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Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations
Published on: August 4, 2016
Identification of target genes for the Ewing's sarcoma EWS/FLI fusion protein by representational difference analysis
B S Braun1, R Frieden, S L Lessnick
1Molecular Biology Institute, University of California, Los Angeles 90024, USA.
Abstract:
The EWS/FLI-1 fusion gene results from the 11;22 chromosomal translocation in Ewing's sarcoma. The product of the gene is one of a growing number of structurally altered transcription factors implicated in oncogenesis. We have employed a subtractive cloning strategy of representational difference analysis in conjunction with a model transformation system to identify genes transcribed in response to EWS/FLI. We have characterized eight transcripts that are dependent on EWS/FLI for expression and two transcripts that are repressed in response to EWS/FLI. Three of the former were identified by sequence analysis as stromelysin 1, a murine homolog of cytochrome P-450 F1 and cytokeratin 15. Stromelysin 1 is induced rapidly after expression of EWS/FLI, suggesting that the stromelysin 1 gene may be a direct target gene of EWS/FLI. These results demonstrate that expression of EWS/FLI leads to significant changes in the transcription of specific genes and that these effects are at least partially distinct from those caused by expression of germ line FLI-1. The representational difference analysis technique can potentially be applied to investigate transformation pathways activated by a broad array of genes in different tumor systems.
Insights
The EWS/FLI-1 fusion gene drives Ewing's sarcoma by altering transcription. Researchers identified specific genes, including stromelysin 1, directly impacted by this oncogenic fusion protein.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Ewing's sarcoma is characterized by the 11;22 chromosomal translocation, leading to the EWS/FLI-1 fusion gene.
- The EWS/FLI-1 fusion protein is a transcription factor implicated in oncogenesis.
- Understanding the downstream transcriptional targets of EWS/FLI-1 is crucial for elucidating Ewing's sarcoma development.
Purpose of the Study:
- To identify genes whose transcription is altered in response to EWS/FLI expression.
- To differentiate the transcriptional effects of EWS/FLI from the germ line FLI-1.
- To explore the potential of representational difference analysis in cancer research.
Main Methods:
- Utilized subtractive cloning strategy of representational difference analysis.
- Employed a model transformation system to study gene expression.
- Performed sequence analysis to identify specific transcripts.
Main Results:
- Identified eight transcripts upregulated and two downregulated by EWS/FLI.
- Stromelysin 1, a murine homolog of cytochrome P-450 F1, and cytokeratin 15 were among the upregulated transcripts.
- Stromelysin 1 induction was rapid, suggesting it as a direct EWS/FLI target gene.
Conclusions:
- EWS/FLI expression significantly alters specific gene transcription.
- These transcriptional changes are partially distinct from those induced by germ line FLI-1.
- Representational difference analysis is a valuable tool for studying oncogenic transformation pathways.
