Bayer School of Natural and Environmental Sciences
John A. Pollock
ETS, Lozenge, Pointed, RUNX, Site directed mutagenesis, Yeast-two hybrid analysis
Lozenge is a transcription factor. It is a member of the Runx1 homology group that includes Runt, Acute Myeloid Leukemia1, and Core Binding Factor alpha1. Expressed in the fruit fly Drosophila, Lozenge contributes to the determination of neuronal and non-neuronal cells by regulating the expression of many fate-determining transcription factors. In addition to Lozenge expression in wide varieties of tissues, it also participates in the pattern formation and specification of the photoreceptors R1/R6 and R7 and cones and pigment cells of the fly eye. A growing number of fate determining transcription factors are influenced by Lozenge during eye development. For example, the directed binding of Lozenge regulates prospero expression, which is required for R7 formation. However, for Lozenge to regulate prospero, a second transcription factor Pointed, a member of Ets-1 family of transcription factors, is also required. In fact, previous researchers have demonstrated that the DNA binding sites for Lozenge and Pointed are in very close proximity to one another.
During eye development alternative splicing of Lozenge removes an Ets interaction domain, a sequence domain that is contained in the fifth exon. My working hypothesis is that Lozenge binds directly to Pointed before binding to the prospero enhancer and that this binding is facilitated by Lozenge Exon 5 alpha helix. Other putative binding sites other than the alpha helix may also be involved in stabilizing the protein-protein interaction. To test this hypothesis I used site directed mutagenesis to alter the amino acid sequence of the putative Lozenge-Pointed interaction domain in the exon 5 alpha helix of Lozenge. The capacity of the protein interaction is then assayed with yeast two-hybrid analysis. Results of yeast two-hybrid analysis showed that a single proline mutation L439P could weaken the Lozenge-Pointed interaction. However, the single mutation L439P is not sufficient to completely disrupt the secondary structure of the alpha helix and consequently the Lozenge-Pointed protein-protein interaction.
Boumaza, L. (2007). Site Directed Mutagenesis of Lozenge: A Yeast Two-hybrid Analysis of Transcription Factor Protein Interaction (Master's thesis, Duquesne University). Retrieved from https://dsc.duq.edu/etd/344