The long-term objective of this application is to elucidate a novel mechanism whereby a leukemia fusion protein disrupts gene expression through uncoupling transcription from splicing. In acute myelogenous leukemia (AMIL), chronic myelogenous leukemia (CML) in blast crisis, and myelodysplastic syndromes, the TLS (translocation liposarcoma) gene is fused to the ERG (ets-related gene) gene through a recurrent t(16;21) translocation. The resultant TLS/ERG fusion protein retains the N-terminal domain of TLS but the C-terminal domain of TLS is replaced by the DNA-binding domain of ERG. TLS/ERG is generally thought to function as a chimeric transcription factor leading to transformation through deregulation of gene transcription, yet our preliminary studies suggest that an alternative mechanism may exist to account for its transformation potential. Wild-type TLS associates with both RNA Polymerase (Pol) II and serine-arginine (SR) splicing factors whereas the TLS/ERG fusion protein inhibits RNA splicing mediated by TLS-associated SR proteins. These observations lead to the hypothesis that TLS functions as an adapter molecule coupling transcription to splicing by binding to RNA Pol II through the N-terminal domain of TLS and recruiting SR splicing factors through the C-terminal domain of TLS, and that TLSIERG disrupts this coupling by binding to RNA Pol II but falling to recruit SR splicing factors. This disruption of TLS-mediated RNA processing may contribute to alterations in growth and differentiation. To test this hypothesis, co-immunoprecipitation experiments will be carried out to confirm the interaction of RNA Pol II with TLS and its SR protein partners in an in vivo cellular context. Mutagenesis studies will be performed to determine a correlation between the ability of TLS/ERG fusion protein to disrupt splicing and its ability to transform fibroblast and myeloid cells. An inducible system will be established to investigate the effects of conditional expression of TLS/ERG and TLS/ERG mutants on cell growth, differentiation, gene transcription and RNA splicing. These studies will further our basic understanding on the coupling of transcription and splicing, and will provide new insight into how an oncogenic fusion protein leads to transformation. It is envisioned that successful completion of this project will unveil potential therapeutic targets in the treatment of human leukemias characterized by the t(16;21) translocation.
