Molecular mechanisms of Hox specificity and functional diversity
Final Report Abstract
My research interests lie in investigating how Hox proteins, a group of conserved and widely expressed transcription factors, pattern the morphological diversification of segments along the antero-posterior body axis of both vertebrates and invertebrates. Using the Drosophila melanogaster Ultrabithorax (Ubx) protein as a model to explore the possible role of posttranslational modifications (PTMs) in Ubx functional diversity, we had previously mapped its acetylation and phosphorylation sites. We found cell context-specific PTM patterns and putative interacting proteins, opening up new lines of research to address how these proteins achieve functional diversity. This proposal aimed to fulfil two major objectives that address currently underexplored aspects of Hox protein function: 1. Understand the significance of phosphorylation for Ubx functional diversity, and 2. Explore the tissue-specific variability of Hox PTMs. For the first objective, Ubx proteins with mutated individual phosphorylation sites were used to confirm phosphorylation at specific sites and to assess their effect on Ubx protein localization, stability, and transcriptional activity in cultured cells. While Ubx localization did not appear to be affected, one specific phosphorylation site seems to be required to extend Ubx half-life. The same site also appears to be required for full transcriptional activity. In vivo, this site may affect the known Ubx function of inducing apoptosis of a specific neuron during embryonic development, but appears to not be important for regulating the segment-specific denticle pattern in the cuticle. In another set of experiments, specific kinases were assessed for their potential roles in Ubx phosphorylation. The results strongly suggest Shaggy (Sgg)/Glycogen synthase kinase 3 (GSK3) and Basket (Bsk)/Jun kinase (JNK) to be most promising candidates for the most prominent phosphorylation site. They both co-immunoprecipitated with Ubx and are likely interacting with it in the cytoplasm of cultured cells. Furthermore, Sgg/GSK3 cannot phosphorylate Ubx when this site is mutated, suggesting that it is directly or indirectly involved in this phosphorylation event. Within scope of the second major aim of the proposal, exploring the tissue-specific variability of Hox PTMs, I succeeded in establishing, i.e. adapting protocols for large-scale isolation of L1 larval brains and haltere imaginal discs of L3 larvae, respectively. These can be applied to collect large samples in order to enrich the Ubx protein for liquid chromatography coupled to tandem mass spectrometry analyses of PTMs in these tissues.
