Tumor-specific retargeting of an oncogenic transcription factor chimera results in dysregulation of chromatin and transcription

Abstract

Chromosomal translocations involving transcription factor genes have been identified in an increasingly wide range of cancers. Some translocations can create a protein “chimera” that is composed of parts from different proteins. How such chimeras cause cancer, and why they cause cancer in some cell types but not others, is not understood. One such chimera is EWS–FLI, the most frequently occurring translocation in Ewing Sarcoma, a malignant bone and soft tissue tumor of children and young adults. Using EWS–FLI and its parental transcription factor, FLI1, we created a unique experimental system to address questions regarding the genomic mechanisms by which chimeric transcription factors cause cancer. We found that in tumor cells, EWS–FLI targets regions of the genome distinct from FLI1, despite identical DNA-binding domains. In primary endothelial cells, however, EWS–FLI and FLI1 demonstrate similar targeting. To understand this mistargeting, we examined chromatin organization. Regions targeted by EWS–FLI are normally repressed and nucleosomal in primary endothelial cells. In tumor cells, however, bound regions are nucleosome depleted and harbor the chromatin signature of enhancers. We next demonstrated that through chimerism, EWS–FLI acquired the ability to alter chromatin. Expression of EWS–FLI results in nucleosome depletion at targeted sites, whereas silencing of EWS–FLI in tumor cells restored nucleosome occupancy. Thus, the EWS–FLI chimera acquired chromatin-altering activity, leading to mistargeting, chromatin disruption, and ultimately, transcriptional dysregulation.

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

“…To test whether FAIRE-enriched regions were likely to harbor regulatory elements, we annotated the sites from each cluster with active and repressive histone modifications. We also tested for association with targeting of numerous transcription factors, including those known to be important in endothelial cell biology, as assessed by ChIP-seq (17,19). (Fig.…”

“…We then adapted HT-FAIRE for high-throughput automation and applied it in a targeted screen. The screen was based on two regions selected from a set of Ewing sarcoma-specific sites that we had previously shown were accessible in multiple Ewing sarcoma cell lines with accessibility that was dependent on continued expression of EWSR1-FLI1 (17). Regions that consistently demonstrate FAIRE enrichment or lack of enrichment across many cell types were included as controls (see Table S3).…”

“…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.…”

Oncogenic ETS fusions deregulate E2F3 target genes in Ewing sarcoma and prostate cancer

“…In a study comparing binding of epitope-tagged EWSR1/FLI1 in an ES cell line (EWSR1502) to FLI1, Patel et al (2012) recently suggested that EWSR1/FLI1 directs chromatin remodeling at a proportion of distant sites. Interestingly, in our study, the predominant transcriptional behavior of genes associated with EWSR1/FLI1 without si- multaneous E2F3 binding was found to be repression.…”

Addendum: Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA

“…• ENCODE dataset 2,3 • Lymphoblastoid cell lines (GM12878, GM12891, GM12892, GM18507, GM19239) • Ewing sarcoma (EWS502) 11 • Normal breast cells (patient-derived) 12 • Renal cell carcinoma (786-O) and breast cancer cells (MCF-7) 13 • Leukemia (patient-derived, TLX + T-ALL) 14 • Colon adenocarcinoma (LoVo) 15 • Prostate cancer (VCaP, LNCaP) 16,17 • Embryonic stem cells (pre-and post-embryoid body cells derived from H9-hESCs) 18 • Lung adenocarcinoma (A549) 19 • Chronic myelogenous leukemia (K562) 20 • Primary lung fibroblasts, astrocytes, and dermal fibroblasts 21…”