Identification of protein interaction between the Drosophila Runx1 transcription factor Lozenge and ETS-1 factor Pointed using site directed mutagenesis and yeast two-hybrid analysis

Many classes of transcriptional regulatory proteins are known to function in both cell proliferation and differentiation. Runx1 proteins one such family of transcription factors, plays critical roles in hematopoiesis, osteogenesis and leukemogenesis and act as promoter organizers that cooperate with other transcription factors such as Ets-1 in the regulation of gene activation or repression. Genes that are regulated by the Runx1-Ets1 complex, frequently have multiple, adjacent consensus binding sites in their promoters. I have investigated a similar interaction in developing fly eye. Lozenge (DmRunx1) and Pointed P2 (DmEts-1) cooperate to upregulate expression of prospero , which has multiple Lz and Ets binding sites. Prospero protein is essential for establishing R7 cell fate in the developing eye. Site directed mutagenesis and yeast two hybrid assay was employed to assess critical residues involved in the Lz-Pnt P2 interaction. Results unequivocally demonstrate that Lz-Pnt P2 interaction occurs independent of their DNA binding sites, implying that the interaction is not mediated by their mutual interaction with DNA. Site directed mutation reveals reduced Lz-Pnt P2 interaction, indicating the relevance of altered amino acids for the contact between the proteins. Interestingly, akin to AML1 (Runx1), Iz is also spliced over the domain important for interaction with Ets-1 proteins. Based on the results obtained in this study, we suggest that splicing produces variants that allow these proteins to either interact with Ets-1 and other proteins to transactivate other genes or to work independently in a divergent role in developmental process.