Fig 2: TEAD is the top selectively enriched motif at GSC-specific open chromatin and TEAD1 is its most highly expressed family member across GBMs a, b Homer de novo motif discovery outlines the 20 most highly enriched TF motifs at chromatin accessibility regions defined by the GSC tumor-specific (a) and developmentally shared (b) differential ATAC-seq peak analyses (motifs in bold show selective enrichment in only one peak set). The TEAD motif (with highest scores for TEAD4 and TEAD1) is the top, selectively enriched motif within differential GSC tumor-specific peaks (in red). See also Supplementary Data 1. c Bar graph of rld-normalized gene expression values for all significantly and uniquely enriched GSC tumor-specific TF motifs, generated from parallel RNA-seq data in E+GSC and E-GBM populations. TEAD1 is the only highly expressed gene (top 25th percentile), which is differentially overexpressed in E+GSCs (*p = 0.0496 for TEAD1, **p = 0.006 for ZNF416, n = 3. Bars represent mean ± SEM). d Violin plot of TEAD1-4 expression in TCGA GBM RNA-seqV2 data (n = 150) shows that TEAD1 is the most highly expressed TEAD family member, followed by TEAD2, TEAD3, and TEAD4. Expression shown is log2(normalized counts + 1), normalized as detailed in methods. p-Values from one-sided Wilcoxon matched pairs test. Bar represents median value. e Bar graph of rld-normalized gene expression values for TEAD1-4 derived from RNA-seq E + GSC data (***p < 0.001; n = 3. Bars represent mean ± SEM)

Fig 3: Expressing the PB1 domain of ARF rescues the proteasome-dependent chronic depletion of AID-tagged proteins. (A) Red genes are activated upon chronic ZNF143 depletion, blue genes are repressed, dark-gray genes are confidently unchanged and expressed at levels comparable with those of changed genes, and light-gray points represent all other genes. ZNF143 (black point) expression is unaffected. (B) Stable expression of the ARF-PB1 domain in HEK293T-TIR1 cells did not change the protein levels of ZNF143 (fifth lane) but increased the stability of ZNF143-AID (seventh lane). (C) TEAD4 levels also increase upon ARF16 expression. (D) Genes that are activated upon chronic ZNF143 depletion in A are, on average, repressed upon ARF rescue, and genes that are repressed upon chronic depletion are activated upon ARF rescue.

Fig 4: CTGF is directly regulated by YAP1 and TEADs in GCa ChIP-PCR and ChIP-qPCR revealed CTGF is a direct target of YAP1. YAP1-IP showed 2.43 times enrichment compared with non-specific IgG-IP for CTGF promoter binding affinity (**, P < 0.001). b Luciferase activities were suppressed by siYAP1 in the constructs which contained CTGF promoter sequences with different fragment size using AGS and NCI-N87 cells (**, P < 0.001). c qRT-PCR and Western blot of CTGF after YAP1 knockdown in AGS, MKN1, MKN28, and NCI-N87 cells (**, P < 0.001). d Immunoprecipitation of FLAG-YAP1 and TEAD1/4 detection in AGS cells (left panel). siTEAD1 or siTEAD4 decreased the luciferase activity after transfection with constructs containing CTGF promoter region in AGS and NCI-N87 cells (middle panel, **, P < 0.001). The Western blot analysis of CTGF, p21/p27, and p-Rb after TEAD1 and TEAD4 knockdown (right panel). e Representative IHC images of CTGF and YAP1 expression in the same GC sample (Case 43, both YAP1 and CTGF show weak expression. Case 77, YAP1 strongly accumulates in the nuclei and CTGF exhibits strong cytoplasmic staining.). Moderate/strong CTGF cytoplasmic staining showed a positive correlation with YAP1 nuclear accumulation (P < 0.001). f YAP1 and CTGF mRNA expression showed a positive correlation (upper panel, P = 0.005) but YAP1 mRNA expression was not significantly associated with TEAD4 expression (lower panel, P = 0.083) in TCGA cohort

Fig 5: ARF rescue promotes faster degradation kinetics upon addition of auxin. ZNF143 (A) and TEAD4 (B) are rapidly depleted upon auxin treatment. (C) A quantitative Western blot measuring the time-dependent degradation of ZNF143-AID in the presence of 500 µM auxin. Note that ZNF143 is chronically depleted in these cells, and a longer exposure was needed to quantify auxin-induced reduction of ZNF143-AID levels. (D) A quantitative Western blot measuring 500 µM auxin-induced ZNF143-AID degradation after ARF16-PB1 rescue. (E) We measured the intensity of ZNF143-AID bands from C and D using densitometry and fit the data using nonlinear regression and a one-phase decay equation.