Fig 1: Hippo pathway inhibition restricts cytokine-driven goblet cell specification, and the NuRD complex regulates SPDEF expression(A) Schematic of HBEC culture and siRNA-mediated knockdown together with cytokine media (CM) supplementation (PnemaCult-EX plus with and without IL-1a, IL-1ß, and IL-13).(B) HBECs were cultured with and without CM and YAP and/or TAZ were depleted by siRNA treatment. qPCR analysis reveals that SPDEF transcripts were minimally affected by individual knockdown of YAP or TAZ. Simultaneous knockdown of YAP/TAZ significantly increased SPDEF transcripts (minimum of n = 3; unpaired t test, ****p < 0.0001).(C) LATS1/2 were depleted by siRNA. qPCR analysis demonstrates significant increases in SPDEF and MUC5AC with cytokine treatment and significant repression following knockdown (n = 4; unpaired t test, *p < 0.05, **p < 0.01, ***p < 0.001).(D) Schematic of experimental design for small molecule inhibition of the Hippo pathway MST kinases in differentiated HBECs. Briefly, HBECs were differentiated in the ALI for 10 days and then the MST inhibitor XMU-MP-1 (10 µM) was added on an ongoing basis for an additional 10 days before the cells were processed for analysis.(E) qPCR analysis of differentiated HBECs treated with XMU-MP-1 (10 µM) demonstrates that MUC5AC and SPDEF were significantly repressed by MST inhibition and CTGF was induced (n = 3; unpaired t test, *p < 0.05, ***p < 0.001).(F and G) qPCR analysis of HBECs transfected with siRNA targeting NuRD complex components. Knockdown of (F) GATAD2A or (G) CHD4 increased SPDEF and MUC5AC expression (n = 5; unpaired t test, *p < 0.05, ***p < 0.001, ****p < 0.0001).(H) ChIP-qPCR analysis shows binding of GATAD2A and CHD4 to the SPDEF promoter in HBECs (n = 3; unpaired t test, **p < 0.01). qPCR amplification of bound chromatin was performed using the same primers as for the TEAD ChIP-qPCR shown in Figure 6B (i.e., NURD complex binding to the TEAD binding site).(I) Schematic outline of the MNase-qPCR sites proximal to the SPDEF start site (indicated by the red lines; A, B, C). Blue line represents the TEAD binding site as identified by ChIP-seq. MNase-qPCR of HBECs transfected with siCTL or YAP/TAZ targeting siRNA (siYap/Taz) collected 72 h following knockdown. Purified DNA was crosslinked and digested with MNase. Amplification by site specific primers revealed a shift of the nucleosomes away from the transcriptional start site (n = 3, *p < 0.05, **p < 0.01).For all plots, data are represented as mean ± SEM. See also Figure S7.