Genome-Wide Histone Modifications and CTCF Enrichment Predict Gene Expression in Sheep Macrophages

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.

Putative Causal Variants Are Enriched in Annotated Functional Regions From Six Bovine Tissues

“…State 4 ("hyperChIPable regions") had all five marks occurring in the same place and was found to varying degrees in all genomic regions looked at. This phenomenon was described in a recent study and was termed a "hyperChIPable" region (Massa et al, 2021). However, this is not consistent with most other published literature which suggests H3K27ac and H3K27Me3, active and inactive marks, respectively, should not occur at the same place in the genome (Tie et al, 2009).…”

“…State 4 had a high probability of observing all 5 marks at once and was enriched in both active and inactive regions but did not show a consistent pattern in any of the regions tested. We defined State 4 as “hyperChIPable regions” as coined in a recent study ( Massa et al, 2021 ).…”

“…State 4 had a high probability of observing all 5 marks at once and was enriched in both active and inactive regions but did not show a consistent pattern in any of the regions tested. We defined State 4 as "hyperChIPable regions" as coined in a recent study (Massa et al, 2021). Analysis of tissue specific genes was hampered by the small numbers involved (Supplementary Table 4); Kidney, lung and spleen only had 14, 11, and six active genes identified as tissue specific.…”

Opportunity to improve livestock traits using 3D genomics

“…While we are making strides in the right directions and annotation of livestock genomes has improved and included some non-coding functional elements (Chanthavixay et al 2020;Halstead et al 2020;Jehl et al 2020;Massa et al 2020;Salavati et al 2020;Kern et al 2021), a centralised and maintained database of gene regulatory elements in livestock species is still lacking. For example, the Eukaryotic Promoter Database (Dreos et al 2017) contains no information on cattle promoters and it does not seem to have been updated within the last 2 years either.…”

“…Tools such as HiCPlus, iEnhancer-ECNN, and the data available in the human enhancer and promoter databases and the currently available livestock epigenomic datasets (Villar et al 2015;Massa et al 2020;Kern et al 2021) are valuable resources to livestock 3D genomics as they may offer valuable insights, given the conservation of 3D architecture among species such as human and livestock.…”