Analysis of AP-1 regulation on Smad3 genome-wide DNA binding sites in resistant basal cell carcinoma

Tumor heterogeneity and lack of knowledge about resistant cell states remain a significant barrier to effective targeted cancer therapies. Basal cell carcinomas (BCCs) uniformly depend on Hedgehog (Hh)/Gli signaling for cell growth. We previously identified a nuclear myocardin-related transcription factor (nMRTF) resistance pathway that amplifies Gli1 activity, but nMRTF cell state and key factors driving its accumulation remain unknown. Epigenetic analysis of isolated nMRTF human tumor subpopulations demonstrates that cooperative AP-1 and TGFß signaling drive nMRTF activation. In this study, we find that AP-1 signaling drives changes in chromatin accessibility leading to differential Smad3 DNA binding and a transcriptional program of upstream activators of Rho, including RhoGEFs, that facilitate nMRTF activity. Overall design: Chromatin immunoprecipitation was performed on murine resistant BCC cell line ASZ001 that were serum starved and treated with 20 uM AP-1 inhibitor T-5224 or 5 ng/ml TGFB3 ligand for 24 hours. 50 million cells were used per replicate. Briefly, cells were fixed with 1% formaldehyde, lysed in modified RIPA buffer, then sonicated using Covaris B208 ultrasonicator. Immunoprecipitation was performed with anti-phospho-Smad2/3 antibody (abcam 52903). Biological duplicate ChIP-seq libraries were generated using the standard protocol for the NEBNext Ultra II DNA Library Prep Kit for Illumina, then sequenced on the Illumina Nextseq (400M) platform. Pre-immunoprecipitation total extract DNA was sequenced as input.