A tandem repeat in TCF25 intron 1 is associated with MC1R activation through dual mechanisms of transcriptional derepression and chromatin looping underlying black plumage in chickens.

Abstract

Plumage pigmentation in chickens offers important insights into the genetic basis of phenotypic diversification. Although melanocortin-1 receptor gene (MC1R) is a central regulator of melanogenesis, the regulatory mechanisms contributing to extended black plumage remain incompletely understood. In this study, we integrated genome-wide association analysis (GWAS) in an F₂ resource population with multi-breed validation (n = 153) cohorts to identify a significant locus on chromosome 11 encompassing MC1R (top SNP P = 1.95 × 10⁻18, genome-wide threshold: 2.85 × 10⁻⁹). Common SNPs in this region did not fully segregate with black plumage, suggesting that non-coding or structural variation may contribute to the phenotype. PacBio HiFi sequencing (n = 6 per pool) and direct genotyping identified a 28-bp tandem repeat in the first intron of TCF25. The 4-copy allele completely co-segregated with black plumage in the populations examined (P = 3.57 × 10⁻105), making it the leading candidate variant associated with this trait. Functional and regulatory analyzes suggested two potential mechanisms linking this repeat-containing region to MC1R regulation. First, reporter assays showed copy-number-dependent regulatory activity of the tandem repeat region, and black-feathered individuals carrying the 4-copy allele exhibited reduced TCF25 expression and elevated MC1R expression in feather bulbs (n = 6). TCF25 overexpression in DF1 cells reduced MC1R and downstream melanogenic gene expression (n = 8), supporting a potential repressive role of TCF25 in the melanogenic pathway. Second, the assay for transposase-accessible chromatin with sequencing (ATAC-seq) and high-throughput chromosome conformation capture (Hi-C) analyzes (each performed in 2 biological replicates) revealed accessible regulatory elements and a black-associated chromatin interaction near the MC1R locus. Candidate accessible regions within this interaction domain showed enhancer activity in reporter assays, suggesting that locus-specific chromatin organization may contribute to enhanced MC1R transcription. Together, these results support a model in which a TCF25 intronic tandem repeat may contribute to black plumage through coordinated changes in TCF25 expression, MC1R transcription, and local chromatin interactions. This study highlights how non-coding structural variation may contribute to adaptive pigmentation traits in chickens.