Hoxc13 polyglycine repeat gain-of-function contributes to mammalian integument evolution by altering targeted genes and interactions

Abstract Background Hoxc13 is a critical transcription factor that regulates the expression of hair keratin genes. Our previous study showed that during mammalian evolution, the Hoxc13 protein acquired a characteristic polyglycine repeat (polyG) insertion. This insertion distinguishes Hoxc13 from its non-mammalian homologs and may contribute to hair production. Methods To investigate the effect of the polyG fragment on the DNA-binding profile of Hoxc13, the normal Hoxc13-W protein and a polyG-deleted Hoxc13-S protein from cashmere goats were synthesized using an in vitro expression system. Their genomic binding profiles were compared using DNA affinity purification sequencing (DAP-seq), genome alignment, pathway enrichment, motif analysis, and molecular docking. Results Hoxc13-W and Hoxc13-S exhibited markedly different genome-wide DNA-binding capacities. Hoxc13-W identified 12,679 binding peaks, whereas Hoxc13-S identified only 3,634, with only 243 peaks shared between the two proteins. Functional enrichment analysis showed that Hoxc13-W specifically bound genes involved in key pathways regulating hair follicle morphogenesis and cycling. In contrast, Hoxc13-S targets were enriched primarily in the axon guidance pathway. Within the Wnt signaling pathway, Hoxc13-W bound 30 genes, whereas Hoxc13-S bound only 5. Motif analysis further demonstrated that the polyG fragment substantially influenced cis-regulatory motifs beyond the core Hoxc13-binding motif (5′-ATAAA-3′). Molecular docking analysis indicated that the polyG fragment altered the N-terminal structure of Hoxc13, thereby affecting its protein-protein interaction capacity. Conclusions Collectively, these findings suggest that the polyG fragment enhances the Hoxc13-mediated gene regulatory network during the evolutionary transition from non-mammalian to mammalian vertebrates and may have contributed to the evolution of mammalian hair-related phenotypic traits.