VGLL2 and TEAD1 fusion proteins drive YAP/TAZ-independent tumorigenesis by engaging p300

Susu Guo
Xiaodi Hu
Jennifer L Cotton
Lifang Ma
Qi Li
Jiangtao Cui
Yongjie Wang
Ritesh P Thakare
Zhipeng Tao
Y Tony Ip
Xu Wu
Jiayi Wang author has email address
Junhao Mao author has email address

Department of Clinical Laboratory, Shanghai Chest Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, United States
Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, United States
Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, United States

https://doi.org/10.7554/eLife.98386.2

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Figures and data

VGLL2-NCOA2 and TEAD1-NCOA2 Induce TEAD-Mediated Transcriptional Activation.
A. Schematic representation of protein structure of VGLL2, TEAD1, NCOA2, VGLL2-NCOA2 and TEAD1-NCOA2. Tondu motif (TDU), TEA DNA binding domain (TEA), YAP binding domain (YBD), basic Helix-Loop-Helix (bHLH), Per-Arnt-Sim domain (PAS), nuclear receptor interaction domain (NID), transcriptional activation domain (TAD). Arrows point to the break points.
B. Immunoblot analysis of YAP^5SA, VGLL2-NCOA2, TEAD1-NCOA2, TAZ^4SA, TEAD1, VGLL2 and NCOA2 in HEK293T cells transfected with the expression constructs carrying the HA tag. The figure shows the representative results of three biological replicates.
C. YAP^5SA, VGLL2-NCOA2, TEAD1-NCOA2, TAZ^4SA, TEAD1, VGLL2, and NCOA2 induce transcriptional activation of TBS (TEAD binding site)-luciferase reporter (TBS-Luc) in HEK293T cells. n = 3. ****, p < 0.0001.
D. Heatmap showing expression levels of the core genes including CTGF, CYR61, ANKRD1 and AMOTL2 significantly regulated in HEK293T cells expressing YAP^5SA, VGLL2-NCOA2 and TEAD1-NCOA2. n=3
E. mRNA levels of CTGF, ANKRD1 and CYR61 in HEK293T cells expressing YAP^5SA, VGLL2-NCOA2, TEAD1-NCOA2, TEAD1, VGLL2 or NCOA2. n=3; ****, p < 0.0001. NS, no significance.

VGLL2-NCOA2 regulates TEAD-dependent reporter activity.
A. Schematic representation of the exons and breaking points of the VGLL2, TEAD1 and NCOA2 genes involved in generating VGLL2::NCOA2 and TEAD1::NCOA2 gene arrangement.
B. EdU staining showing cell proliferation of HEK293T cells transfected with VGLL2-NCOA2 or TEAD1-NCOA2. Bar chart showing the percentage of EdU-positive cells. Scale bars, 200 μm, n=3; **, p < 0.01.
C. Immunoblot analysis of VGLL2-NCOA2, VGLL2-NCOA2^ΔVGLL2 and VGLL2-NCOA2^ΔNCOA2 in HEK293T cells transfected with the expression constructs carrying the V5 tag.
D. TBS-Luc reporter activity in HEK293T cells expressing VGLL2-NCOA2, VGLL2-NCOA2^ΔVGLL2, and VGLL2-NCOA2^ΔNCOA2. n = 3. ****, p < 0.0001.
E. Immunoblot analysis of VGLL2-NCOA2-V5 in HEK293T cells transfected with different amount of expression constructs.
F. VGLL2-NCOA2 promotes the activation of TBS-Luc reporter in dose-dependent manner. n = 3. ***, p < 0.001; ****, p < 0.0001.
G-H. Relative mRNA levels of CTGF, ANKRD1, and CYR61 in HEK293T cells expressing VGLL2-NCOA2 (G) or TEAD1-NCOA2 (H) with HA (5’ end), FLAG (3’ end) or V5 (3’ end) tag. n=3; NS, no significance.

VGLL2-NCOA2 and TEAD1-NCOA2-induced transcription does not require YAP and TAZ.
A. Co-IP assay showing VGLL2-NCOA2 binding to TEAD1 but not YAP^5SA. YAP^5SA-Flag or TEAD1-Flag was co-expressed with VGLL2-NCOA2-HA in HEK293T cells and immunoprecipitated with an anti-HA antibody.
B. VGLL2-NCOA2 binds to endogenous TEAD but not YAP/TAZ. Endogenous YAP/TAZ and TEAD proteins in HEK293T cells were detected by anti-YAP/TAZ and panTEAD antibodies, respectively.
C. Co-IP assay showing endogenous YAP/TAZ binding to TEAD1 but not TEAD1-NCOA2. TEAD1-Flag or TEAD1-NCOA2-Flag was expressed in HEK293T cells and immunoprecipitated with an anti-Flag antibody. Endogenous YAP/TAZ proteins were detected by anti-YAP/TAZ antibodies.
D. The activity of TBS-Luc reporter in HEK293T cells expressing YAP^5SA, VGLL2-NCOA2, or TEAD1-NCOA2, with TEAD inhibitor CP1 (5 μM) treatment or co-expression of TEAD-ENR repressor construct. n=3; ****, p < 0.0001. NS, no significance.
E. Schematic representation of TEAD-ENR. TEA DNA-binding domain (TEA) and Engrailed repressor domain (ENR).
F. Immunoblot analysis of TEAD-ENR expression in HEK293T cells.
G. Co-IP assay showing YAP/TAZ were not essential for VGLL2-NCOA2 binding to endogenous TEAD. VGLL2-NCOA2-HA was expressed in HEK293T cells with or without YAP/TAZ knockdown and immunoprecipitated with an anti-HA antibody.
H. Relative mRNA levels of CTGF, ANKRD1, and CYR61 in HEK293T cells with YAP/TAZ knockdown and expressing VGLL2-NCOA2 or TEAD1-NCOA2. n=3; ****, p < 0.0001.

Characterization of VGLL2-NCOA2- and YAP-controlled transcriptional and chromatin landscapes.
A. Intersection of ATAC-seq (n=2), RNA-seq (n=3), and CUT&RUN (n=2) datasets in HEK293T cells expressing VGLL2-NCOA2 or YAP^5SA.
B. Venn diagrams showing the overlaps of ATAC-seq peaks, CUT&RUN peaks, and differentially regulated genes from RNA-seq in HEK293T cells expressing VGLL2-NCOA2 or YAP^5SA.
C. KEGG pathway enrichment analysis of ATAC-seq peaks identified in HEK293T cells expressing VGLL2-NCOA2 or YAP^5SA. The "Hippo signaling pathway" is highlighted in red.
D. Distribution of CUT&RUN binding sites for VGLL2-NCOA2 and YAP^5SA.
E. Motif enrichment analysis of VGLL2-NCOA2 and YAP^5SA CUT&RUN Peaks.
F. Genomic tracks showing VGLL2-NCOA2 and YAP^5SA occupancy at the CTGF, ANKRD1, and CYR61 loci.

Analysis of VGLL2-NCOA2, TEAD1-NCOA2 and YAP^5SA -induced transcriptomes.
A. Volcano maps of RNA-seq data sets of HEK293T cells expressing VGLL2-NCOA2, TEAD1-NCOA2, or YAP^5SA. Red dots represent upregulated mRNAs. Blue dots represent downregulated mRNAs. p < 0.05, Log₂FoldChange > 1 or < -1.
B. Veen diagram showing the overlaps of differentially regulated genes identified by RNA-seq in HEK293T cells expressing VGLL2-NCOA2, TEAD1-NCOA2, or YAP^5SA.
C. KEGG pathway enrichment analysis of differentially regulated genes identified by RNA-seq in HEK293T cells expressing VGLL2-NCOA2, TEAD1-NCOA2 or YAP^5SA. “Hippo signaling pathway” is highlighted in red.

ATAC-seq and CUT&RUN data characterization in VGLL2-NCOA2 and YAP^5SA-expressing cells.
A. Heatmaps of TEAD-motif containing ATAC-seq peaks in HEK293T cells expressing VGLL2-NCOA2 or YAP^5SA.
B. Distribution of ATAC-seq peaks in HEK293T cells expressing VGLL2-NCOA2 or YAP^5SA. For VGLL2-NCOA2 ATAC-seq peaks, Promoter (<=1kb) 50.57%, Promoter (1−2kb) 6.59%, 5’ UTR 0.13%, 3’ UTR 1.44%, Exon 1.52%, Intron 18.39%, and Distal and Intergenic 21.35%. For YAP^5SA ATAC-seq peaks, Promoter (<=1kb) 51.4%, Promoter (1−2kb) 6.89%, 5’ UTR 0.13%, 3’ UTR 1.45%, Exon 1.58%, Intron 17.28%, and Distal and Intergenic 21.26%. n=2.
C. VGLL2-NCOA2 promotes chromatin accessibility. Scatter diagrams of ATAC-seq peaks show more up-regulated (red) than downregulated (blue) in HEK293T cells transfected with VGLL2-NCOA2 or YAP^5SA.
D. Heatmaps of VGLL2-NCOA2 and YAP^5SA CUT&RUN peaks.
E. VGLL2-NCOA2 and YAP genomic occupancy. Scatter diagrams showing more up-regulated CUT&RUN peaks (red) than down-regulated CUT&RUN peaks (blue) of VGLL2-NCOA2 and YAP^5SA.
F. KEGG pathway enrichment analysis of VGLL2-NCOA2 and YAP^5SA CUT&RUN peaks. “Hippo signaling pathway” is highlighted in red.

VGLL2-NCOA2 and TEAD1-NCOA2 engage p300 epigenetic regulators.
A. Diagram showing the BioID proteomic analyses of BirA*-VGLL2-NCOA2, BirA*-TEAD1-NCOA2, BirA*-YAP^5SA, and BirA*-TAZ^4SA.
B. Co-IP assay showing endogenous p300 binding to VGLL2-NCOA2 and TEAD1-NCOA2 but not YAP^5SA.
C. KEGG enrichment analysis of p300 CUT&RUN peaks in control HEK293T cells and HEK293T cells expressing VGLL2-NCOA2 or TEAD1-NCOA2. The "Hippo signaling pathway" is highlighted in red.
D. Motif enrichment analysis of p300 CUT&RUN peaks in control HEK293T cells and HEK293T cells expressing VGLL2-NCOA2 or TEAD1-NCOA2.
E. Genomic tracks showing p300 occupancy at the CYR61, ANKRD1, CTGF, and CREB3 loci in control HEK293T cells and HEK293T cells expressing VGLL2-NCOA2 or TEAD1-NCOA2.

p300 is required for VGLL2-NCOA2- and TEAD1-NCOA2-induced tumorigenesis in vitro.
A. Co-IP assay showing the NCOA2 fusion part of VGLL2-NCOA2 was essential for p300 binding. VGLL2-NCOA2-V5, VGLL2-NCOA2^ΔNCOA2-V5 and VGLL2-NCOA2^ΔVGLL2-V5 was expressed in HEK293T cells and immunoprecipitated with an anti-V5 antibody. Endogenous p300 proteins were detected by anti-p300 antibody.
B-D. mRNA levels of Ctgf, Ankrd1, and Cyr61 in C2C12 cells expressing YAP^5SA, VGLL2-NCOA2, or TEAD1-NCOA2 with or without treatment of A485 (5 μM). n=3; ****, p < 0.0001. NS, no significance.
E. Representative image of colony formation of C2C12 cells expressing YAP^5SA, VGLL2-NCOA2, or TEAD1-NCOA2 with or without treatment of A485 (5 μM) for 2 weeks. Scale bars, 100 μm.
F-G. Colony size (F) and number of colonies (G) formed by C2C12 cells expressing YAP^5SA, VGLL2-NCOA2, or TEAD1-NCOA2 with or without treatment of A485 (5 μM). **, p < 0.01. ****, p < 0.0001. NS, no significance.

p300 is essential for VGLL2-NCOA2- and TEAD1-NCOA2-induced tumorigenesis in vivo.
A. Representative H&E and IHC staining of Desmin and Ki67 in C2C12-control allograft, C2C12-VGLL2-NCOA2 tumor allograft, and C2C12-TEAD1-NCOA2 tumor allograft. Scale bars, 200 μm.
B. Immunoblot analysis of VGLL2-NCOA2-FLAG and TEAD1-NCOA2-FLAG expression in C2C12 cells, detected by an anti-FLAG antibody.
C-D. Allograft leg volume of C2C12-VGLL2-NCOA2 (C) and C2C12-TEAD1-NCOA2 (D) after intramuscular injection into the leg of Nude mice with or without intraperitoneal injection of A485 (100 mg/kg). The error bars represent the mean leg volume ± SEM. n = 6. ****, p < 0.0001.
E. Representative H&E and IHC staining of Ki67 in C2C12-VGLL2-NCOA2 and C2C12-TEAD1-NCOA2 tumor allografts with or without A485 (100 mg/kg) treatment. Scale bars, 200 μm.
F. Percentage of Ki67-positive cells in Figure 6E. n=6; ***, p < 0.001.
G-H. mRNA levels of Ctgf, Ankrd1, and Cyr61 in C2C12-VGLL2-NCOA2 and C2C12-TEAD1-NCOA2 tumor allografts with or without A485 (100 mg/kg) treatment. **, p < 0.01. ***, p < 0.001. ****, p < 0.0001.

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