Structural and functional studies on human FLI-1 and EWS-FLI-1 oncoproteins.

The human FLI-1 and EWS genes are involved in the formation of EWS-FLI-1 fusion gene due to chromosomal translocations in Ewing's sarcoma and related tumors. In addition, murine FLI-1 was implicated in the erythroleukemias induced by Friend murine leukemia virus (F-MuLV). In this thesis, several important issues regarding the potential mechanisms in FLI-1-involved tumorigenesis have been addressed. The optimum FLI-1 binding sequence was determined using binding site selection strategy. It shows greater specificity at 5$\sp\prime$ end of the GGAA core site than the selected binding sequences of other ETS proteins. EWS-FLI-1 displayed similar DNA binding specificity and affinity as FLI-1. However, the N-terminal regions of EWS-FLI-1 and FLI-1 displayed differential transactivation capabilities in S. cerevisiae. Moreover, GAL4-FLI-1 inhibited yeast colony formation while GAL4-EWS-FLI-1 did not, suggesting that these two proteins are functionally distinct. FLI-1 and EWS genes were also differentially regulated in human T cells during transition from quiescent to cycling state. Therefore, EWS-FLI-1 might contribute to the pathogenesis of Ewing's tumors as an aberrant transcription factor, and/or by deregulating the expression of FLI-1 DNA binding activity relative to cell cycle. The oncogenicity of FLI-1 or EWS-FLI-1 in lymphoid tissues is being pursued using a transgenic approach. To understand the structural basis of DNA binding specificity of ETS proteins, the solution structure of FLI-1 ETS domain was determined by using multidimensional NMR approach. It consists of three $\alpha$-helices and a four-stranded antiparallel $\beta$-sheet, similar to structures of the class of helix-turn-helix DNA binding motifs first found in the catabolite gene activator protein of E. coli. The residues in the third helix which are involved in the DNA contacts have been identified by mutagenesis and NMR experiments, suggesting that the third helix is the DNA recognition helix. Our data suggest that in comparison to structurally related proteins, the ETS domain uses a new variation of the helix-turn-helix motif for binding to DNA as a monomer.PhDMicrobiology and ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/104662/1/9542902.pdfDescription of 9542902.pdf : Restricted to UM users only