Figure 8.

YAP interacts with NFIB, KLF5, and NKX2-1 to regulate gene expression

(A) Immunoprecipitation assay of HBEC3 cells co-expressing (S127A)YAP-FLAG and GFP-NFIB-HA constructs demonstrating YAP and NFIB co-precipitate.

(B) HBEC3 cells co-expressing (S127A)YAP, KLF5, and NKX2-1 activate KLF5 promoter luciferase activity.

(C) HBEC3 cells co-transfected with (S127A)YAP, NKX2-1, and KLF5 activate AGER promoter luciferase.

(D) HBEC3 cells co-expressing empty pCMV vector, KLF5, NKX2-1, NFIB, and (S127A)YAP increase AGER RNA assessed by qRT-PCR.

(E) AGER RNA was measured in HBEC3 cells co-transfected with pCMV empty vector (control), (S127A)YAP, NFIB, KLF5, and NKX2-1 with siRNAs targeting TEADs 1-4. AGER induction is partially inhibited by TEAD siRNAs. Graphs are representative of multiple (N > 3) experiments.

(F) A schematic of the AGER promoter and luciferase assay with locations of predicted TEAD, NKX2-1, and NFIB binding sites that were mutated to assess DNA binding of respective transcription factors to the AGER promoter.

(G) HBEC3 cells expressing NKX2-1, KLF5, and YAP in the presence of AGER luciferase constructs with five site mutations in predicted binding sites of TEAD (TEADΔ), NKX2-1 (NKX2-1Δ) or NFIB (NFIBΔ). Altering the predicted binding sites of NKX2-1 (p < 0.05), TEAD (p < 0.0001), or NFIB (p < 0.0001) significantly reduced AGER promoter activation by YAP, KLF5, and NKX2-1.

(H) A schematic of NFIB, KLF5, NKX2-1, YAP, and TEAD interacting on the AGER promoter to induce AGER transcription. See Figure S5 for further information and Figure S6 for full size co-immunoprecipitation blots. ∗Indicates p<0.05 as determined by Two-way ANOVA followed by Sidak's multiple comparisons test. ∗∗p<0.001, ∗∗∗p<0.0001.