The role of the first mammalian N-term. methyltransferase, NRMT, in tumorigenesis

第一个哺乳动物 N 项的作用。

• 批准号: 8328713
• 负责人: Christine E Schaner-Tooley
• 金额: $ 8.79万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-05 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8328713
• 关键词: A Mouse; Acetylation; Advisory Committees; Affect; Aneuploidy; Antibodies; Apoptotic; Binding; Biological; Biological Assay; Biological Process; Biology; Cell Cycle; Cell Line; Cell Proliferation; Cell Separation; Cell physiology; Chromatin; Committee Members; Consensus Sequence; Critiques; Cytoplasm; DNA; DNA Binding; DNA repair protein; DNA-Binding Proteins; Data; Defect; Development; Educational workshop; Elements; Embryo; Environment; Epithelial Cells; Exhibits; Fatty acid glycerol esters; Fellowship; Fibroblasts; Flow Cytometry; Fluorescence Resonance Energy Transfer; Fourier Transform; Future; Gene Combinations; Gene Targeting; Genes; Genetic; Gland; Glioblastoma; Goals; Growth; Harvest; Histology; Human; Image; Immunoprecipitation; In Vitro; Individual; Institution; Knockout Mice; Lead; Learning; Malignant Neoplasms; Mammary gland; Mass Spectrum Analysis; Measures; Medicine; Mentors; Methods; Methylation; Methyltransferase; Microarray Analysis; Microscopy; Mitotic; Modeling; Modification; Monitor; Mono-S; Mus; Mutation; N-terminal; Neoplasm Metastasis; Nuclear; Oncogene Proteins; Operative Surgical Procedures; Paraffin Embedding; Patients; Pattern; Pharmaceutical Preparations; Phase; Phenotype; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; Reagent; Research; Retinoblastoma Protein; Role; Running; Series; Signal Pathway; Site; Slide; System; Techniques; Testing; Therapeutic Agents; Time; Tissue Sample; Training; Transgenic Organisms; Transplantation; Tumor Suppressor Genes; Tumor Suppressor Proteins; Tumor Tissue; Universities; Virginia; Work; Writing; career development; cellular imaging; chromatin immunoprecipitation; design; experience; in vitro Assay; in vivo; interest; leukemia; malignant breast neoplasm; mass spectrometer; meetings; mouse model; mutant; myosin light chain 2; programs; protein function; research study; skills; stoichiometry; tool; tumor; tumor progression; tumorigenesis

DESCRIPTION (provided by applicant): During my postdoctoral fellowship, I successfully purified and identified the first mammalian N-terminal methyltransferase, NRMT. Shown by microarray analysis to have reduced expression/copy number in human breast cancers, I have also illustrated NRMT knockdown results in increased cell proliferation and a multi- spindle phenotype. Given that NRMT knockdown results in these two cancer prone phenotypes, I intend to further study how it is regulated, how N-terminal methylation affects substrate function, and how NRMT misregulation leads to tumorigenesis. My goal after completion of my postdoctoral work is to obtain a tenure- track position at an academic research institution and establish an independent research program. Successful completion of my research goals includes training in a mouse mammary transplant model. As I have no previous mouse research experience, the additional training time provided by a K99 will be imperative for learning these skills. Obtaining a K99, will also allow for further training in mass spectrometry, fluorescent- activating cell sorting, and FRET analysis. The long-term goal of my research is to use the NRMT mouse model to dissect the signaling pathways that lead to breast cancer. As personalized medicine treatments become increasingly necessary, I want to create a model where combinations of genes can be easily manipulated simultaneously. These models can be used to test which current drug treatments are most effective for patients with specific combinations of mutations and to screen for new and more effective therapeutic agents. Research The list of genes involved in tumorigenesis is quite extensive, however, many of their biological functions remain unknown. One such gene, Mettl11a (now renamed NRMT), has been shown to be under-expressed in breast cancers, but has only recently been identified as the first mammalian N-terminal methyltransferase. Reduction of NRMT also results in a multi-spindle phenotype, and the associated aneuploidy is often considered to contribute to cancer progression. As NRMT is a newly discovered protein, the goals of this proposal are to understand the role of N-terminal methylation on protein and cellular function, in order to study how its misregulation leads to tumorigenesis. The first two aims, determining whether N-terminal methylation is constitutive and determining whether N-terminal methylation universally alters the DNA binding of its substrates, are designed to better understand the basic biology of N-terminal methylation. These aims will involve mass spectrometry, fluorescent-activating cell sorting, FRET analysis, and construction of a knockout mouse. The objective of the third aim is to determine the role of NRMT in development and tumorigenesis. A mouse mammary transplant model will be established for assaying if the multi-spindle NRMT knockdown phenotype leads to developmental defects and/or tumorigenesis. The key element of the career development aspect of this proposal will be learning the mouse system needed for successful completion of the third aim. The experiments of aims one and two have been designed for the downtime between surgeries and gland or tumor harvesting. Environment The University of Virginia is well equipped for completion of all three aims of this proposal. The on-campus mass spectrometry facility, of advisory committee member Dr. Don Hunt, has a Fourier transform mass spectrometer necessary for distinguishing N-terminal methylation from acetylation. The UVA Flow Cytometry Core will provide the training necessary for all fluorescent-activating cell sorting experiments. The UVA Gene Targeting and Transgenic Facility will create the NRMT knockout mouse. The Research Histology Core will paraffin embed and make slides of all normal mouse tissue and tumors. My advisory committee member Dr. Amy Bouton will aid in interpretation of the histology. The expertise and reagents of advisory committee member Dr. Todd Stukenberg will aid in characterization of the NRMT multi-spindle phenotype. The UVA W.M. Keck Center for Cellular Imaging supplies state-of-the art imaging facilities for immunofluorescent imaging of mouse tissue samples and training in FRET analysis. The Macara lab currently has three expert mouse biologists available for training in mouse handling and surgical techniques. In addition, UVA offers numerous courses aimed at career development/training, including the UVA transgenic methods and applications workshop, the Flow Cytometry Training Workshop, the Workshop on FRET Microscopy, and a monthly career development seminar series offered by the Office of Postdoctoral Professional Development.

描述（由申请人提供）：在我的博士后研究金期间，我成功纯化并确定了第一个哺乳动物N末端甲基转移酶NRMT。通过微阵列分析显示，在人类乳腺癌中的表达/拷贝数降低，我还说明了NRMT敲低导致细胞增殖增加和多纺锤表型的增加。鉴于NRMT敲低导致这两种癌症表型导致，我打算进一步研究其如何调节，N末端甲基化如何影响底物功能以及NRMT的正调如何导致肿瘤发生。我的博士后工作完成后，我的目标是在学术研究机构获得任期轨道地位，并建立独立的研究计划。成功完成我的研究目标包括对小鼠乳腺移植模型进行培训。由于我以前没有鼠标研究经验，因此K99提供的额外培训时间对于学习这些技能至关重要。获得K99，还将允许在质谱法，荧光激活细胞排序和FRET分析方面进行进一步的训练。我的研究的长期目标是使用NRMT小鼠模型来剖析导致乳腺癌的信号传导途径。随着个性化的医学治疗变得越来越必要，我想创建一个模型，其中很容易同时操纵基因的组合。这些模型可用于测试哪种当前药物治疗对具有特定突变组合的患者最有效，并筛选出新的，更有效的治疗剂。研究肿瘤发生涉及的基因清单非常广泛，但是，其许多生物学功能仍然未知。一种这样的基因Mettl11a（现已更名为NRMT）在乳腺癌中被证明不足，但直到最近才被鉴定为第一个哺乳动物N末端甲基转移酶。 NRMT的还原还会导致多主轴表型，并且相关的非整倍性通常被认为有助于癌症进展。由于NRMT是一种新发现的蛋白质，因此该提案的目标是了解N末端甲基化对蛋白质和细胞功能的作用，以研究其不正调如何导致肿瘤发生。确定N末端甲基化是否是组成型的，并确定N端甲基化是否普遍改变其底物的DNA结合，旨在更好地理解N末端甲基化的基本生物学。这些目标将涉及质谱，荧光激活细胞分选，FRET分析和敲除小鼠的构造。第三个目的的目的是确定NRMT在发育和肿瘤发生中的作用。如果多主轴NRMT敲低表型导致发育缺陷和/或肿瘤发生，则将建立小鼠乳腺移植模型。该建议的职业发展方面的关键要素将是学习成功完成第三个目标所需的鼠标系统。目的的实验是针对手术与腺体或肿瘤收集之间的停机时间设计的。环境弗吉尼亚大学能够完成该提案的所有三个目标。顾问委员会成员Don Hunt博士的校园质谱设施具有傅立叶变换质谱仪，可将N末端甲基化与乙酰化区分开。 UVA流式细胞仪核心将为所有荧光激活细胞分选实验提供必要的训练。 UVA基因靶向和转基因设施将创建NRMT敲除鼠标。研究组织学核心将嵌入石蜡并制造所有正常小鼠组织和肿瘤的载玻片。我的咨询委员会成员艾米·布顿（Amy Bouton）博士将帮助解释组织学。咨询委员会成员Todd Stukenberg博士的专业知识和试剂将有助于表征NRMT多主轴表型。 UVA W.M.凯克（Keck）细胞成像中心提供了最先进的成像设施，用于对小鼠组织样品进行免疫荧光成像和FRET分析的训练。 Macara Lab目前有三位专家生物学家可用于鼠标处理和手术技术的培训。此外，UVA提供了许多针对职业发展/培训的课程，包括UVA转基因方法和应用研讨会，流式细胞仪培训研讨会，FRET显微镜研讨会以及由后期专业专业发展办公室提供的每月职业发展研讨会系列。