Fig. 7

KAT2B regulates NF2 expression via recruiting SP1 to the promoter region of NF2 gene. (A) Venn diagram displays the overlapping genes of the potential transcription factors (TFs) for NF2 (from SwissRegulon) and KAT2B interactors (from BioGrid). (B, C) Protein immunoprecipitation was performed to validate the TFs interacted with KAT2B. B, the proteins were immunoprecipitated by KAT2B antibody; C, the proteins were immunoprecipitated by YY1 or SP1 antibody. IgG was used as negative control. (D) GST pull-down assay detecting the direct interaction between KAT2B and SP1. (E) GC-rich motif of NF2 promoter and ChIP-qPCR primer design. The GC-rich motifs at the promoter region of NF2 were predicted by Eukaryotic Promoter Database (cut-off p value: 0.001). Arrows with p#1–4 indicate the four primer sets that were used for the ChIP-qPCR analysis. Primer pairs #2 and #3 (p#2 and #3) were used to amplify the two GC motif regions; primer pairs #1 and #4 (p#1 and #4) were used to amplify non-GC motif regions. (F) ChIP-qPCR assay to show the enrichment of SP1 in the promoter region of NF2 (n = 4). (G) ChIP-quantitative real-time PCR analyses to examine the effect of KAT2B overexpression on the binding of SP1 to NF2 gene promoter (by using NF2 ChIP-qPCR primer pair 3 (p#3)) (n = 4). Rabbit immunoglobulin G was used as negative control. (H) The level of NF2 mRNA was analyzed by quantitative real-time PCR in vector control cells or KAT2B overexpressed cells with or without SP1 depletion (n = 4). (I) Western blotting for KAT2B, SP1 and NF2 in control cells or KAT2B overexpressed cells with or without SP1 depletion. (J) Schematic presentation highlighting the mechanisms of KAT2B in CCA. KAT2B inhibits CCA cell growth via interaction with SP1 to promote the expression of NF2 and thus activate Hippo pathway, which leads to subsequent inhibition of the oncogenic YAP signaling. **P < 0.01