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The oncogenic EWS-FLI1 protein binds in vivo GGAA microsatellite sequences with potential transcriptional activation function.

Guillon N, Tirode F, Boeva V, Zynovyev A, Barillot E, Delattre O - PLoS ONE (2009)

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.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Paris, France.

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.

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Related in: MedlinePlus

Long distance EWS-FLI1 binding on GGAA microsatellites results in significant gene expression activation.A. Proportion of EWS-FLI1-bound regions, as compared to the proportion of random regions, around EWS-FLI1 regulated genes. The proportion of EWS-FLI1-bound regions as a function of the distance to the transcription start sites of EWS-FLI1-up or -down regulated genes (solid lines) is shown. As a control, a similar function is indicated for 1500 randomly chosen regions (dashed line). B. Gene Set Enrichment Analysis (GSEA) of genes flanking EWS-FLI1-bound microsatellites. The 94 genes flanking the 80 microsatellites>9R regions (upper panel) as well as the 144 genes flanking the non-microsatellites regions (lower panel) were used as gene set. The expression dataset resulted from previously described EWS-FLI1 inhibition experiments of A673 and SK-N-MC Ewing cell lines [37], [40], ranked using the signal-to-noise algorithm. A strong enrichment of genes flanking EWS-FLI1 bound GGAA microsatellites among EWS-FLI1 up-regulated genes is observed (upper panel). C–F. Regions upstream of EWS-FLI1 up-regulated genes are enriched in GGAA-microsatellites. The number of microsatellites with either 3 to 9 GGAA repeats (grey line) or more than 9 repeats (black line) was calculated for each 1 Kb window from 1 Kb to 1 Mb upstream of the transcription start sites. The numbers of GGAA repeats along DNA are shown for (C) 17000 known genes (control distribution), (D) 582 EWS-FLI1-up-regulated genes, (E) 558 EWS-FLI1-down-regulated genes and (F) 561 genes that are expressed in A673 and SK-N-MC cell lines but not regulated by EWS-FLI1. The control distribution shown in C is also indicated on part D, E and F.
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pone-0004932-g003: Long distance EWS-FLI1 binding on GGAA microsatellites results in significant gene expression activation.A. Proportion of EWS-FLI1-bound regions, as compared to the proportion of random regions, around EWS-FLI1 regulated genes. The proportion of EWS-FLI1-bound regions as a function of the distance to the transcription start sites of EWS-FLI1-up or -down regulated genes (solid lines) is shown. As a control, a similar function is indicated for 1500 randomly chosen regions (dashed line). B. Gene Set Enrichment Analysis (GSEA) of genes flanking EWS-FLI1-bound microsatellites. The 94 genes flanking the 80 microsatellites>9R regions (upper panel) as well as the 144 genes flanking the non-microsatellites regions (lower panel) were used as gene set. The expression dataset resulted from previously described EWS-FLI1 inhibition experiments of A673 and SK-N-MC Ewing cell lines [37], [40], ranked using the signal-to-noise algorithm. A strong enrichment of genes flanking EWS-FLI1 bound GGAA microsatellites among EWS-FLI1 up-regulated genes is observed (upper panel). C–F. Regions upstream of EWS-FLI1 up-regulated genes are enriched in GGAA-microsatellites. The number of microsatellites with either 3 to 9 GGAA repeats (grey line) or more than 9 repeats (black line) was calculated for each 1 Kb window from 1 Kb to 1 Mb upstream of the transcription start sites. The numbers of GGAA repeats along DNA are shown for (C) 17000 known genes (control distribution), (D) 582 EWS-FLI1-up-regulated genes, (E) 558 EWS-FLI1-down-regulated genes and (F) 561 genes that are expressed in A673 and SK-N-MC cell lines but not regulated by EWS-FLI1. The control distribution shown in C is also indicated on part D, E and F.

Mentions: Among the 246 EWS-FLI1 specific regions, 146 were localized in intergenic regions, 13 in exons, 79 in gene introns and 8 in promoters. These EWS-FLI1 binding sites were very frequently located far away from any transcription unit, with a mean distance to transcription start sites of 242 Kb and up to 3 Mb. To address the issue of a potential link between EWS-FLI1 bound regions and EWS-FLI1 regulated transcription, we compared the distances of the 246 EWS-FLI1-specific regions or of randomly picked regions to the nearest EWS-FLI1 regulated gene. We used a previously published list of EWS- FLI1 regulated genes that were identified through shRNA inhibition experiments in A673 and SK-N-MC Ewing cell lines [37]. This list contains 557 and 577 genes that are down- or up-regulated by EWS-FLI1, respectively (fold change>/2/ with a Welsh p-value<0.01). Figure 3A shows the percentage of EWS-FLI1-bound or random regions with an EWS-FLI1-modulated gene at a given distance. It is interesting to note that about 43% of the 246 EWS-FLI1 bound regions have the transcription start site of an EWS-FLI1-up-regulated gene within 1 Mb (as compared to 27% for random regions) and 60% within 2 Mb (46% for random). The increased proportion of EWS-FLI1-down-regulated genes located within 1 or 2 Mb of EWS-FLI1 regions is less obvious (31% as compared to 24% for random regions and 47% as compared to 42%, respectively). These results indicated that the 246 EWS-FLI1 bound regions were significantly closer to EWS-FLI1-regulated genes than randomly selected regions (Mann-Whitney p-value<10−16). However, no correlation between expression level of genes and their distance to microsatellites>9R could be found. To further analyze the link between EWS-FLI1 transcriptional expression modulation and EWS-FLI1-bound microsatellites, GSEA analyses were performed [39]. As expression dataset, we used the afore-mentioned published data [37], [40], ranked using the signal-to-noise metric. The gene set contained the genes flanking the 80 regions containing the microsatellites>9R. As shown on the upper panel of Figure 3B, the gene set is overrepresented at the left edge that contains EWS-FLI1 up-regulated genes. Indeed, among the 94 genes flanking the microsatellites>9R, 30 were at the leading edge (Z-score = 8.6, Fisher p-value = 2.1×10−11). GSEA analysis carried on the regions bound by EWS-FLI1 that do not contain GGAA microsatellite is shown on Figure 3B, lower panel. This shows that relative enrichments are observed at both edges, however the GSEA overall statistics do not reach significance. This analysis demonstrated that EWS-FLI1 up-regulated genes are significantly enriched in the vicinity of EWS-FLI1-bound microsatellites with more than 9 GGAA repeats therefore suggesting that microsatellites>9R are associated with a function of EWS-FLI1 in transcription activation.


The oncogenic EWS-FLI1 protein binds in vivo GGAA microsatellite sequences with potential transcriptional activation function.

Guillon N, Tirode F, Boeva V, Zynovyev A, Barillot E, Delattre O - PLoS ONE (2009)

Long distance EWS-FLI1 binding on GGAA microsatellites results in significant gene expression activation.A. Proportion of EWS-FLI1-bound regions, as compared to the proportion of random regions, around EWS-FLI1 regulated genes. The proportion of EWS-FLI1-bound regions as a function of the distance to the transcription start sites of EWS-FLI1-up or -down regulated genes (solid lines) is shown. As a control, a similar function is indicated for 1500 randomly chosen regions (dashed line). B. Gene Set Enrichment Analysis (GSEA) of genes flanking EWS-FLI1-bound microsatellites. The 94 genes flanking the 80 microsatellites>9R regions (upper panel) as well as the 144 genes flanking the non-microsatellites regions (lower panel) were used as gene set. The expression dataset resulted from previously described EWS-FLI1 inhibition experiments of A673 and SK-N-MC Ewing cell lines [37], [40], ranked using the signal-to-noise algorithm. A strong enrichment of genes flanking EWS-FLI1 bound GGAA microsatellites among EWS-FLI1 up-regulated genes is observed (upper panel). C–F. Regions upstream of EWS-FLI1 up-regulated genes are enriched in GGAA-microsatellites. The number of microsatellites with either 3 to 9 GGAA repeats (grey line) or more than 9 repeats (black line) was calculated for each 1 Kb window from 1 Kb to 1 Mb upstream of the transcription start sites. The numbers of GGAA repeats along DNA are shown for (C) 17000 known genes (control distribution), (D) 582 EWS-FLI1-up-regulated genes, (E) 558 EWS-FLI1-down-regulated genes and (F) 561 genes that are expressed in A673 and SK-N-MC cell lines but not regulated by EWS-FLI1. The control distribution shown in C is also indicated on part D, E and F.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654724&req=5

pone-0004932-g003: Long distance EWS-FLI1 binding on GGAA microsatellites results in significant gene expression activation.A. Proportion of EWS-FLI1-bound regions, as compared to the proportion of random regions, around EWS-FLI1 regulated genes. The proportion of EWS-FLI1-bound regions as a function of the distance to the transcription start sites of EWS-FLI1-up or -down regulated genes (solid lines) is shown. As a control, a similar function is indicated for 1500 randomly chosen regions (dashed line). B. Gene Set Enrichment Analysis (GSEA) of genes flanking EWS-FLI1-bound microsatellites. The 94 genes flanking the 80 microsatellites>9R regions (upper panel) as well as the 144 genes flanking the non-microsatellites regions (lower panel) were used as gene set. The expression dataset resulted from previously described EWS-FLI1 inhibition experiments of A673 and SK-N-MC Ewing cell lines [37], [40], ranked using the signal-to-noise algorithm. A strong enrichment of genes flanking EWS-FLI1 bound GGAA microsatellites among EWS-FLI1 up-regulated genes is observed (upper panel). C–F. Regions upstream of EWS-FLI1 up-regulated genes are enriched in GGAA-microsatellites. The number of microsatellites with either 3 to 9 GGAA repeats (grey line) or more than 9 repeats (black line) was calculated for each 1 Kb window from 1 Kb to 1 Mb upstream of the transcription start sites. The numbers of GGAA repeats along DNA are shown for (C) 17000 known genes (control distribution), (D) 582 EWS-FLI1-up-regulated genes, (E) 558 EWS-FLI1-down-regulated genes and (F) 561 genes that are expressed in A673 and SK-N-MC cell lines but not regulated by EWS-FLI1. The control distribution shown in C is also indicated on part D, E and F.
Mentions: Among the 246 EWS-FLI1 specific regions, 146 were localized in intergenic regions, 13 in exons, 79 in gene introns and 8 in promoters. These EWS-FLI1 binding sites were very frequently located far away from any transcription unit, with a mean distance to transcription start sites of 242 Kb and up to 3 Mb. To address the issue of a potential link between EWS-FLI1 bound regions and EWS-FLI1 regulated transcription, we compared the distances of the 246 EWS-FLI1-specific regions or of randomly picked regions to the nearest EWS-FLI1 regulated gene. We used a previously published list of EWS- FLI1 regulated genes that were identified through shRNA inhibition experiments in A673 and SK-N-MC Ewing cell lines [37]. This list contains 557 and 577 genes that are down- or up-regulated by EWS-FLI1, respectively (fold change>/2/ with a Welsh p-value<0.01). Figure 3A shows the percentage of EWS-FLI1-bound or random regions with an EWS-FLI1-modulated gene at a given distance. It is interesting to note that about 43% of the 246 EWS-FLI1 bound regions have the transcription start site of an EWS-FLI1-up-regulated gene within 1 Mb (as compared to 27% for random regions) and 60% within 2 Mb (46% for random). The increased proportion of EWS-FLI1-down-regulated genes located within 1 or 2 Mb of EWS-FLI1 regions is less obvious (31% as compared to 24% for random regions and 47% as compared to 42%, respectively). These results indicated that the 246 EWS-FLI1 bound regions were significantly closer to EWS-FLI1-regulated genes than randomly selected regions (Mann-Whitney p-value<10−16). However, no correlation between expression level of genes and their distance to microsatellites>9R could be found. To further analyze the link between EWS-FLI1 transcriptional expression modulation and EWS-FLI1-bound microsatellites, GSEA analyses were performed [39]. As expression dataset, we used the afore-mentioned published data [37], [40], ranked using the signal-to-noise metric. The gene set contained the genes flanking the 80 regions containing the microsatellites>9R. As shown on the upper panel of Figure 3B, the gene set is overrepresented at the left edge that contains EWS-FLI1 up-regulated genes. Indeed, among the 94 genes flanking the microsatellites>9R, 30 were at the leading edge (Z-score = 8.6, Fisher p-value = 2.1×10−11). GSEA analysis carried on the regions bound by EWS-FLI1 that do not contain GGAA microsatellite is shown on Figure 3B, lower panel. This shows that relative enrichments are observed at both edges, however the GSEA overall statistics do not reach significance. This analysis demonstrated that EWS-FLI1 up-regulated genes are significantly enriched in the vicinity of EWS-FLI1-bound microsatellites with more than 9 GGAA repeats therefore suggesting that microsatellites>9R are associated with a function of EWS-FLI1 in transcription activation.

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.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Paris, France.

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.

Show MeSH
Related in: MedlinePlus