EWS-FLI1 regulates a transcriptional program in cooperation with Foxq1 in mouse Ewing sarcoma

Abstract

EWS-FLI1 constitutes an oncogenic transcription factor that plays key roles in Ewing sarcoma development and maintenance. We have recently succeeded in generating an ex vivo mouse model for Ewing sarcoma by introducing EWS-FLI1 into embryonic osteochondrogenic progenitors. The model well recapitulates the biological characteristics, small round cell morphology, and gene expression profiles of human Ewing sarcoma. Here, we clarified the global DNA binding properties of EWS-FLI1 in mouse Ewing sarcoma. GGAA microsatellites were found to serve as binding sites of EWS-FLI1 albeit with less frequency than that in human Ewing sarcoma; moreover, genomic distribution was not conserved between human and mouse. Nevertheless, EWS-FLI1 binding sites within GGAA microsatellites were frequently associated with the histone H3K27Ac enhancer mark, suggesting that EWS-FLI1 could affect global gene expression by binding its target sites. In particular, the Fox transcription factor binding motif was frequently observed within EWS-FLI1 peaks and Foxq1 was identified as the cooperative partner that interacts with the EWS portion of EWS-FLI1. Trib1 and Nrg1 were demonstrated as target genes that are co-regulated by EWS-FLI1 and Foxq1, and are important for cell proliferation and survival of Ewing sarcoma. Collectively, our findings present novel aspects of EWS-FLI1 function as well as the importance of GGAA microsatellites.

Keywords: EWS-FLI1; Ewing sarcoma; Foxq1; GGAA microsatellite; Trib1.

Figure 1

Global EWS‐FLI1 binding in mouse Ewing sarcoma. A, Histology of mouse Ewing sarcoma, ES49, used for ChIP‐Seq analysis. Hematoxylin and eosin staining (top). FLAG‐tagged EWS‐FLI1 expression was detected by western blotting. CIC‐DUX4 sarcoma (CDS4)17 was used as a negative control (bottom). B, Global distribution of EWS‐FLI1‐binding sites in relation to known genes. C, Distribution of EWS‐FLI1‐binding peaks and histone H3K27Ac peaks in relation to transcriptional start sites (TSS) of known genes. D, Density plots of signal intensities of EWS‐FLI1 and H3K27Ac peaks from TSS. E, Total H3K27Ac ChIP‐Seq signal in units of reads per million in enhancer regions for all enhancers in the ES49 cell line. Enhancers are ranked by increasing H3K27Ac ChIP‐Seq signal. F, AME suite motif analysis carried out on EWS‐FLI1 binding sites. Ets binding motifs (Elf3 and ELF5) and GGAA microsatellites (EWS‐FLI1) were efficiently detected. G, EWS‐FLI1 binds to GGAA microsatellites at Pvt1 and Sdk1 loci. H3K27Ac signals accompanying these binding sites are highlighted in yellow. Arrows indicate the transcriptional orientation of each gene. H, Venn diagrams for EWS‐FLI1 (FLAG) and H3K27Ac ChIP‐Seq signals and up‐ or downregulated genes depicted by microarray analysis

Figure 2

Detection of FOX binding motifs and upregulation of Foxq1 in Ewing sarcoma. A, AME suite motif analysis identification of enrichment of FOX binding motifs within EWS‐FLI1‐binding peaks. B, Heatmap of expression for 44 murine Fox family genes in mouse Ewing sarcoma (mES) and embryonic superficial zone (eSZ).5 Six Fox family genes are highlighted in the box with fold changes of expression compared to eSZ. C, Growth suppression of ES49 cells by siRNA‐mediated gene silencing (top). Data represent the means ± SEM of 3 independent experiments (*P < .01). Efficiencies of gene silencing were assessed as relative gene expression to Gapdh mRNA by real‐time qPCR (bottom). Data represent the means ± SEM of 3 independent experiments (*P < .02, **P < .001). D, Expression of FOXQ1,FOXF1, and FOXB1 in human sarcoma. Gene expression levels were compared on microarray data obtained from the Gene Expression Omnibus (GEO) database. ES, Ewing sarcoma; ASPS, alveolar soft part sarcoma; OS, osteosarcoma; RMS, rhabdomyosarcoma; SS, synovial sarcoma. Numbers of each sarcoma case examined are indicated. E, Expression of FOXQ1,FOXF1, and FOXB1 in human sarcoma cell lines. U937, monocytic leukemia; KAS, clear cell sarcoma; EWS, Ewing sarcoma with EWS‐FLI1; KH, Ewing sarcoma with EWS‐ERG. Normalized gene expression levels to GAPDH mRNA are indicated as the average of normalized intensity values obtained by real‐time qPCR. F, Growth suppression of EWS human Ewing sarcoma cells by FOXQ1 silencing (left). Efficiency of gene silencing was assessed as relative gene expression to Gapdh mRNA by real‐time qPCR (center). Data represent the means ± SEM of 3 independent experiments (*P < .02, **P < .001). Efficiency of FOXQ1 silencing was confirmed by western blotting (right)

Figure 3

EWS‐FLI1 and Foxq1 coregulate target genes. A, EWS‐FLI1 binding peaks at Trib1 (top) and Nrg1 (bottom) loci. Sequences around the peaks are shown in yellow boxes and GGAA microsatellites and FOX binding motifs are indicated in red or blue, respectively. Arrows indicate transcription orientation of each gene. B, Luciferase reporter assay. The DNA fragments indicated in (A) were introduced into the pGL3 promoter vector. Relative luciferase activities are shown in the presence or absence of Foxq1 and/or EWS‐FLI1 (EF) (left and center). Data represent the means ± SEM of 3 independent experiments (*P < .002, **P < .001). Protein expression of Foxq1 and EWS‐FLI1 is shown (right). C, Effect of Foxq1 or EWS‐FLI1 silencing on Trib1 and Nrg1 expression in mouse Ewing sarcoma as detected by real‐time qPCR. Expression levels were normalized to Gapdh mRNA. Data represent the means ± SEM of 3 independent experiments (*P < .002, **P < .001). D, Effect of FOXQ1 or EWS‐FLI1 silencing on TRIB1 and NRG1 in human Ewing sarcoma as detected by real‐time qPCR. Expression levels were normalized to GAPDH mRNA. Data represent the means ± SEM of 3 independent experiments (*P < .05, **P < .01)

Figure 4

EWS‐FLI1 interacts with Foxq1 through its EWS region. A, ES49 cells were transiently transfected with HA‐tagged Foxq1. Cell lysates were immunoprecipitated with an anti‐HA monoclonal antibody and immunoblotted with rabbit polyclonal anti‐FLAG or anti‐HA antibodies (left). Expression of HA‐tagged Foxq1 was assessed by immunoblotting whole cell lysates with an anti‐HA antibody. Gapdh serves as loading control (right). B, 293T cells were transiently transfected with HA‐tagged Foxq1 and HA‐tagged EWS‐FLI1, EWS or FLI1. The cell lysates were immunoprecipitated with mouse monoclonal anti‐HA (left) or anti‐FLAG (right) antibodies and immunoblotted with indicated antibodies

Figure 5

Trib1 and Nrg1 are involved in Ewing sarcoma proliferation and survival. A, Growth inhibition of ES49 cells by gene silencing (left). Cell numbers were counted 48 h after transfection of siRNAs. Relative cell growth compared with control siRNA infection is indicated. Data represent the means ± SEM of 3 independent experiments (*P < .02, **P < .01). Efficiencies of gene silencing was assessed as relative gene expression to Gapdh mRNA by real‐time qPCR (right). Data represent the means ± SEM of 3 independent experiments (*P < .001). B, Growth inhibition of EWS human Ewing sarcoma cells by gene silencing (left). Cell numbers were counted 48 h after transfection of siRNAs. Relative cell growth compared with control siRNA infection is indicated. Data represent the means ± SEM of 3 independent experiments (*P < .01, **P < .001). Efficiencies of gene silencing were assessed as relative gene expression to GAPDH mRNA by real‐time qPCR (right). Data represent the means ± SEM of 3 independent experiments (*P < .05, **P < .001). C, Effects of Trib1 or Nrg1 knockdown on DNA synthesis or apoptosis in ES49 cells. DNA synthesis and apoptosis were quantitated by measuring frequencies of EdU‐ or Annexin V‐positive cells by FACS, respectively. Data represent the means ± SEM of 3 independent experiments (*P < .05, **P < .01). D, Effects of TRIB1 or NRG1 knockdown on DNA synthesis or apoptosis in EWS cells. DNA synthesis and apoptosis were quantitated by measuring frequencies of EdU‐ or Annexin V‐positive cells by FACS, respectively. Data represent the means ± SEM of 3 independent experiments (*P < .05, **P < .01). E, Immunoblotting shows expression of C/EBPβ in ES49 cells upon Trib1 knockdown. The LAP isoform of C/EBPβ is indicated. F, TRIB1 and NRG1 expression in human sarcoma samples assessed by real‐time qPCR. ES, Ewing sarcoma; CCS, clear cell sarcoma; DFSP, dermatofibrosarcoma protuberans; EMS, extraskeletal myxoid chondrosarcoma; LPS, liposarcoma