Investigate FOXA2, NKX2-1 and P300 regulated transcription program by RNA-seq in prostate cancer cells.
Emerging evidence suggests that the epigenetic state, including the DNA methylation, chromatin accessibility and histone modification, is substantially remodeled, leading to transcriptional reprogramming during the transformation of castration resistant adenocarcinoma (CRPC-Adeno) to neuroendocrine (CRPC-NE) lineage and LUAD to LUAD-SCLC lineage . However, the underlying mechanism is largely unknown. Here, we found FOXA2 transdifferentiates prostate adenocarcinoma away from an AR+/AR gene signature positive (AR+/ARS+) luminal cell lineage to an embryonic and neuroendocrine-lineage by inducing a population of AR+/ARS- transient cells through NKX2-1. Mechanistically, as a pioneer factor, FOXA2 primes NE lineage enhancers by inducing regional chromatin accessibility and DNA demethylation at early stage of transformation. Whereas, once the expression of NKX2-1 is induced by FOXA2, FOXA2 interacts with NKX2-1, mediates gene promoter and enhancer looping and cooperatively recruits P300/CBP complex to NE lineage enhancers, thereby activates NE lineage gene transcription. Interestingly, similar mechanism is also observed in SCLC cancer, suggesting FOXA2 and NKX2-1 are general regulators orchestrating the epigenetic landscape of NE lineage cancer. Finally, therapeutic targeting P300/CBP with CCS1477 which is in clinical trial for lethal PCa significantly impairs NEPC tumor growth. Overall design: 1. To delineate the transcription program regulated by FOXA2 during NEPC transformation, we performed bulk RNA-seq in LNCaP FOXA2 time course cells (FOXA2-D0, D2, D7, D14, D21 and D28) and 22Rv1 cells with FOXA2 overexpression for 4 weeks.  2. To examine whether FOXA2 driven embryonic and neuron lineage transcription program was mediated by NKX2-1, we performed RNA-seq in FOXA2 stable transformed LNCaP cells (LuNE) with FOXA2 or NKX2-1 KO alone, or both KO using gRNAs. 3.To determine NKX2-1 and FOXA2 driven embryonic and NEPC like transcription program whether also regulated by P300/CBP in LuNE cells,  we performed RNA-seq in LuNE cells with control,  P300, CBP alone or P300 and CBP double KD or LuNE cells treated with P300/CBP inhibitor CCS1477.