A comparative analysis of chromatin accessibility in cattle, pig, and mouse tissues

Abstract

Background: Although considerable progress has been made towards annotating the noncoding portion of the human and mouse genomes, regulatory elements in other species, such as livestock, remain poorly characterized. This lack of functional annotation poses a substantial roadblock to agricultural research and diminishes the value of these species as model organisms. As active regulatory elements are typically characterized by chromatin accessibility, we implemented the Assay for Transposase Accessible Chromatin (ATAC-seq) to annotate and characterize regulatory elements in pigs and cattle, given a set of eight adult tissues.

Results: Overall, 306,304 and 273,594 active regulatory elements were identified in pig and cattle, respectively. 71,478 porcine and 47,454 bovine regulatory elements were highly tissue-specific and were correspondingly enriched for binding motifs of known tissue-specific transcription factors. However, in every tissue the most prevalent accessible motif corresponded to the insulator CTCF, suggesting pervasive involvement in 3-D chromatin organization. Taking advantage of a similar dataset in mouse, open chromatin in pig, cattle, and mice were compared, revealing that the conservation of regulatory elements, in terms of sequence identity and accessibility, was consistent with evolutionary distance; whereas pig and cattle shared about 20% of accessible sites, mice and ungulates only had about 10% of accessible sites in common. Furthermore, conservation of accessibility was more prevalent at promoters than at intergenic regions.

Conclusions: The lack of conserved accessibility at distal elements is consistent with rapid evolution of enhancers, and further emphasizes the need to annotate regulatory elements in individual species, rather than inferring elements based on homology. This atlas of chromatin accessibility in cattle and pig constitutes a substantial step towards annotating livestock genomes and dissecting the regulatory link between genome and phenome.

Keywords: Cattle; Chromatin accessibility; Comparative epigenomics; Functional annotation; Pig.

Fig. 1

Experimental design and ATAC-seq quality metrics. a Overview of tissues collected from adult male cattle and pigs for ATAC-seq, wherein the Tn5 transposase preferentially cuts DNA at accessible sites and simultaneously inserts sequencing adapters. b Scatterplots showing Pearson correlation of normalized genome-wide ATAC-seq signal (reads per million; RPM) between biological replicates for brain cortex and lung. c Heatmaps depicting normalized ATAC-seq signal at all TSS, sorted by signal intensity. Signal shown for brain cortex and lung. d Normalized ATAC-seq signal in cattle tissues at the GAPDH locus (a housekeeping gene), as well as several genes with tissue-specific activity. Pink bars indicate that signal exceeded the viewing range (up to 20 RPM). e Normalized ATAC-seq signal in pig tissues at the same genes. f PCA of normalized ATAC-seq signal in consensus open chromatin identified in cattle. G) PCA of normalized ATAC-seq signal in consensus open chromatin identified in pig

Fig. 2

Open chromatin localization and differential accessibility. a Distribution of cattle and pig consensus open chromatin relative to genomic features. Because peaks often span multiple features, peaks were categorized based on 1 bp overlap with features in the following order: first as TSS (±50 bp), then as promoter (2 kb upstream of TSS), transcription termination site (TTS) (±50 bp), 5′ untranslated region (UTR), 3′ UTR, coding sequence (CDS), intronic, and if no features were overlapped, peaks were considered intergenic. b Distribution of consensus peak activity, ranging from tissue-specific (accessible in only one tissue) to ubiquitous (accessible in all sampled tissues). Consensus peaks that were accessible in a single tissue were further broken down by tissue. c Distribution of consensus peak activity for regions containing CTCF motifs

Fig. 3

Characterization of tissue-specific consensus open chromatin. a Number of consensus peaks demonstrating tissue-specific accessibility in each tissue and species. b Normalized ATAC-seq signal (RPM) at regions demonstrating tissue-specific accessibility in cattle and pig tissues. Tissue-specific peaks were first grouped by corresponding tissue, then ordered by signal intensity. Peaks were scaled to 500 bp, and signal is shown 500 bp upstream and downstream. c Distribution of tissue-specific open chromatin relative to gene annotations. d Enrichment of known TF binding motifs in tissue-specific open chromatin. Motifs sorted by TF family. Sets of tissue-specific open chromatin for each species are grouped by tissue. Increasing size and color intensity indicate increasing enrichment for a given motif

Fig. 4

Conservation of regulatory element accessibility in cattle, pig, and mouse. a Summary of pairwise open chromatin conservation. Circles reflect number of consensus ATAC-seq peaks in each species. Arrow width reflects proportion of consensus peaks that could be projected to the other species. Lighter section of arrows reflect proportion of regions that could be mapped which demonstrated conserved accessibility in at least one tissue in both species. b Of regions that could be projected to all three species, number of regions with conserved accessibility in all three species and in ungulates laid over a phylogenetic tree reflective of evolutionary distance in millions of years (MYA). c Genomic distribution of regions with conserved accessibility in all three species, relative to the mouse gene annotation. Brief summary of enriched gene ontology (GO) terms for genes marked by conserved open chromatin at their TSS in every tissue. d Consensus peaks, consensus peaks with conserved sequence (that could be mapped to all three species), and consensus peaks with conserved sequence and conserved accessibility in all three species at the MEF2A locus. Tracks show normalized ATAC-seq signal. Conserved promoter open chromatin highlighted in green. Conserved distal (putative enhancer) open chromatin highlighted in yellow