The goal of this proposal is to understand the molecular mechanisms that direct patterned expression of the regulatory genes during early development. To this end, Dr. Pick has chosen to focus her attention on the proximal enhancer from the segmentation gene ftz. Dr. Pick has identified a core element of 323 bp in this enhancer which directs expression of the ftz promoter or linked heterologous promoters in seven stripes in transgenic embryos. There are two cis-acting modules within this core enhancer, both of which are required for in vivo expression. One of these sites is repeated three times within the core enhancer and contains binding sites for four different proteins: Adf-1, TTK, FTZ-F1, and fEBP9. Dr. Pick's working hypothesis is that combinatorial interactions of these multiple factors that bind to the enhancer element contribute to the striking restriction of expression patterns (formation of stripes) in vivo. As the first aim, Dr. Pick will study four proteins that bind to three subregions of the 323 bp element. Dr. Pick has demonstrated that these sites serve a redundant function in inducing striped expression of the reporter gene. Three of the factors are previously identified proteins and clones already exist. Dr. Pick will isolate the fourth, fEBP9, using the standard biochemical purification method, sequence the peptides and clone the gene based on deduced nucleotide sequences. As the second part of the first aim, Dr. Pick plans to characterize the phenotypes caused by Adf-1 and ttk mutations. The role of ttk will be examined not only for zygotic function but also its maternal contribution by making germ line clones.
The second aim of the proposal is to investigate how Ftz cofactors modulate the in vivo activities of Ftz protein. Dr. Pick will characterize physical interactions between FTZ and FTZ-F1, a putative cofactor of FTZ. She will map domains in the FTZ and FTZ-F1 proteins that are required for their interaction. In collaboration with Dr. J.B.A. Ross, she will also determine intermediate species of the FTZ/FTZ-F1/DNA ternary complex. The equilibrium intermediates will be identified using analytical ultracentrifugation and fluorescence anisotropy. Finally, she will characterize the new putative cofactor of FTZ, called FIP2, which has been identified in a yeast two-hybrid screen. She will determine its protein localization in vivo and characterize its property as cofactor of FTZ DNA binding. The fip-2 gene has been mapped to a chromosomal region rich in existing mutations. If one of these is a fip-2 mutation, a phenotypic analysis will be carried out.
