Convergence of YAP/TAZ, TEAD and P63 activity directs premalignant lung gene expression [ChIP-seq]

Bronchial premalignant lesions (PMLs) are composed of expanding bronchial basal cells that can progress to lung squamous cell carcinoma (LUSC) by evading immune responses. Despite ongoing efforts that have mapped gene expression and cell diversity across bronchial PML pathologies, signaling and transcriptional events driving malignancy are poorly understood. Evidence has suggested key roles for the Hippo pathway effectors YAP and TAZ and associated TEAD and TP63 transcription factor families in bronchial basal cell biology and LUSC. In this study we mapped target genes regulated by these factors by combined RNA-seq and ChIP-seq in primary human bronchial epithelial cells, revealing a converged transcriptional network that is strongly associated with bronchial PML progression. Our observations suggest that YAP/TAZ-TEAD-TP63 associate to cooperatively promote basal cell proliferation and repress signals associated with interferon responses and immune cell communication. Directly repressed targets include MHC Class II factors expressed in bronchial epithelium that correlate with adaptive immune Th1 cell activities known to contribute to the immunosuppressive microenvironment in lung cancers. Our findings provide a molecular mechanism for gene regulation in progressive PMLs that contributes to immune evasion, offering potential new avenues for lung cancer interception. Overall design: HBECs for ChIP were cultured in Pneumocult EX Plus media and cross-linked in 1mM EGS in PBS for 30min followed by a 1% formaldehyde treatment for 10 min. Fixation was subsequently neutralized with 0.125M glycine in PBS. Harvested chromatin was isolated as single samples from each patient line, sonicated using the Bioruptor UCD- 200 and the incubated with the following antibodies at 4 °rnight: Rabbit anti-Yap (Abcam, Cat# ab52771, 3ug), Rabbit anti-TEAD (AvivaSysBio, Cat# ARP38276, 1ug), and Mouse anti-P63 (Biocare # CM163, 5ug). Immunoprecipitated complexes were collected by Protein A/G Magnetic beads (Pierce, 8802). Samples were washed with low salt buffer (20mM Tris, 140mM NaCl, 1mM EDTA, 0.1% NaDeoxycholate, 0.1% SDS, 1% Triton X-100), followed by a high salt buffer (20mM Tris, 500mM NaCl, 1mM EDTA, 0.5% NaDeoxycholate, 1% Triton X-100), and a LiCl buffer (20mM Tris, 1mM EDTA, 0.1% NaDeoxycholate, 1% Triton X-100, 250mM LiCl). Chromatin was de-crosslinked overnight at 65C and purified using the Qiaquick PCR purification kit (Qiagen, 28104). For ChIP-seq, the purified DNA was ligated to specific adaptors and sequenced using DNB-seq, performed by BGI, to a depth of 40 million reads.