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

描述（由申请人提供）：在博士后期间，我成功纯化并鉴定了第一个哺乳动物n端甲基转移酶NRMT。微阵列分析显示，在人类乳腺癌中表达/拷贝数减少，我也说明了NRMT敲低导致细胞增殖增加和多纺锤体表型。鉴于NRMT敲低导致这两种癌症易感表型，我打算进一步研究它是如何被调节的，n端甲基化如何影响底物功能，以及NRMT错误调节如何导致肿瘤发生。完成博士后工作后，我的目标是在学术研究机构获得终身职位，并建立一个独立的研究项目。成功完成我的研究目标包括在小鼠乳腺移植模型中进行训练。由于我以前没有老鼠研究的经验，K99提供的额外训练时间对于学习这些技能是必不可少的。获得K99，也将允许进一步的培训，质谱，荧光活化细胞分选，和FRET分析。我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通路。随着个性化医学治疗变得越来越有必要，我想创建一个模型，在这个模型中，基因的组合可以很容易地同时被操纵。这些模型可用于测试当前哪种药物治疗对具有特定突变组合的患者最有效，并筛选新的和更有效的治疗药物。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尚不清楚。其中一个这样的基因，Mettl11a（现在更名为NRMT），已经被证明在乳腺癌中表达不足，但直到最近才被确定为第一个哺乳动物n端甲基转移酶。NRMT的减少也会导致多纺锤体表型，而相关的非整倍体通常被认为有助于癌症的进展。由于NRMT是一种新发现的蛋白质，本课题的目标是了解n端甲基化在蛋白质和细胞功能中的作用，以研究其错误调控如何导致肿瘤发生。前两个目标，确定n端甲基化是否构成和确定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端甲基化的基本生物学。这些目标将包括质谱分析、荧光激活细胞分选、FRET分析和基因敲除小鼠的构建。第三个目的是确定NRMT在发展和肿瘤发生中的作用。我们将建立小鼠乳腺移植模型，分析多纺锤体NRMT敲低表型是否会导致发育缺陷和/或肿瘤发生。本提案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。目标一和目标二的实验是为手术和腺体或肿瘤收获之间的停机时间而设计的。弗吉尼亚大学为完成这一提案的三个目标做好了充分的准备。咨询委员会成员唐·亨特博士的校园质谱分析设备有一台傅里叶变换质谱仪，用于区分n端甲基化和乙酰化。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培训。UVA基因靶向和转基因设施将创建NRMT敲除小鼠。研究组织学核心将对所有正常小鼠组织和肿瘤进行石蜡包埋和切片。我的顾问委员会成员艾米·伯顿博士将协助解释组织学。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多纺锤体表型。UVA W.M. Keck细胞成像中心提供最先进的成像设备，用于小鼠组织样本的免疫荧光成像和FRET分析培训。Macara实验室目前有三名老鼠生物学专家，可以接受老鼠处理和手术技术方面的培训。此外，弗吉尼亚大学还提供了许多针对职业发展/培训的课程，包括弗吉尼亚大学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，以及由博士后专业发展办公室提供的每月职业发展系列研讨会。

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.