Characterizing the Repressor/Activator Complex Myb-Muvb in Gene Regulation

描述（由申请人提供）：在果蝇D. melanogaster中，绒毛膜发育的特定基因时间受到Myb-MuvB复合物的严格调控，该复合物由许多转录因子组成。这种对基因表达的严格控制依赖于通过翻译后修饰（PTMs）形成的复杂的蛋白质信号通路，这种基因表达的放松通常与肿瘤发生和其他疾病有关。近年来，通过经典的生物化学和分子遗传学技术，研究人员对Myb-MuvB复合体中几种蛋白的功能进行了研究。我们发现这个复合体是非常独特的，因为它在特定的发育阶段起着激活作用，在其他阶段起着抑制作用。本提案的第一个长期目标是继续使用分子遗传学技术（例如RNAi， //p-out突变体等）来揭示复合物中未表征蛋白的功能（例如Lin-52）。Botchan博士的实验室已经阐明了该复合体中许多蛋白质的功能，例如确定Mip130和Myb之间维持生存能力的关键相互作用。通过使用既定的方案并与其他博士后研究人员密切合作，我将描述Lin-52在该复合体中的作用，因为初步工作表明这种蛋白质也是生存所必需的。第二个目标是使用现场高分辨率ltq -傅立叶变换质谱仪来表征可能对Myb-MuvB的抑制/激活作用至关重要的翻译后修饰（PTMs）。通过毛细管LC/LTQ-FTMS/MS，初步研究发现了Myb-MuvB蛋白成员上新的磷酸化位点。这是非常重要的第一步，因为它表明了我们的分析方法的可行性：在从相对较少的果蝇胚胎中纯化的非常罕见的复合物上检测到ptm。最后一个长期目标是确定Myb-MuvB抑制或激活位点周围的组蛋白H3和H4 PTMs。这将包括共免疫沉淀Myb-MuvB：核小体复合物，组蛋白的酸提取，以及随后通过LC/LTQ-FTMS/MS表征组蛋白PTMs。本研究将结合分子遗传学和蛋白质组学来阐明独特的基因激活/抑制复合物Myb-MuvB的功能。通过充分表征新发现的蛋白质在该复合体中的作用以及蛋白质修饰是否在其功能中发挥作用，我们将揭示基因调控的重要信息，这是许多人类疾病的焦点。

DESCRIPTION (provided by applicant): 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.