Comparative epigenomics reveals the impact of ruminant-specific regulatory elements on complex traits

Abstract

Background: Insights into the genetic basis of complex traits and disease in both human and livestock species have been achieved over the past decade through detection of genetic variants in genome-wide association studies (GWAS). A majority of such variants were found located in noncoding genomic regions, and though the involvement of numerous regulatory elements (REs) has been predicted across multiple tissues in domesticated animals, their evolutionary conservation and effects on complex traits have not been fully elucidated, particularly in ruminants. Here, we systematically analyzed 137 epigenomic and transcriptomic datasets of six mammals, including cattle, sheep, goats, pigs, mice, and humans, and then integrated them with large-scale GWAS of complex traits.

Results: Using 40 ChIP-seq datasets of H3K4me3 and H3K27ac, we detected 68,479, 58,562, 63,273, 97,244, 111,881, and 87,049 REs in the liver of cattle, sheep, goats, pigs, humans and mice, respectively. We then systematically characterized the dynamic functional landscapes of these REs by integrating multi-omics datasets, including gene expression, chromatin accessibility, and DNA methylation. We identified a core set (n = 6359) of ruminant-specific REs that are involved in liver development, metabolism, and immune processes. Genes with more complex cis-REs exhibited higher gene expression levels and stronger conservation across species. Furthermore, we integrated expression quantitative trait loci (eQTLs) and GWAS from 44 and 52 complex traits/diseases in cattle and humans, respectively. These results demonstrated that REs with different degrees of evolutionary conservation across species exhibited distinct enrichments for GWAS signals of complex traits.

Conclusions: We systematically annotated genome-wide functional REs in liver across six mammals and demonstrated the evolution of REs and their associations with transcriptional output and conservation. Detecting lineage-specific REs allows us to decipher the evolutionary and genetic basis of complex phenotypes in livestock and humans, which may benefit the discovery of potential biomedical models for functional variants and genes of specific human diseases.

Keywords: Epigenetic regulation; GWAS enrichment; Liver; Regulatory elements; Ruminant evolution.

Fig. 1

Summary and characterization of 137 epigenetic and gene expression data in six mammals. A Datasets analyzed by this study. B The number of regulatory regions (promoters and enhancers) identified in the liver of each species. C Fold enrichments of regulatory elements (REs) for 14 chromatin states previously predicted in cattle and pig liver [19]. These chromatin states mainly represented enhancers (CTCF/Enhancer, Active_Enhancer, and Primed_Enhancer), promoters (CTCF/Promoter, Active_Promoter, Promoter, Poised_Promoter), repressed regions (Insulator, Low_Signal, and Polycomb_Repressed), open regions (Active_Element), and TSS-proximal regions (CTCF/TSS, Active_TSS, and Flanking TSS). D The percentages of REs overlapped with public data in cattle liver (blue) and newly annotated in this study (orange). E The sample clustering based on pairwise Spearman correlation of gene expression. F Similarity of sample clustering patterns across different omics data types using Rand index

Fig. 2

Evolutionary changes of regulatory elements (REs) and gene expression across mammals. Phylogenetic trees were built using the neighbor-joining method for gene expression of 9796 one-to-one orthologous genes (A), H3K27ac signals (B), H3K4me3 signals (C), chromatin accessibility (D), and DNA methylation level (E) in REs (enhancers and promoters) of orthologous genes. F Comparisons of total branch lengths of the phylogenetic trees across five omics data types using 1000 bootstrapping test. “****” indicates P < 0.0001

Fig. 3

The dynamic of regulatory elements (REs) in the liver during ruminant evolution. A The fractions of REs that are highly conserved (AC), ruminant-specific (RS), and cattle-specific (CS) in cattle. B Specificity of liver REs determined by comparing to the other seven cattle tissues-adipose, cortex, cerebellum, hypothalamus, lung, spleen, and muscle. C Histone mark signals (H3K4me3 for cattle-specific promoters (CSP), H3K27ac for cattle-specific enhancers (CSE)), DNA methylation levels, and chromatin accessibility of tissue-specific CSE and CSP across eight cattle tissues. D GREAT Gene Ontology (GO) terms enrichment for six lineage-specific REs (FDR <0.01). E H3K27ac and H3K4me3 ChIP-seq profiles of PON1 gene (right) for six species, cattle, sheep, goat, pig, human, and mouse, at the loci of ruminant-specific enhancers (RSE). The expression levels of the PON1 gene (left) in the 28 organ systems of cattle. TPM, transcripts per kilobase million. F Transcription factor (TF) motifs enriched in six lineage-specific REs. The bubble plot shows the enrichment of TF motifs in six lineage-specific REs, and the heatmap shows the chromatin accessibility of TF promoters across five mammals. G The fraction of RE peaks harboring canonical PPARA motif (JASPAR – MA1148.1) as a function of distance from RE peaks to the summits

Fig. 4

Regulatory elements (REs) drive interspecies transcriptional conservation during evolution. A Comparison of total branch lengths for the phylogenetic trees across four categories of one-to-one orthologues genes based on the 1000 bootstrapping test. Enhancer, Promoter, Both, and None indicate genes with only enhancers, only promoters, both enhancers, and promoters, without REs, respectively. Box plots showing the median phastCons scores (B) and pLI score (C) for four categories of one-to-one orthologues genes. D The number of associated enhancers and promoters contributes to interspecies transcriptional stability. E The association of gene stability and conserved REs, including highly conserved enhancers (ACE) and highly conserved promoters (ACP). “**” indicates P < 0.01; “***” indicates P < 0.001; “****” indicates P < 0.0001

Fig. 5

Regulatory elements (REs) are enriched for genomic variants of gene expression and complex traits. The fold enrichment of REs for genomic variants [39] (A), expression QTLs (eQTLs) detected from cattle liver [9] (B), and six categories of QTLs (including 489 complex traits) downloaded from the cattle QTL database [43] (C), based on 1000 bootstrapping test. D The P values of SNPs inside and outside of REs from GWAS summary datasets for protein percentage in cattle [3]. “*” indicates P < 0.05; “**” indicates P < 0.01; “****” indicates P < 0.0001; “ns” indicates P >0.05

Fig. 6

GWAS signal enrichment analysis of regulatory elements (REs) for complex traits and diseases. GWAS signals enrichment analysis of the characterized REs for 44 and 52 complex traits in cattle (A) and humans (B), respectively. “Blue” represents traits that were only significantly enriched in cattle-specific REs (CS-REs) in A. “*” indicates P < 1.0e−5. ACE, ACP, RSE, RSP, CSE, CSP, HMPSP, HMPSE, HSE, and HSP stand for all-conserved enhancers, all-conserved promoters, ruminant-specific enhancers, ruminant-specific promoters, cattle-specific enhancers, cattle-specific promoters, human-pig-mouse-specific enhancers, human-pig-mouse-specific promoters, human-pig-specific enhancers, human-pig-specific promoters, human-specific enhancers, human-specific promoters, respectively. C, D Pearson’s correlations of enrichment degrees (−log₁₀P) across 52 human traits calculated by using the stratified linkage disequilibrium score regression (LDSC) and the count-based marker-set test

Fig. 7

A cattle-specific enhancer of DGAT1 was associated with protein percentage in cattle. A The top panel is the Manhattan plot of protein percentage in cattle [3]. The bottom panel shows H3K27ac (green) and H3K4me3 (purple) profiles within the DGAT1 locus across six species. B H3K27ac (green) and H3K4me3 (purple) profiles within the DGAT1 locus across eight tissues in cattle. C The PEER-corrected expression level of DGAT1 is significantly associated with three genotypes of rs384957047 in cattle liver [9]. D The relative luciferase activity of the recombinant plasmids constructed with DGAT1_T and DGAT1_C of rs384957047. “**” indicates P < 0.01; “***” indicates P < 0.001