EWS/FLI and its downstream target NR0B1 interact directly to modulate transcription and oncogenesis in Ewing's sarcoma

Abstract

Most Ewing's sarcomas harbor chromosomal translocations that encode fusions between EWS and ETS family members. The most common fusion, EWS/FLI, consists of an EWSR1-derived strong transcriptional activation domain fused, in-frame, to the DNA-binding domain-containing portion of FLI1. EWS/FLI functions as an aberrant transcription factor to regulate genes that mediate the oncogenic phenotype of Ewing's sarcoma. One of these regulated genes, NR0B1, encodes a corepressor protein, and likely plays a transcriptional role in tumorigenesis. However, the genes that NR0B1 regulates and the transcription factors it interacts with in Ewing's sarcoma are largely unknown. We used transcriptional profiling and chromatin immunoprecipitation to identify genes that are regulated by NR0B1, and compared these data to similar data for EWS/FLI. Although the transcriptional profile overlapped as expected, we also found that the genome-wide localization of NR0B1 and EWS/FLI overlapped as well, suggesting that they regulate some genes coordinately. Further analysis revealed that NR0B1 and EWS/FLI physically interact. This protein-protein interaction is likely to be relevant for the development of Ewing's sarcoma because mutations in NR0B1 that disrupt the interaction have transcriptional consequences and also abrogate oncogenic transformation. Taken together, these data suggest that EWS/FLI and NR0B1 physically interact, coordinately modulate gene expression, and mediate the transformed phenotype of Ewing's sarcoma.

Figure 1

NR0B1 target genes contribute to the EWS/FLI gene expression signature. A, B, C, Venn diagrams representing the overlap between the overall NR0B1 and EWS/FLI gene expression signatures, and down- and up-regulated genes, respectively. The Chi square-determined p-value is indicated.

Figure 2

Coordinate occupancy of NR0B1 and EWS/FLI at specific genomic loci. A, Probe enrichment pattern for NR0B1 and EWS/FLI ChIP-chip derived from two separate biological samples per experimental condition. Relative genomic positioning of probes was determined by the Integrated Genome Browser software program (Affymetrix). Probe p-values were determined by the Agilent ChIP Analytics program. NR0B1, KCNN2, and HSPA4L are examples of overlapping probe patterns, while GPR101 represents distinct probe distribution. B, ChIP of the NR0B1 intronic region using the indicated antibodies. Data are plotted as fold enrichment of the intron compared to the enrichment of a negative control gene (ALB). Error bars indicate standard deviation of three independent experiments. Asterisks signify p < 0.05. C, Venn diagram representing the overlap between ChIP-chip identified bound NR0B1 and EWS/FLI gene targets. The Chi square-determined p-value is indicated.

Figure 3

NR0B1 and EWS/FLI directly interact. A, Schematic of wild-type and mutant NR0B1 constructs. NR0B1 consists of an amino-terminal domain (amino acids 1 – 253) that contains 3½ alanine-glycine rich repeating units harboring three LXXLL motifs, and a carboxyl-terminal domain (amino acids 253 – 470) that is homologous to other nuclear hormone receptors ligand binding domains. B, Western blot analysis of input and co-immunoprecipitation samples of 293EBNA cells transfected with wild-type EWS/FLI and NR0B1. Immunoprecipitation experiments and western blots were performed with the indicated antibodies. C, Co-immunoprecipitation of 293EBNA transfected with EWS/FLI and various NR0B1 mutant constructs, using the indicated antibodies. WT indicates the NR0B1 wild-type allele. N.5 and C.5 represent the amino- and carboxy-terminal domains of NR0B1, respectively. N.5 LXXLL indicates amino-terminal NR0B1 with all three LXXLL motifs mutated. All NR0B1 constructs are 3x-FLAG-tagged. Input samples were utilized to ensure appropriate expression of all constructs tested. D, Co-immunoprecipitation of 293EBNA transfected with NR0B1 and EWS/FLI parental proteins EWSR1 or FLI1, using the indicated antibodies.

Figure 4

NR0B1 and EWS/FLI co-localize to the nucleus. A, Immunofluorescence of EWS502 Ewing's sarcoma cells infected with the indicated cDNA constructs and detected with the indicated antibody. Nuclei are shown by DAPI staining and 293EBNA cells are shown as a negative control. B, C, A673 cells infected or 293EBNA transfected, respectively, with the indicated cDNA constructs and subfractionated. Western blots were performed using the designated antibodies. Tubulin is utilized as a control for the cytoplasmic fraction, while mSin3A is used as a control for the nuclear fraction.

Figure 5

NR0B1 and EWS/FLI interact on chromatin and their co-expression influences transcription. A, Sequential ChIP of NR0B1 and EWS/FLI in A673 and TC71 Ewing's sarcoma cells. Using our “knockdown/rescue” approach, endogenous NR0B1 was first replaced with a 3x-FLAG-tagged-NR0B1 allele. NR0B1 was immunoprecipitated first with anti-FLAG antibody, the isolated NR0B1-associated chromatin mixture was then subject to anti-FLI immunoprecipitation. A673 and TC71 cells (lacking any FLAG construct) and non-specific antibodies were used as controls. Data are plotted as fold enrichment for the NR0B1 intronic region compared to the average enrichment of a negative control gene (ALB). Bars are representative of four of six independent experiments. B, Luciferase assays in 293EBNA co-transfected with the ~700bp NR0B1 intronic response element upstream of a minimal promoter and the indicated cDNA constructs. Relative luciferase activity is the ratio of firefly luciferase activity to Renilla luciferase activity (to control for transfection efficiency). Error bars indicate standard deviations, and the asterisk indicates p < 0.05.

Figure 6

The NR0B1-EWS/FLI interacting domain is critical for transformation. A, Soft agar colony formation in A673 and TC71 Ewing's sarcoma cells infected with the indicated constructs. NR0B1 LXXLL Mutant cDNA represents the full-length triple LXXLL NR0B1 mutant allele. B, Quantification of colonies formed in soft agar. Error bars indicate standard deviations of duplicate assays.