The oncogenic EWS-FLI1 protein binds in vivo GGAA microsatellite sequences with potential transcriptional activation function.
Bottom Line: Most bound sites are found outside promoter regions.Importantly, in vivo EWS-FLI1-bound microsatellites are significantly associated with EWS-FLI1-driven gene activation.Put together, these results point out the likely contribution of microsatellite elements to long-distance transcription regulation and to oncogenesis.
Affiliation: Institut Curie, Paris, France.
The fusion between EWS and ETS family members is a key oncogenic event in Ewing tumors and important EWS-FLI1 target genes have been identified. However, until now, the search for EWS-FLI1 targets has been limited to promoter regions and no genome-wide comprehensive analysis of in vivo EWS-FLI1 binding sites has been undertaken. Using a ChIP-Seq approach to investigate EWS-FLI1-bound DNA sequences in two Ewing cell lines, we show that this chimeric transcription factor preferentially binds two types of sequences including consensus ETS motifs and microsatellite sequences. Most bound sites are found outside promoter regions. Microsatellites containing more than 9 GGAA repeats are very significantly enriched in EWS-FLI1 immunoprecipitates. Moreover, in reporter gene experiments, the transcription activation is highly dependent upon the number of repeats that are included in the construct. Importantly, in vivo EWS-FLI1-bound microsatellites are significantly associated with EWS-FLI1-driven gene activation. Put together, these results point out the likely contribution of microsatellite elements to long-distance transcription regulation and to oncogenesis.
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Mentions: In order to characterize EWS-FLI1 consensus binding sites, over-representation of sequence motifs was searched for. Frequencies of every possible 4–8 bp long oligomer were assessed in the 246 EWS-FLI1 specific regions compared to their respective frequencies in the human genome. A clear over-representation of oligomers containing GGAA motifs was observed (results obtained for 6-mer motifs are displayed in Fig. 1A). More precisely, 104 regions presented microsatellite sequences consisting of 3 or more GGAA-containing tandem repeats: (GGAA)n, (GGAAN)n or (GGAANN)n. The other 142 regions did not contain such microsatellites. Both types of regions were found in A673 and SK-N-MC cell lines (Fig. 1B), indicating that neither type of region was cell specific. The RegionMiner and MatInspector softwares (Genomatix) were used to assess whether the two types of EWS-FLI1 specific regions were enriched in bona fide ETS factor binding sites. Regions containing microsatellites did not show any additional ETS consensus over-representation after repeat filtration (Table S2). In contrast, a clear over-representation of ETS family binding motifs was observed in the EWS-FLI1-specific regions that do not contain microsatellite sequences (Table 2). These regions also presented very frequent combination of two ETS sites or of ETS site with consensus sites for other transcription factors (Table 3). These non-microsatellite EWS-FLI1 specific regions were also analyzed with the MEME software that defines position weight matrices giving frequency distributions of each base at each position . As shown in Figure 1C, MEME retrieved a consensus sequence highly similar to an ETS binding sequence.