Transcriptomic Analysis Identifies CDKN2A-Associated Transcriptional Alterations in Primary Melanocytes of Lueyang Black-Boned Chicken.

Pronounced melanin enrichment characterizes the Lueyang black-boned chicken, yet the transcriptional basis underlying this pigmentation phenotype remains incompletely understood. We established a standardized in vitro culture system for primary avian melanocytes to investigate CDKN2A-associated transcriptional changes related to melanocyte cellular states and pigmentation-related gene expression. Primary melanocytes were isolated and characterized by morphology, ferrous sulfate staining, and TRP2 immunofluorescence, with passages P2-P4 showing relatively stable cellular characteristics. Using CDKN2A overexpression and knockdown models combined with RNA-seq analysis, we identified transcriptional alterations associated with CDKN2A modulation. Notably, CDKN2A overexpression was associated with increased expression of cell cycle-related genes (e.g., CDKN1A) and melanogenesis-associated transcriptional markers, particularly MITF, suggesting an association between CDKN2A expression and pigment-related transcriptional programs. Conversely, CDKN2A knockdown produced opposite expression patterns. Gene set enrichment analysis (GSEA) further indicated that high CDKN2A expression was associated with enrichment of melanogenesis and MAPK signalling pathways, whereas CDKN2A knockdown was associated with enrichment of Wnt signalling pathways. By minimizing potential in vivo paracrine effects, this in vitro model suggested that CDKN2A-associated transcriptional changes might reflect alterations related to cell cycle status and melanocyte cellular states. Although this study provided transcript-level evidence, direct functional assays of melanin production were not performed; therefore, these findings should be interpreted as correlative rather than definitive evidence of CDKN2A-associated pigmentation regulation. These findings provide a transcriptomic resource for investigating the relationship between cell-state regulation and pigmentation-associated programs in avian melanocytes.