In the fruit fly D. melanogaster, the specific timing of genes for the development of the chorion is tightly regulated by the Myb-MuvB complex, which consists of many transcriptional factors. This tight control of gene expression - the laxing of which is often involved in oncogenesis and other diseases - relies on intricate protein signalling pathways via posttranslational modifications (PTMs). Recently, the functional aspects of several proteins in the Myb-MuvB complex were elucidated by classical biochemical and molecular genetics techniques. It was found that this complex is very unique as it plays an activating role during specific stages of developmental and at other stages, a repressive role. The first long-term goal of this proposal is to continue using molecular genetics techniques (e.g., RNAi, //p-out mutants, etc) to uncover the function of uncharacterized proteins in the complex (e.g., Lin-52). Dr. Botchan's laboratory has already elucidated the function of many proteins in this complex, such as determining the critical interaction between Mip130 and Myb to maintain viability. By using established protocols and working closely with other Postdoctoral researchers, I will characterize the role of Lin-52 in this complex, as preliminary work suggests this protein is also required for viability. The second goal is to use an on-site high-resolution LTQ-Fourier Transform Mass Spectrometer to characterize the posttranslational modifications (PTMs) that may be pivotal to the repressor/activator roles of Myb-MuvB. Preliminary work (presented herein) has identified novel phosphorylation sites on Myb-MuvB protein members using capillary LC/LTQ-FTMS/MS. This is a very important first step since it shows the feasibility of our analytical approach: the detection of PTMs on a very rare complex purified from relatively few Drosophila embryos. The last long-term objective is to identify histone H3 and H4 PTMs around the sites that Myb-MuvB represses or activates. This will involve co- immunoprecipitating Myb-MuvB:nucleosome complexes, acid extraction of the histones, and subsequent characterization of histone PTMs by LC/LTQ-FTMS/MS. This research will couple molecular genetics with proteomics to elucidate the function of the unique gene activator/repressor complex Myb-MuvB. By fully characterizing the role of newly discovered proteins in this complex and whether protein modification plays a role in its function, we will uncover vital information on gene regulation, which is the focal point of many human diseases. [unreadable] [unreadable] [unreadable]
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