Abstract
Alveolar macrophages function in innate and adaptive immunity, wound healing, and homeostasis in the lungs dependent on tissue-specific gene expression under epigenetic regulation. The functional diversity of tissue resident macrophages, despite their common myeloid lineage, highlights the need to study tissue-specific regulatory elements that control gene expression. Increasing evidence supports the hypothesis that subtle genetic changes alter sheep macrophage response to important production pathogens and zoonoses, for example, viruses like small ruminant lentiviruses and bacteria like Coxiella burnetii. Annotation of transcriptional regulatory elements will aid researchers in identifying genetic mutations of immunological consequence. Here we report the first genome-wide survey of regulatory elements in any sheep immune cell, utilizing alveolar macrophages. We assayed histone modifications and CTCF enrichment by chromatin immunoprecipitation with deep sequencing (ChIP-seq) in two sheep to determine cis-regulatory DNA elements and chromatin domain boundaries that control immunity-related gene expression. Histone modifications included H3K4me3 (denoting active promoters), H3K27ac (active enhancers), H3K4me1 (primed and distal enhancers), and H3K27me3 (broad silencers). In total, we identified 248,674 reproducible regulatory elements, which allowed assignment of putative biological function in macrophages to 12% of the sheep genome. Data exceeded the FAANG and ENCODE standards of 20 million and 45 million useable fragments for narrow and broad marks, respectively. Active elements showed consensus with RNA-seq data and were predictive of gene expression in alveolar macrophages from the publicly available Sheep Gene Expression Atlas. Silencer elements were not enriched for expressed genes, but rather for repressed developmental genes. CTCF enrichment enabled identification of 11,000 chromatin domains with mean size of 258 kb. To our knowledge, this is the first report to use immunoprecipitated CTCF to determine putative topological domains in sheep immune cells. Furthermore, these data will empower phenotype-associated mutation discovery since most causal variants are within regulatory elements.
Highlights
Livestock researchers are identifying functional variants outside of genes as associated with valuable production traits, supporting the need to molecularly annotate regulatory elements (Ibeagha-Awemu and Zhao, 2015; Zhao et al, 2015; Wang et al, 2017)
We identified significant regions bound by CTCF with native ChIP-seq (N-ChIP) that revealed insulators and allowed the first preliminary estimates of chromatin domains in sheep alveolar macrophages
We found in sheep macrophages as Barski et al (2007) found in human T-cells, that locations with CTCF enrichment were enriched for multiple histone methylation marks found at domain boundaries
Summary
Livestock researchers are identifying functional variants outside of genes as associated with valuable production traits, supporting the need to molecularly annotate regulatory elements (Ibeagha-Awemu and Zhao, 2015; Zhao et al, 2015; Wang et al, 2017). DNA regulatory elements are sequences associated with a reproducible biological function that can control gene expression through epigenetic modifications (Birney et al, 2007). Little is known regarding in vivo tissue annotation of regulatory elements in livestock species (Villar et al, 2015; Zhao et al, 2015; Wang et al, 2017, 2018; Naval-Sanchez et al, 2018; Nguyen et al, 2018; Fang et al, 2019; Hall et al, 2020; Kingsley et al, 2020). The FAANG consortium recognized this need and formed a global network of researchers for epigenetic discovery in food animal species (Andersson et al, 2015; Tuggle et al, 2016; Giuffra and Tuggle, 2019)
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