Functional Dissection of TP63 Dual-Promoter Regulatory Elements Reveals Context-Dependent Responsiveness to FOXA2, NKX2-1, SOX9, and TP63-Related Inputs.

The TP63 locus generates TAp63 and ΔNp63 isoforms through alternative promoter usage, but the cis-regulatory features that confer differential promoter responsiveness remain incompletely defined. Here, we functionally dissected distal TAp63-associated and proximal ΔNp63-associated TP63 promoter regions using reporter-based assays in A549 cells. Rabbit TP63 promoter fragments were selected from distal and proximal promoter-associated regions and analyzed by serial deletion and site-directed mutagenesis. Deletion mapping identified discrete promoter intervals that positively or negatively affected reporter activity, while motif perturbation revealed promoter-context-dependent contributions of predicted FOXA1/2, NKX2-1, SOX9, and TP63-related regulatory elements. Endogenous ChIP-qPCR at corresponding human TP63 promoter-associated regions provided local enrichment evidence for selected transcription-factor-associated signals, and FOXA2 or SOX9 depletion shifted endogenous TAp63 and ΔNp63 protein abundance. These findings identify candidate TP63 promoter intervals and transcription-factor-responsive motifs that differentially affect dual-promoter reporter output. The study provides a promoter-level framework for investigating how TP63 promoter responsiveness is influenced by promoter architecture, motif composition, and cellular regulatory background within a heterologous reporter context, while highlighting the need for future validation in more physiological human epithelial systems.