The Oncogenic EWS-FLI1 Protein Binds In Vivo GGAA Microsatellite Sequences with Potential Transcriptional Activation Function

Noëlle Guillon,Olivier Delattre,Emmanuel Barillot,Valentina Boeva,Franck Tirode,Andrei Zynovyev,Laszlo Tora

doi:10.1371/journal.pone.0004932

Abstract

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.

Highlights

Ewing tumors, the second most frequent bone tumors in teenagers and young adults, show specific translocations fusing the 59 part of EWS to the 39 sequence encoding the DNA binding domain of an ETS factor [1,2]
Immunoprecipitation experiments were conducted in SK-N-MC and A673, two Ewing cell lines that express type 1 EWS-FLI1, and in MON, a malignant rhabdoid tumor (MRT) cell line
The antibody that was used is directed against the C-terminus part of FLI1. It could theoretically immunoprecipitate wild type FLI1, this protein is expressed in none of the three afore-mentionned cell lines

Summary

Introduction

The second most frequent bone tumors in teenagers and young adults, show specific translocations fusing the 59 part of EWS to the 39 sequence encoding the DNA binding domain of an ETS factor [1,2]. Various experimental procedures, including SELEX experiments and mapping of promoters regulated by EWS-FLI1, have shown that ETS factors bind purine-rich sequences with a GGAA/T core consensus sequence, surrounded by nucleotides that contribute to the specificity of each factor [8,9,10,11]. This was recently highlighted by a large-scale study of the properties of ETS factors promoter occupancy showing that DNA binding may be divided into two complementary mechanisms [12]. The second process would involve more specific mechanisms, with the recognition of less typical binding sites, possibly in cooperation with other DNAbinding factors

Methods

Results

Conclusion