Fig 2: Proteomic analysis of TEAD3 identifies DNA-damage-associated proteins as TEAD-interacting partners. A, human TEAD proteins 1 to 4 contain a conserved nuclear localization signal (NLS). Protein schematic and sequence alignment of the NLS of D. melanogaster protein Scalloped to human TEAD proteins. B, schematic of the TEAD3 NLS mutant generated by mutation of seven NLS residues to asparagine. C, subcellular fractionation of MYC-WT and MYC-NLS mutant TEAD3. Cyto. = cytoplasm, NP = nucleoplasm, Chrom. = Chromatin. This is a representative image from three or more replicates. D, heatmap showing the enriched Hallmark gene sets from MSigDB (FDR q-value <0.05) for WT and NLS mutant TEAD3 interactions in both cell lines. The enriched hallmark gene sets were sorted by the q-value of Detroit WT, and the top 15 gene sets are shown in the heatmap. E, scatter plot representing the abundance (Average Sum PSMs, log2 scale) and Specificity (SAINT Log Odds Score, direction ∗ log2 scale of absolute Log Odds Score) for all identified proteins by AP-MS in the TEAD3 WT compared to negative control. Interactions with a SAINT score >0.99, average sum PSMs >10, a SAINT score >0.99, and a Bayesian false discovery rate (BFDR) < 0.001 were classified as significant interactions (red), while the others were marked as background (light gray). F, dotplot representing the PSMs for selected hits, marked in (E), per cell line and IP for each replicate. AP-MS, affinity purification mass spectrometry; PRKDC, DNA-dependent protein kinase, catalytic subunit; PSM, peptide spectral match; PARP1, poly(ADP-ribose) polymerase 1; RIF1, Rap1-interacting factor 1; TEADs, TEA domain family members 1 to 4; XRCC6, X-ray repair cross-complementing protein 6; XRCC5, X-ray repair cross-complementing protein 5; YAP, Yes-associated protein.

Fig 3: TEAD1/2 loss and YAP/TAZ loss have distinct impacts on subpallial neural progenitor lineage progression. (A) Immunostaining on a E13.5 brain section, highlighting an apical progenitor (AP), a subapical progenitor (SAP), and a basal progenitor (BP) in the MGE. (B and C) Quantification of the proportion of pH3+ cells found at different locations. (D) A schematic of proposed progenitor states during subpallial neural progenitor lineage progression. (E) Immunostaining and quantification of progenitors in the states defined by OLIG2 and ASCL1 immunosignal. N = 6 control mice with 3 from each line, N = 3 dKO mice. Areas in dashed boxes are enlarged in the images to the right. MGE, medial ganglionic eminence. LGE, lateral ganglionic eminence. Values are mean ± SEM. Each data point represents an individual animal. Two-sided unpaired t-test. Unmarked comparisons (vs. control) did not show significant difference (P > 0.05).

Fig 4: TEADs inhibit RUNX2 activity independent of YAP binding.(A) Effects of YAP on Runx2-activated 6xOSE2-luciferase activity in C3H10T1/2 cells. (B) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, YAP, and TEAD4. (C) Schematic illustration of TEAD4 and TEAD4Y429H mutation. (D) 3xSd-luciferase activity was determined in HEK-293T cells cotransfected with YAP, TEAD4, and TEAD4Y429H. (E) Coimmunoprecipitation experiments of RUNX2, TEAD4, and TEAD4Y429H in HEK-293T cells. The arrow indicated IgG heavy chain. (F) Effects of TEAD4 on RUNX2-activated 6xOSE2-luciferase activity in C3H10T1/2 cells. (G) Effects of TEAD4Y429H on RUNX2-activated 6xOSE2-luciferase activity in C3H10T1/2 cells. (H) Cells isolated from WT mice were infected with GFP or TEAD shRNAs, TEAD4, or TEAD4Y429H lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (I) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (J) Relative mRNA levels of Runx2, Alp, Col1α1, Osterix, Tead1, and Yap were quantified by RT-PCR. In (A), (B), (D), (F), (G), (I), and (J), data were presented as means ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Student’s t test.

Fig 5: Multiple TEADs are expressed in Schwann cells of WT and Tead1 cKO mice.(A) Immunostaining and quantification of TEAD1 +SCs on longitudinal sections of P22 WT and Tead1 cKO SNs. SC nuclei are identified by Sox10. All cell nuclei are marked by DAPI. n=3 mice per genotype, ****p<0.0001, Student’s unpaired t-test. Scale bar: 20 μm. (B) Western blotting of P50 WT and Tead1 cKO SN lysates with anti-TEAD1, -TEAD2, -TEAD3, -TEAD4, and -pan TEAD. TEAD expression is normalized to that of β-actin as an internal control, and WT expression is arbitrarily given the value 1. n=3 mice per genotype. ns, p>0.05; *p≤0.05, **p≤0.005, ***p≤0.001, Student’s unpaired t-test. (C) Western blotting of P50 WT and Tead1 cKO SN lysates with anti-YAP, -phospho-YAPs (Ser127 and Ser397), and -TAZ. YAP/TAZ expression is normalized to that of β-actin as an internal control, and WT expression is arbitrarily given the value 1. n=3 mice per genotype. *p≤0.05, ***p≤0.001, ****p<0.0001, Student’s unpaired t-test. (D) Immunostaining of YAP/TAZ +SCs on longitudinal sections of P50 WT and Tead1 cKO SNs. SC nuclei are identified by Sox10. All cell nuclei are marked by DAPI. n=3 mice per genotype, ns, p>0.05, Student’s unpaired t-test. Scale bar: 20 μm. Figure 1—source data 1.This zip archive contains source files for graphs in Figure 1A, B, C and D and uncropped labeled or unlabeled blots of Figure 1B and D.