Oncogenic Fusion Protein EWS/FLI1 Down-regulates Gene Expression by Both Transcriptional and Posttranscriptional Mechanisms

Ann Park,Christopher T Denny,Jennifer L Anderson,Kelly A France

doi:10.1074/jbc.m111.225433

Ann Park, Christopher T Denny + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m111.225433

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Abstract

Ewing family tumors are characterized by a translocation between the RNA binding protein EWS and one of five ETS transcription factors, most commonly FLI1. The fusion protein produced by the translocation has been thought to act as an aberrant transcription factor, leading to changes in gene expression and cellular transformation. In this study, we investigated the specific processes EWS/FLI1 utilizes to alter gene expression. Using both heterologous NIH 3T3 and human Ewing Family Tumor cell lines, we have demonstrated by quantitative pre-mRNA analysis that EWS/FLI1 repressed the expression of previously validated direct target genes at the level of transcript synthesis. ChIP experiments showed that EWS/FLI1 decreases the amount of Pol II at the promoter of down-regulated genes in both murine and human model systems. However, in down-regulated target genes, there was a significant disparity between the modulation of cognate mRNA and pre-mRNAs, suggesting that these genes could also be regulated at a posttranscriptional level. Confirming this, we found that EWS/FLI1 decreased the transcript half-life of insulin-like growth factor binding protein 3, a down-regulated direct target gene in human tumor-derived Ewing's sarcoma cell lines. Additionally, we have shown through reexpression experiments that full EWS/FLI1-mediated transcriptional repression requires intact EWS and ETS domains. Together these data demonstrate that EWS/FLI1 can dictate steady-state target gene expression by modulating both transcript synthesis and degradation.

Highlights

The Ewing family tumors (EFTs)2 are a family of malignant diseases that have traditionally been labeled Ewing’s sarcoma, primitive neuroectodermal tumors, and Askin tumors [1]
Using both heterologous NIH 3T3 and human Ewing Family Tumor cell lines, we have demonstrated by quantitative pre-mRNA analysis that EWS/FLI1 repressed the expression of previously validated direct target genes at the level of transcript synthesis
If levels of pre-mRNA are unchanged whereas mRNA is differentially regulated, the data would suggest that there is no change in the rate of RNA synthesis and that gene regulation is occurring at the level of RNA stability

Summary

Introduction

The Ewing family tumors (EFTs)2 are a family of malignant diseases that have traditionally been labeled Ewing’s sarcoma, primitive neuroectodermal tumors, and Askin tumors [1]. Using both heterologous NIH 3T3 and human Ewing Family Tumor cell lines, we have demonstrated by quantitative pre-mRNA analysis that EWS/FLI1 repressed the expression of previously validated direct target genes at the level of transcript synthesis.

Results

Conclusion