Microsatellites as EWS/FLI response elements in Ewing's sarcoma

Abstract

The ETS gene family is frequently involved in chromosome translocations that cause human cancer, including prostate cancer, leukemia, and sarcoma. However, the mechanisms by which oncogenic ETS proteins, which are DNA-binding transcription factors, target genes necessary for tumorigenesis is not well understood. Ewing's sarcoma serves as a paradigm for the entire class of ETS-associated tumors because nearly all cases harbor recurrent chromosomal translocations involving ETS genes. The most common translocation in Ewing's sarcoma encodes the EWS/FLI oncogenic transcription factor. We used whole genome localization (ChIP-chip) to identify target genes that are directly bound by EWS/FLI. Analysis of the promoters of these genes demonstrated a significant over-representation of highly repetitive GGAA-containing elements (microsatellites). In a parallel approach, we found that EWS/FLI uses GGAA microsatellites to regulate the expression of some of its target genes including NR0B1, a gene required for Ewing's sarcoma oncogenesis. The microsatellite in the NR0B1 promoter bound EWS/FLI in vitro and in vivo and was both necessary and sufficient to confer EWS/FLI regulation to a reporter gene. Genome wide computational studies demonstrated that GGAA microsatellites were enriched close to EWS/FLI-up-regulated genes but not down-regulated genes. Mechanistic studies demonstrated that the ability of EWS/FLI to bind DNA and modulate gene expression through these repetitive elements depended on the number of consecutive GGAA motifs. These findings illustrate an unprecedented route to specificity for ETS proteins and use of microsatellites in tumorigenesis.

Fig. 1.

The GGAA microsatellites in the NR0B1 and FCGRT promoters are the EWS/FLI response elements. (A) TC71 Ewing's sarcoma cells cotransfected with a 1.6-kb NR0B1 promoter luciferase vector and either EF-2-RNAi (targeting EWS/FLI) or ERG-RNAi (negative control). The “control vector” does not contain NR0B1 promoter elements. The error bars the figure indicate SDs, and asterisks indicate P < 0.05. (B) 293EBNA cells cotransfected with the indicated NR0B1 promoter luciferase vectors (containing the indicated amount of promoter sequence upstream of the transcriptional start site) and an EWS/FLI (or empty control) cDNA expression vector. The control vector does not contain NR0B1 promoter elements. (C) NR0B1 promoter with GGAA microsatellite indicated. The microsatellite contains 25 GGAA repeats. (D) Luciferase assays in 293EBNA cells with the full 102-bp NR0B1 microsatellite upstream of a minimal promoter element. The control vector does not contain the microsatellite, but does contain the minimal promoter element. (E and F) Luciferase reporter assays by using 293EBNA cells and either the indicated FCGRT promoter deletion constructs or the isolated FCGRT GGAA microsatellite upstream of a minimal promoter, respectively. Of note, the FCGRT microsatellite is present at approximately −1.6 kb upstream of the transcriptional start site.

Fig. 2.

EWS/FLI occupies GGAA microsatellite containing promoters in vivo. Chromatin immunoprecipitation of the indicated promoters from A673 Ewing's sarcoma cells by using antibodies against FLI (which recognizes EWS/FLI), ETS1, or ELK1. Data are plotted as fold enrichment for each region compared to the average enrichment of two negative control genes. The error bars indicate SEMs of two to five independent experiments.

Fig. 3.

Enrichment of GGAA microsatellites in the promoters of EWS/FLI-up-regulated genes. (A) Cumulative portion of microsatellites (GGAA or GGAT) plotted as a function of distance between the microsatellites and the closest 5′ gene edge. (B) Correlation between EWS/FLI-up- and -down-regulated genes (in red and blue, respectively) and microsatellite distance analyzed by Fisher's exact test in the A673 Ewing's sarcoma cell line. Significant correlations cross over the “Bonferroni line” (see Methods). N.B., only up-regulated genes vs. GGAA microsatellites can be seen at the scales used.

Fig. 4.

Ability of EWS/FLI to bind and activate via GGAA repetitive regions depends on the number of consecutive GGAA motifs. (A) Sequences of the oligonucleotides used for these analyses. The GGAA repeats are underlined. (B) Left shows an EMSA with a DNA duplex containing seven consecutive GGAA motifs. A specific EWS/FLI band is present when 3xFLAG-EWS/FLI from nuclear extracts is included. This specific band is supershifted with anti-FLAG antibody and competed with DNA duplex (I) containing a high-affinity ETS site but is not competed with DNA duplex (II) containing a PU.1 site that does not bind EWS/FLI (14). A control extract that does not contain EWS/FLI produces only nonspecific binding (indicated by “ns”). Right shows EMSA with DNA duplexes containing the indicated number of consecutive GGAA motifs and 3xFLAG-EWS/FLI. The positions of specific EWS/FLI-bound complexes are indicated. (C) Luciferase assays in 293EBNA cells with 36-bp sequence containing the indicated number of consecutive GGAA motifs (as indicated in A) upstream of a minimal promoter. The error bars indicate SDs, and asterisks indicate P < 0.05.