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

第一个哺乳动物 N 项的作用。

基本信息

批准号：
8731642
负责人：
Christine E Schaner-Tooley
金额：
$ 22.7万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-05 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8731642
关键词：
A Mouse Acetylation Advisory Committees Affect Aneuploidy Antibodies Apoptotic Binding Biological Biological Assay Biological Process Biology Breast Epithelial Cells Cell Cycle Cell Line Cell Proliferation Cell Separation Cell physiology Chromatin Committee Members Consensus Sequence Cytoplasm DNA DNA Binding DNA repair protein DNA-Binding Proteins Data Defect Development Educational workshop Elements Embryo Environment Exhibits Fatty acid glycerol esters Fibroblasts Flow Cytometry Fluorescence Resonance Energy Transfer Fourier Transform Future Gene Combinations Gene Targeting Genes Genetic Gland Glioblastoma Goals Growth Harvest Histology Image Immunoprecipitation In Vitro Knockout Mice Learning Mammary gland Mass Spectrum Analysis Measures 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 Post-Translational Protein Processing Process Proteins Reagent Research Retinoblastoma Protein Role Running Series Site Slide System Techniques Testing Therapeutic Agents Tissue Sample Training Transgenic Organisms Transplantation Tumor Suppressor Genes Tumor Suppressor Proteins Tumor Tissue Universities Virginia career development cellular imaging chromatin immunoprecipitation design in vitro Assay in vivo interest leukemia malignant breast neoplasm mass spectrometer mouse model mutant myosin light chain 2 protein function research study skills stoichiometry tool tumor tumor progression tumorigenesis

项目摘要

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.

参与肿瘤发生的基因列表相当广泛，然而，它们的许多生物学功能仍然未知。其中一种基因 Mettl11a（现已更名为 NRMT）已被证明在乳腺癌，但最近才被鉴定为第一个哺乳动物 N 末端甲基转移酶。 NRMT 的减少也会导致多纺锤体表型，并且相关的非整倍性通常是被认为有助于癌症进展。由于 NRMT 是一种新发现的蛋白质，因此该研究的目标建议了解 N 末端甲基化对蛋白质和细胞功能的作用，以便研究它的失调如何导致肿瘤发生。前两个目的，确定N端是否甲基化是组成型的，并确定 N 端甲基化是否普遍改变其 DNA 结合底物，旨在更好地了解 N 末端甲基化的基本生物学。这些目标将涉及质谱分析、荧光激活细胞分选、FRET 分析和敲除构建老鼠。第三个目标是确定 NRMT 在发育和肿瘤发生中的作用。一个将建立小鼠乳腺移植模型来检测多轴NRMT敲低是否有效表型导致发育缺陷和/或肿瘤发生。职业发展的关键要素该提案的一个方面将是学习成功完成第三个目标所需的鼠标系统。目标一和二的实验是为了手术和腺体之间的停机时间而设计的。肿瘤收获。环境弗吉尼亚大学完全有能力完成该提案的所有三个目标。校园内咨询委员会成员 Don Hunt 博士的质谱设备具有傅里叶变换质量区分 N 末端甲基化和乙酰化所需的光谱仪。 UVA 流式细胞术 Core 将提供所有荧光激活细胞分选实验所需的培训。 UVA基因靶向和转基因设施将创建 NRMT 基因敲除小鼠。研究组织学核心将石蜡包埋所有正常小鼠组织和肿瘤并制作玻片。我的顾问委员会成员 Dr. 艾米·布顿 (Amy Bouton) 将帮助解释组织学。顾问委员会的专业知识和试剂成员 Todd Stukenberg 博士将帮助表征 NRMT 多纺锤体表型。长波紫外线 W.M.凯克细胞成像中心提供最先进的免疫荧光成像设施小鼠组织样本成像和 FRET 分析训练。 Macara实验室目前拥有三名专家小鼠生物学家可以接受小鼠处理和手术技术的培训。此外，UVA还提供许多旨在职业发展/培训的课程，包括 UVA 转基因方法和应用研讨会、流式细胞术培训研讨会、FRET 显微镜研讨会和博士后专业发展办公室每月提供职业发展研讨会系列。