Gene function & annotation

Trend-Level GWAS SNPs Gain Functional Support Through Epigenomic Prioritization Using CRISPRa in Chicken

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Abstract

Genome-wide association studies (GWAS) often identify SNPs that fall below conservative genome-wide significance thresholds due to limited sample sizes, polygenic trait architectures, and small effect sizes. Nevertheless, trend-level SNPs may still hold biological relevance when interpreted in the context of functional genomic evidence. In this study, we conducted a GWAS for alanine content in Korean native chicken breast muscle and selected SNPs with -log10(P-value) ≥ 3 as trend-level candidates for downstream evaluation. Our aim was to determine whether these non-significant but suggestive loci harbor regulatory potential by integrating muscle-specific epigenomic information with CRISPR activation (CRISPRa)-based functional validation. We first analyzed chicken muscle epigenomic data produced by the Functional Annotation of Animal Genomes (FAANG) consortium, including DNase-seq, H3K27ac, H3K4me1, and H3K4me3. Overlap of these datasets with the trend-level SNPs revealed three candidates positioned within putative regulatory elements. Among them, a SNP located in the intron of ARHI2 showed a strong H3K27ac peak and chromatin signatures characteristic of an active enhancer in muscle tissue. This SNP-containing region was therefore selected for functional interrogation. To assess its regulatory capacity, the locus was activated using CRISPRa in DF1 chicken fibroblast cells. Bulk RNA sequencing following activation identified 4,307 differentially expressed genes (DEGs; FDR < 0.01), including 2,075 upregulated and 2,232 downregulated genes. Functional enrichment analysis indicated that upregulated genes were associated with oxidative phosphorylation, mitochondrial electron transport, ATP synthesis, and glutathione metabolism, reflecting enhanced oxidative and antioxidant processes. In contrast, downregulated genes were enriched in ribosome biogenesis, rRNA processing, translational initiation, and protein metabolic pathways, suggesting reduced anabolic protein synthesis. Motif scanning using FIMO revealed E-box sequences within the enhancer region, compatible with binding by bHLH-LZ transcription factors such as MITF and USF1. These TFs are known to coordinate metabolic reprogramming by promoting oxidative metabolism while attenuating ribosomal and translational programs. Overall, this study demonstrates that trend-level GWAS SNPs, when combined with muscle-specific epigenomic data, can reveal functional regulatory elements that may be overlooked under strict statistical thresholds. CRISPRa-based perturbation further enables direct assessment of the downstream transcriptional consequences of non-coding variants. Our findings highlight a regulatory element within the ARHI2 intron that influences metabolic pathways associated with alanine content in chicken breast muscle, offering an improved framework for functional interpretation of non-coding GWAS signals in livestock.

Keywords: 2026

How to Cite:

Cho, E., Han, J., Kim, T., Kim, J. & Lee, J., (2026) “Trend-Level GWAS SNPs Gain Functional Support Through Epigenomic Prioritization Using CRISPRa in Chicken”, World Congress on Genetics Applied to Livestock Production Digital Archive 2026(1): 2286714. doi: https://doi.org/10.31274/wcgalp.24101

Rights: 1