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. 2021 Apr 29;10:e62857. doi: 10.7554/eLife.62857

Figure 8. The ATAC complex is critical for the oncogenic transcriptomes of the fusion proteins.

(A) Principal component analysis showing the effect of knock-down of YEATS2 or ZZZ3 on the TAZ-CAMTA1 and YAP-TFE3 transcriptomes. (B) Genes with at least a two-fold decrease in at least two of the four conditions (YT shYEATS2, YT shZZZ3, TC shYEATS2, and TC shZZZ3); top genes most down-regulated according to aforementioned filter. N/A indicates gene was not differentially expressed in the data set. ND indicates there was no decrease in expression. (C) Extracellular matrix-related downregulated genes in part B validated by quantitative RT-PCR. (D) Scatter plot of two types of pathway enrichment evidence: probability of over-representation (pORA) and probability of accumulation (pAcc) as calculated by iPathwayGuide for SW872 YAP-TFE3 cells with knock-down of YEATS2 or ZZZ3. (E) Intersection of differentially expressed genes after knock-down of YEATS2 in SW872 YAP-TFE3 cells with open chromatin regions unique to YAP-TFE3 expressing cells (not present with empty vector). (F) Intersection of differentially expressed genes after knock-down of ZZZ3 in SW872 YAP-TFE3 cells with open chromatin regions unique to YAP-TFE3 expressing cells (not present with empty vector). (G) Working model of TAZ-CAMTA1:ATAC complex. (H) Vitronectin expression in epithelioid hemangioendothelioma (EHE) compared to epithelioid angiosarcoma (E–AS) and epithelioid hemangioma (EH). Vitronectin expression could be more focal (top example of EHE) and secreted in the ECM between neoplastic cells (demonstrated by arrow) or more diffusely expressed in the ECM (bottom example of EHE). Overall survival curves as a function of YEATS2 (I) and ZZZ3 RNA expression (J) across different histological types of sarcoma utilizing RNA-Seq expression data for The Cancer Genome Atlas (TCGA). RNA-Seq experiments in SW872 cells were performed using biological duplicates for each of the conditions. For gene expression data, the population was set to the total number of recovered genes with the mean of five counts across all samples. ATAC-Seq experiments in SW872 cells were performed using biological triplicates for each of the conditions (expression constructs). For ATAC-Seq analysis, the population was set as the total number of genes annotated across all conditions. For quantitative RT-PCR, standard deviation was calculated from fold change values for each triplicate. Error bars were used to define one standard deviation. For Kaplan-Meier curves significance was determined by Log-rank (Mantel-Cox) test. Each experiment was repeated at least twice. For all panels, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

Figure 8—source data 1. Differentially expressed genes in SW872 cells expressing TAZ-CAMTA1 or YAP-TFE3 after YEATS2 or ZZZ3 knock-down.
elife-62857-fig8-data1.xlsx (462.1KB, xlsx)

Figure 8.

Figure 8—figure supplement 1. Validation of mass spectrometry/RNAi screen.

Figure 8—figure supplement 1.

(A) Knock-down of YEATS2 in SW872 TAZ-CAMTA1 cells by qRT-PCR. (B) Soft agar assay for SW872 TAZ-CAMTA1 cells after YEATS2 knock-down. (C) Knock-down of ZZZ3 in SW872 TAZ-CAMTA1 cells evaluated by western blot. (D) Soft agar assay after ZZZ3 knock-down in SW872 TAZ-CAMTA1 cells. (E) Co-immunoprecipitation experiment (Co-IP) in HEK293 cells expressing Flag-TAZ-CAMTA1 or Flag-TAZ control. Myc-KAT14 is transiently transfected. Flag-TAZ-CAMTA1 demonstrates an enrichment for Myc-KAT14 relative to IgG control after Flag pull-down. In contrast, the Flag-TAZ control does not demonstrate enrichment of Myc-KAT14 relative to IgG control after Flag pull-down. (F) Co-IP in HEK293 cells expressing Flag-YAP-TFE3. Myc-KAT14 is transiently transfected. (G) Co-IP in SW872 cells expressing TAZ-CAMTA1. Myc-KAT14 is transiently transfected. Each experiment was repeated at least twice. For all panels, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.
Figure 8—figure supplement 2. The ATAC complex modulates expression of the MAPK signaling pathway, the PI3K signaling pathway, and extracellular matrix (ECM) proteins.

Figure 8—figure supplement 2.

(A) Knock-down of YEATS2 in YAP-TFE3 expressing SW872 cells inhibited expression of components of MAPK signaling by iPathwayGuide analysis. (B) Upregulation of PI3K-Akt signaling in SW872 cells expressing YAP-TFE3. (C) Knock-down of YEATS2 in SW872 YAP-TFE3 cells resulted in downregulation of the PI3K signaling pathway. (D) Knock-down of ZZZ3 in SW872 YAP-TFE3 cells resulted in downregulation of the PI3K signaling pathway. (E) Expression of COL1A1 and CTGF in the ECM matrix surrounding the neoplastic cells in EHE 1. Arrow shows perinuclear expression/cytoplasmic expression of CTGF. (F) Expression of FBLN5 in the cytoplasm of EHE 2.