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

基因调控中抑制子/激活子复合物 Myb-Muvb 的表征

• 批准号: 7652447
• 负责人: James Joseph Pesavento
• 金额: $ 5.17万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-02 至 2010-07-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7652447
• 关键词: Acids; Affect; Animal Model; Area; Binding Sites; Biochemical; Biochemistry; Blood capillaries; Cells; Cellular biology; Chorion; Chromosomes, Human, Pair 3; Complex; Core Protein; DNA Binding; DNA biosynthesis; DNA replication origin; Detection; Development; Differentiated Gene; Disease; Drosophila genus; Drosophila melanogaster; E2F2 transcription factor; Embryo; Fellowship; Fourier Transform; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Techniques; Genetic Transcription; Genome; Goals; Histone H3; Histones; Investigation; Laboratories; Learning; Link; Mass Spectrum Analysis; Mediating; Modeling; Modification; Molecular; Molecular Biology Techniques; Molecular Genetics; Mutagenesis; Nature; Nucleosomes; Oocytes; Organism; Pathway interactions; Pattern; Phosphorylation Site; Play; Post-Translational Protein Processing; Pre-Replication Complex; Process; Proteins; Proteomics; Protocols documentation; RNA Interference; Replication Initiation; Replication-Associated Process; Research; Research Personnel; Resolution; Role; Signal Pathway; Signal Transduction; Site; Staging; System; Techniques; Time; Training; Work; capillary; histone modification; human MPP1 protein; human disease; mass spectrometer; member; mutant; novel; protein complex; recombinational repair; tool; transcription factor; tumorigenesis

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.

描述（由申请人提供）：在果蝇D。在黑腹果蝇中，由许多转录因子组成的Myb-MuvB复合体严格调节基因发育绒毛膜的特定时间。这种对基因表达的严格控制--其松懈通常与肿瘤发生和其他疾病有关--依赖于通过翻译后修饰（PTM）的复杂蛋白质信号传导途径。最近，在Myb-MuvB复合物的几个蛋白质的功能方面阐明了经典的生物化学和分子遗传学技术。人们发现，这种复合物是非常独特的，因为它在特定的发展阶段起着激活作用，在其他阶段起着抑制作用。该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）以揭示复合物中未表征的蛋白质的功能（例如，Lin-52）。Botchan博士的实验室已经阐明了这种复合物中许多蛋白质的功能，例如确定Mip 130和Myb之间维持活力的关键相互作用。通过使用已建立的方案并与其他博士后研究人员密切合作，我将描述Lin-52在这种复合物中的作用，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的阻遏物/激活物作用至关重要的翻译后修饰（PTM）。初步工作（本文所述）已经确定了新的磷酸化位点的Myb-MuvB蛋白成员使用毛细管LC/LTQ-FTMS/MS。这是一个非常重要的第一步，因为它显示了我们的分析方法的可行性：检测PTM的一个非常罕见的复杂的纯化相对较少的果蝇胚胎。最后一个长期目标是确定Myb-MuvB抑制或激活的位点周围的组蛋白H3和H4 PTM。这将涉及共免疫沉淀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PTM。这项研究将分子遗传学与蛋白质组学相结合，以阐明独特的基因激活剂/阻遏物复合物Myb-MuvB的功能。通过充分表征新发现的蛋白质在该复合物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。