental impact of animal-source foods, which are vital for nutrition in developing countries. Climate change and pandemics like COVID-19 significantly affect food security, increasing the need for improvements in food production and sustainability. Chicken, cattle, and pigs are major contributors to global food production, providing economically and nutritionally valuable protein. Understanding the genetic basis of complex traits in these animals is essential for continued genetic improvement to meet rising demands while using fewer animals. These species also contribute significantly to our understanding of evolutionary biology, human development, disease, and medicine. Most causative genetic variants associated with complex traits are located in non-coding regions that regulate gene expression. Human and mouse catalogs of regulatory elements (REs) have been critical in identifying genetic variants linked to health and disease. While some studies have explored the evolution of regulatory sequences in non-model species, much remains unknown about the extent of epigenomic and regulatory conservation, particularly across large evolutionary distances. This study, a pilot project of the Functional Annotation of Animal Genomes (FAANG) consortium, presents a functional annotation of the chicken, pig, and cattle genomes across eight tissues. Comparative analysis with human and mouse ENCODE data reveals that while the sequence and position of REs, particularly enhancers, show low conservation, tissue-specific patterns of transcription factor motif enrichment are highly conserved. The study aims to provide a valuable data resource for agricultural research and comparative epigenomics.

The authors reference several key studies in their introduction. ENCODE and modENCODE projects are highlighted for their contributions to understanding regulatory elements in humans and mice. Previous research on the evolution of regulatory sequences in non-model and non-mammalian species is acknowledged, emphasizing the gap in knowledge regarding the extent of epigenomic and regulatory conservation across large evolutionary distances. Specific papers are cited for studies investigating the evolution of regulatory sequences in various species, including birds and mammals. The FAANG consortium's previous work on multi-species annotation of transcriptome and chromatin structure in domesticated animals is also referenced.

The study employed a genome-wide functional annotation approach using eight tissues (liver, lung, spleen, skeletal muscle, subcutaneous adipose, cerebellum, brain cortex, and hypothalamus) from sexually mature male chickens, pigs, and cattle. Six epigenetic data types were profiled: four histone modifications (H3K4me3, H3K27ac, H3K4me1, H3K27me3), CTCF binding (using ChIP-seq), and chromatin accessibility (using DNase-seq for chickens and ATAC-seq for pigs and cattle). Transcriptome sequencing (RNA-seq) was performed to correlate gene expression with regulatory region activity. A total of 240 ChIP-seq libraries were generated and sequenced, along with DNase-seq and ATAC-seq libraries. Stringent data quality standards, mirroring ENCODE consortium criteria, were applied. Data reproducibility was verified using hierarchical clustering and principal component analysis (PCA). ChromHMM was used to predict genome-wide chromatin states, categorizing genomic regions into 14 distinct states based on combinations of ChIP-seq marks. Regulatory elements (REs) were identified and classified as TSS proximal, genic, or intergenic. Comparative epigenomic analysis was performed by mapping RE coordinates between the five species (chicken, pig, cattle, human, and mouse) using Ensembl alignments. Transcription factor footprinting was conducted to identify potential transcription factor binding events within REs. Target gene prediction was performed by correlating gene expression with the enrichment of histone modifications or open chromatin at enhancers. Topologically associated domains (TADs) were predicted using CTCF-binding sites. Finally, an overlap analysis was performed between identified REs and sequence variants associated with complex traits in dairy cattle from a previous GWAS study.

The study identified a significant number of regulatory elements (REs) in chicken, pig, and cattle genomes. Chicken genomes exhibited approximately half the number of REs compared to pigs and cattle, primarily due to fewer genic and intergenic REs. Most active REs co-occurred with open chromatin regions. Comparative analysis across five species revealed low conservation in the sequence and position of REs, particularly enhancers, but high conservation in tissue-specific patterns of transcription factor motif enrichment. A core set of REs was conserved across all five species, associated with genes involved in basic metabolic processes. Target gene prediction showed that chicken enhancers regulate a larger number of genes compared to mammalian enhancers, suggesting increased multifunctionality. PCA analysis indicated stronger clustering of REs by tissue than by species, highlighting tissue-specific conservation of regulatory features. Overlap with dairy cattle GWAS data showed that SNPs within REs had a higher density of lower p-values compared to SNPs outside REs, for traits like milk protein and fat content. This suggests that RE annotations can significantly reduce the search space for causative variants in complex traits. These findings highlight a balance between positional conservation and functional conservation of regulatory elements, emphasizing the importance of tissue-specific regulatory mechanisms across diverse vertebrate species.

The findings demonstrate that despite low positional conservation of regulatory elements (REs), especially enhancers, across large evolutionary distances, there's significant functional conservation in tissue-specific transcription factor binding and target gene regulation. This suggests a core set of evolutionarily stable REs that play essential roles in basic metabolic processes. The observation that chicken enhancers regulate more genes than mammalian enhancers indicates potential differences in regulatory complexity between avian and mammalian systems. The integration of the RE annotations with existing GWAS data for dairy cattle effectively narrows the search for causative variants, showcasing the practical applications of this research in agricultural genomics. This study significantly advances our understanding of comparative epigenomics and provides a valuable resource for future studies exploring complex traits and evolutionary biology.

This research presents a comprehensive functional annotation of regulatory elements across three important agricultural species (chicken, pig, and cattle), providing a valuable resource for both comparative genomics and agricultural research. The findings highlight the functional conservation of regulatory mechanisms despite low positional conservation, particularly in tissue-specific transcription factor enrichment and target gene regulation. The study's data has immediate applications in refining GWAS studies by identifying more likely causative variants within REs. Future research could expand upon this work by including more tissues, developmental stages, and species, utilizing single-cell sequencing technologies to explore the complexities of gene regulation at a cellular level.

The study focused solely on male animals, limiting the generalizability of the findings to both sexes. The use of correlative analysis for target gene prediction may introduce some uncertainty, and additional experimental validation of the predicted interactions would be beneficial. The reliance on CTCF ChIP-seq data for TAD prediction, due to the unavailability of Hi-C data, could introduce some limitations in accuracy. Further, the analysis predominantly uses data from a single developmental stage, potentially hindering the understanding of developmental dynamics of these regulatory elements.