The Cutaneous Biology of MAGE Transcription Factors

MAGE 转录因子的皮肤生物学

基本信息

批准号：
8385968
负责人：
B Jack Longley
金额：
$ 20.32万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8385968
关键词：
Affect Aggressive Clinical Course Animal Care and Use Committees Apoptosis Binding Biological Assay Biology Boxing Cell Cycle Regulation Cell Line Cell Survival Cell physiology ChIP-seq Characteristics Chemistry Chromatin Chromatin Structure Clinical Cosmetics Critical Pathways Cutaneous DNA Sequence Development Drug Delivery Systems Family Funding Future Gene Activation Gene Expression Gene Expression Regulation Gene Targeting Genes Germ Cells Goals Growth Human Immunoblotting In Vitro Individual Luciferases Malignant Neoplasms Mediating Melanocytic nevus Melanoma Cell Molecular Bank Molecular Profiling Mus Neoplastic Cell Transformation Nevus Outcome Pathway Analysis Pathway interactions Patients Pattern Pharmaceutical Chemistry Production Protein Binding Proteins Protocols documentation RNA RNA Interference Reporter Genes Repression Resistance Resources Role Scaffolding Protein Screening procedure Series Signal Pathway Testing Tumor Suppressor Proteins Two-Hybrid System Techniques Ubiquitination United States National Institutes of Health Validation Work Zinc Fingers base cell growth chemotherapy gene repression genome-wide human ZNF45 protein in vivo inhibitor/antagonist insight melanocyte melanoma melanoma-associated antigen metaplastic cell transformation novel novel strategies research study selective expression skills small hairpin RNA small molecule therapy development transcription factor tumor ubiquitin ligase

项目摘要

DESCRIPTION (provided by applicant): MAGE proteins are normally expressed only in developing germ cells but are aberrantly expressed in some nevi, most melanomas, and many malignancies. Their selective expression makes them nearly tumor specific targets but the dominant paradigm regards MAGE expression as a functionally irrelevant byproduct of cellular transformation. This project challenges this paradigm by proposing that MAGE regulate cell growth and contribute to tumor development by regulating KAP1, a scaffolding protein that in turn mediates gene regulation by the large family of KRAB domain containing zinc finger transcription factors (KZNFs). The long term goals are: 1. To show how MAGE proteins contribute to cell growth and survival by functioning as master regulators of KZNFs, and 2. To develop novel therapies for malignancies based on interfering with MAGE expression or function. The Specific Aims are: Aim 1: To identify and manipulate MAGE effects on gene regulation in human melanocytes. To determine MAGE effects in melanocytes, we will correlate MAGE expression with genome wide KAP1 binding, chromatin compaction, and gene repression using ChIP-seq and RNA-seq of normal human melanocytes, with and without ectopic MAGE expression. Controls will use low passage human melanoma cell lines, with and without shRNA mediated MAGE knockdown, and chromatin from melanocytic nevi removed for cosmetic purposes. Analysis will include comprehensive gene annotation, clustering, and network analysis for multi-parametric identification of global effects of MAGE on cellular processes and critical downstream components. Validation will test the role of downstream network components with assays for proliferation, survival and cell cycle regulation, by selective inhibition of pathway components with RNAi. To begin to develop and validate small molecules that inhibit MAGE effects, MAGE knockdown effects on KAP1 binding and gene expression profiles will be compared to the effects of small molecule MAGE inhibitors. Aim 2: To determine mechanisms for differential MAGE effects on different genes. We have discovered that MAGE proteins can enhance or decrease KZNF and KAP1 mediated repression of specific genes. To determine mechanisms for differential MAGE effects on gene regulation, we will test the hypothesis that differences in the composition of KRAB domains determines whether MAGE expression increases, decreases, or has no effect on KZNF mediated gene repression. These experiments will test MAGE effects with a comprehensive series of KRAB domains for: a. KAP1- KRAB binding in a mammalian two hybrid assay, b. KAP1 mediated gene repression in a luciferase reporter gene assay, and c. KAP1 mediated KZNF ubiquitination by immunoblotting. PUBLIC HEALTH RELEVANCE: MAGE expression has been correlated with aggressive clinical course, the acquisition of resistance to chemotherapy and poor clinical outcome in many types of tumors. This project will provide novel insights into mechanisms and patterns of MAGE effects that underline transition from normal melanocyte to aggressive melanoma. Developing therapies based on MAGE functions will save lives and have a huge economical impact.

描述（由申请人提供）：法师蛋白通常仅在发育中的生殖细胞中表达，但在某些NEVI，大多数黑色素瘤和许多恶性肿瘤中都异常表达。它们的选择性表达使它们几乎是肿瘤特定靶标的，但主要范式将MAGE表达视为细胞转化的功能无关的副产品。该项目通过提出法师调节细胞生长并通过调节KAP1来促进肿瘤的发展，从而挑战了这一范式，这是一种脚手架蛋白，这又介导了含有锌指转录因子（KZNFS）的大型krab结构域调节基因调节的基因调节。长期目标是：1。通过充当KZNF的主要调节剂，以表明法师蛋白如何促进细胞生长和生存，而2。基于干扰法师表达或功能的新型恶性肿瘤。具体目的是：目标1：识别和操纵法师对人黑色素细胞基因调节的影响。为了确定黑素细胞中的MAGE效应，我们将使用正常人黑色素细胞的chip-seq和RNA-Seq与基因组宽KAP1结合，染色质压实和基因抑制相关联，具有和不具有异位MAGE表达。对照组将使用低通道的人类黑色素瘤细胞系，有或没有SHRNA介导的法术敲低，而从黑色素细胞NEVI中取出的染色质则用于化妆品。分析将包括综合基因注释，聚类和网络分析，以识别法师对蜂窝过程和关键下游成分的全球影响的多参数识别。验证将通过选择性抑制RNAi的途径成分来测试具有增殖，存活和细胞周期调节测定法的下游网络组件的作用。为了开始开发和验证抑制MAGE效应的小分子，将将对KAP1结合和基因表达谱的作用与小分子MAGE抑制剂的作用进行比较。目标2：确定差异法对不同基因的差异作用的机制。我们发现法师蛋白可以增强或减少KZNF和KAP1介导的特定基因抑制作用。为了确定差异法对基因调节的影响的机制，我们将检验以下假设：KRAB结构域组成的差异决定MAGE表达是否增加，降低或对KZNF介导的基因抑制没有影响。这些实验将通过一系列krab域测试法师效应： KAP1-在哺乳动物两种混合分析中的Krab结合，b。 KAP1介导的荧光素酶报告基因测定中的基因抑制，c。 KAP1通过免疫印迹介导的KZNF泛素化。公共卫生相关性：法师表达与积极的临床过程，对化学疗法的抵抗力获得，在许多类型的肿瘤中抗药性不佳。该项目将提供对从正常黑素细胞到侵袭性黑色素瘤过渡的MAGE效应机制和模式的新见解。基于法师职能的制定疗法将挽救生命并产生巨大的经济影响。