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.

Long noncoding RNA EWSAT1-mediated gene repression facilitates Ewing sarcoma oncogenesis

“…Consistent with previous microarray experiments, RNAseq analysis identified a large number of genes repressed by EWS-FLI1 (8,16). Direct activation of gene expression by EWS-FLI1 occurs via binding to a consensus motif near promoters or by binding to a distal microsatellite repeat (57,58). To date, the mechanisms responsible for EWS-FLI1-mediated repression remain unclear.…”

High-throughput small molecule screen identifies inhibitors of aberrant chromatin accessibility

“…Translocations with other ETS genes are detected in most of the remaining tumors, yielding similarly functioning fusion proteins (16). We recently found that, despite conservation of the ETS DNA binding domain, the fusion oncoprotein uniquely localizes to specific microsatellite regions (17,18). Using formaldehyde-assisted isolation of regulatory elements (FAIRE), a biochemical strategy to enrich for nucleosome-depleted regions of chromatin, we demonstrated that EWSR1-FLI1 binding was necessary to maintain nucleosome depletion at these sites.…”

Genomic tools for dissecting oncogenic transcriptional networks in human leukemia

“…In addition, even in the context of well characterized matrices, pattern matching methods do not take into account the possible effect of other proteins in the transcriptional complex, which may redirect or facilitate the binding of the transcription factor under study to DNA sequences not represented in the consensus matrix. [42][43][44] In this regard, a recent study on the E2F-binding specificity confirmed the fact that only a small percentage of the regions bound in vivo by E2F factors actually contain the E2F consensus motif. 45 Pattern detection methods search for recurring and overrepresented DNA motifs in specific subsets of sequences, such as genomic regions identified by ChIP-chip or ChIP-seq, as bound by a given transcription factor.…”

“…45 Pattern detection methods search for recurring and overrepresented DNA motifs in specific subsets of sequences, such as genomic regions identified by ChIP-chip or ChIP-seq, as bound by a given transcription factor. 42,46 Pattern detection techniques can be used to identify putative DNA-binding motifs for transcription factors without previous knowledge of their DNA-binding properties and for the discovery of neighbor cooperating motifs specifically enriched in coregulated sets of genes.…”