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. 2021 May 20:12:628849.
doi: 10.3389/fgene.2021.628849. eCollection 2021.

Characterizing Genetic Regulatory Elements in Ovine Tissues

Kimberly M Davenport  1 , Alisha T Massa  2 , Suraj Bhattarai  3 , Stephanie D McKay  3 , Michelle R Mousel  4   5 , Maria K Herndon  2 , Stephen N White  2   4   6 , Noelle E Cockett  7 , Timothy P L Smith  8 , Brenda M Murdoch  1   6 ; Ovine FAANG Project Consortium
Affiliations

Characterizing Genetic Regulatory Elements in Ovine Tissues

Kimberly M Davenport et al. Front Genet. .

Abstract

The Ovine Functional Annotation of Animal Genomes (FAANG) project, part of the broader livestock species FAANG initiative, aims to identify and characterize gene regulatory elements in domestic sheep. Regulatory element annotation is essential for identifying genetic variants that affect health and production traits in this important agricultural species, as greater than 90% of variants underlying genetic effects are estimated to lie outside of transcribed regions. Histone modifications that distinguish active or repressed chromatin states, CTCF binding, and DNA methylation were used to characterize regulatory elements in liver, spleen, and cerebellum tissues from four yearling sheep. Chromatin immunoprecipitation with sequencing (ChIP-seq) was performed for H3K4me3, H3K27ac, H3K4me1, H3K27me3, and CTCF. Nine chromatin states including active promoters, active enhancers, poised enhancers, repressed enhancers, and insulators were characterized in each tissue using ChromHMM. Whole-genome bisulfite sequencing (WGBS) was performed to determine the complement of whole-genome DNA methylation with the ChIP-seq data. Hypermethylated and hypomethylated regions were identified across tissues, and these locations were compared with chromatin states to better distinguish and validate regulatory elements in these tissues. Interestingly, chromatin states with the poised enhancer mark H3K4me1 in the spleen and cerebellum and CTCF in the liver displayed the greatest number of hypermethylated sites. Not surprisingly, active enhancers in the liver and spleen, and promoters in the cerebellum, displayed the greatest number of hypomethylated sites. Overall, chromatin states defined by histone marks and CTCF occupied approximately 22% of the genome in all three tissues. Furthermore, the liver and spleen displayed in common the greatest percent of active promoter (65%) and active enhancer (81%) states, and the liver and cerebellum displayed in common the greatest percent of poised enhancer (53%), repressed enhancer (68%), hypermethylated sites (75%), and hypomethylated sites (73%). In addition, both known and de novo CTCF-binding motifs were identified in all three tissues, with the highest number of unique motifs identified in the cerebellum. In summary, this study has identified the regulatory regions of genes in three tissues that play key roles in defining health and economically important traits and has set the precedent for the characterization of regulatory elements in ovine tissues using the Rambouillet reference genome.

Keywords: ChIP-seq; FAANG; WGBS; epigenetics; functional genomics; histone modifications; methylation; sheep.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The percent of the total number of peaks normalized per Mb on each chromosome for (A) H3K4me3, (B) H3K27ac, (C) H3K4me1, (D) H3K27me3, and (E) CTCF averaged from all four animals (F1, F2, M1, and M2).
FIGURE 2
FIGURE 2
Signal of H3K4me3 ChIP-seq peaks 2 kb upstream and downstream of transcription start sites (TSS) identified by CAGE assays. (A) Liver H3K4me3 signal (from F1, M1, and M2 consensus peaks) near TSS annotated in the reference genome, (B) spleen H3K4me3 signal (from F2, M1, and M2 consensus peaks) near annotated transcription start sites (TSS), and (C) cerebellum H3K4me3 signal (from F1, M1, and M2 consensus peaks) near annotated TSS.
FIGURE 3
FIGURE 3
Integrative genomics viewer (IGV) screenshot of sequence pileup normalized with the input control for active and repressive histone marks and DNA methylation in two representative samples (M1 and M2) for (A) positive control Albumin (ALB) gene in the liver, (B) positive control Solute carrier family 11 member 1 (SLC11A1) in the spleen, (C) positive control Paired box 6 (PAX6) in the cerebellum, and (D) negative control REC8 gene (REC8) in all three tissues.
FIGURE 4
FIGURE 4
Principal component analysis plot based on CG methylation. Four animals are labeled as F1, F2, M1, and M2. The cerebellum, liver, and spleen samples are labeled as C, L, and S, respectively.
FIGURE 5
FIGURE 5
(A) Methylation level at CG compared with non-CG sites in the liver, spleen, and cerebellum and (B) methylation level at non-CG (CHG and CHH) sites in each tissue enlarged.
FIGURE 6
FIGURE 6
Chromatin state description and ChromHMM heatmap with histone mark signal overlap consensus from M1 and M2 compared with the number of hypermethylated regions and hypomethylated region consensus per Mb for M1 and M2 for the (A) liver, (B) spleen, and (C) cerebellum.
FIGURE 7
FIGURE 7
Percent of the genome in the liver, spleen, and cerebellum (from M1 and M2) assigned to each category of quiescent/low (gray), CTCF (black), repressed enhancer (blue), poised enhancer (green), active enhancer (gold), and promoter (red) depicted visually in panel (A) the bar graph and numerically in panel (B) the table.
FIGURE 8
FIGURE 8
Percent of overlapping promoter (red), active enhancer (gray), poised enhancer (green), and repressed enhancer (blue) chromatin state categories and hypermethylated (purple) and hypomethylated (orange) regions between the liver, spleen, and cerebellum tissues of the consensus categories from M1 and M2. The total number of chromatin states for each tissue is displayed in black horizontal bars.
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References

    1. Afgan E., Baker D., Batut B., van den Beek M., Bouvier D., Čech M., et al. (2018). The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 46 537–544. 10.1093/nar/gky379 - DOI - PMC - PubMed
    1. Albert F. W., Kruglyak L. (2015). The role of regulatory variation in complex traits and disease. Nat. Rev. Genet. 16 197–212. 10.1038/nrg3891 - DOI - PubMed
    1. Al-Mamun H. A., Clark S. A., Kwan P., Gondro C. (2015). Genome-wide linkage disequilibrium and genetic diversity in five populations of Australian domestic sheep. Genet. Sel. Evol. 47:90. 10.1186/s12711-015-0169-6 - DOI - PMC - PubMed
    1. Allan J., Fraser R. M., Owens-Hughes T., Keszenman-Pereyra D. (2012). Micrococcal nuclease does not substantially bias nucleosome mapping. J. Mol. Biol. 417 152–164. 10.1016/j.jmb.2012.01.043 - DOI - PMC - PubMed
    1. Andersson L., Archibald A. L., Bottema C. D., Brauning R., Burgess S. C., Burt D. W., et al. (2015). Coordinated international action to accelerate genome-to-phenome with FAANG, the functional annotation of animal genomes project. Gen. Biol. 16:57. 10.1186/s13059-015-0622-4 - DOI - PMC - PubMed

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