Protein degradation is integrated, alongside transcriptional and other post-transcriptional regulation, into the regulation of gene expression during development. Our ability to dissect the mechanisms underlying this integration has advanced significantly with the discovery of F-box proteins, which act as specificity factors that target particular substrates for ubiquitin-mediated degradation. Our primary goals are to understand (i) how F-box proteins are regulated, (ii) what kinds of gene regulatory functions can be performed by these proteins, and (iii) how degradation substrates that share a common F-box protein are functionally related to each other. As a model system, we are dissecting the function of Partner of paired (Ppa), a newly-identified F-box protein implicated in the regulation of Drosophila segmentation and nuclear cycle during embryogenesis. We propose to examine Ppa function in Drosophila and zebrafish.
Our specific aims are: (1) To determine whether Ppa has transcriptional repression activity. We will test the hypothesis that, in addition to regulating degradation of the Paired transcription factor, Drosophila Ppa also represses the ability of Paired to activate transcription of target genes. We will test Ppa deletion constructs in vivo, and test if Ppa's putative transcriptional repression domain functions with a heterologous DNA-binding activity. We will also determine whether zebrafish Ppa has repression activity. (2) To characterize post-transcriptional regulation of Ppa. Our preliminary expression analysis suggests that Drosophila Ppa protein expression is regulated at the post-transcriptional level in order to up-regulate Ppa protein in cells with high levels of substrate requiring degradation. Using ectopic expression studies, deletion analysis, and protein-synthesis inhibition, we will assess the substrate dependence of this regulation, and whether it might occur through stabilization of the Ppa protein. We will also test whether Ppa up-regulation by one substrate leads to changes in expression of other Ppa substrates. (3) To identify new Ppa substrates. Potential new substrates of Ppa will be identified using two-hybrid tests of known segmentation proteins and a two-hybrid screen for new candidates. Ppa-dependent degradation of potential substrates will be tested using in vivo expression assays (see Aim 1). By examining Drosophila and zebrafish Ppa in parallel, we will assess which properties of Ppa are conserved across taxa, and thereby provide a more general understanding of how F-box-mediated protein degradation can be integrated into gene regulatory networks in development.
