Identification and characterization of GGAA microsatellites as novel EWS-FLI regulatory elements in Ewing's sarcoma.

摘要

Ewing's sarcoma is an aggressive bone-associated tumor of children and young adults. Most cases of Ewing's sarcoma express the EWS/FLI fusion protein due to a chromosomal translocation. EWS is a protein of unknown function, while FLI is an ETS family member. EWS/FLI functions as an aberrant transcription factor to dysregulate genes involved in tumorigenesis. Understanding the mode of gene regulation employed by EWS/FLI may provide molecular insights into the transformed phenotype of Ewing's sarcoma.;Further, I elucidated a novel mechanism for EWS/FLI target gene regulation via GGAA microsatellites: EWS/FLI binds GGAA microsatellites as a homo-dimer. Moreover, this homotypic interaction between two EWS/FLI molecules localized to the ETS domain of FLI. Interestingly, while the FLI ETS domain was sufficient to bind GGAA microsatellite, the amino-terminal EWS domain was necessary for reporter gene regulation via GGAA microsatellites.;GSTM4 was next identified as another GGAA microsatellite driven gene required for Ewing's sarcoma oncogenesis. Most importantly Ewing's sarcoma patients with higher GSTM4 expression levels in their tumors had poor survival outcomes than those with lower GSTM4 expression levels.;Taken together my work has identified and characterized GGAA microsatellites as novel EWS/FLI regulatory elements. I have elucidated a new mechanism of ETS protein interaction with a nonconsensus ETS DNA-binding sequence allowing for identification of approaches to inhibit these emergent properties of EWS/FLI, thereby leading to development of new therapeutic avenues for this highly malignant disease.;In this work I made the unprecedented discovery that EWS/FLI regulates some of its targets by binding to GGAA microsatellites. This was surprising because these microsatellites do not conform to known EWS/FLI binding sites, nor have they been previously implicated in cancer development. My work demonstrates that GGAA microsatellites are enriched near the transcriptional start site of EWS/FLI up-regulated genes, and are bound by EWS/FLI in vivo. Furthermore, EWS/FLI regulation via GGAA microsatellites is critical for Ewing's sarcoma development because up-regulation of NR0B1, a gene absolutely required for Ewing's sarcoma oncogenesis, is dependent on the GGAA microsatellite in its promoter. These findings suggest a new paradigm for cancer-relevant gene regulation by EWS/FLI, and perhaps other ETS family members as well.