The Cutaneous Biology of MAGE Transcription Factors

MAGE 转录因子的皮肤生物学

• 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
• 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

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）：MAGE蛋白通常仅在发育中的生殖细胞中表达，但在一些痣、大多数黑色素瘤和许多恶性肿瘤中异常表达。它们的选择性表达使它们几乎是肿瘤特异性靶标，但主流范式将 MAGE 表达视为细胞转化的功能上无关的副产品。该项目挑战了这一范式，提出 MAGE 通过调节 KAP1 来调节细胞生长并促进肿瘤发展，KAP1 是一种支架蛋白，反过来又通过含有锌指转录因子 (KZNF) 的 KRAB 结构域大家族介导基因调节。长期目标是： 1. 展示 MAGE 蛋白如何通过充当 KZNF 的主调节因子来促进细胞生长和存活，以及 2. 基于干扰 MAGE 表达或功能开发针对恶性肿瘤的新疗法。具体目标是： 目标 1：识别和操纵 MAGE 对人类黑素细胞基因调控的影响。为了确定 MAGE 在黑素细胞中的作用，我们将使用正常人黑素细胞（有或没有异位 MAGE 表达）的 ChIP-seq 和 RNA-seq，将 MAGE 表达与全基因组 KAP1 结合、染色质压缩和基因抑制相关联。对照将使用低传代人黑色素瘤细胞系，有或没有 shRNA 介导的 MAGE 敲低，并出于美容目的而去除黑素细胞痣的染色质。分析将包括全面的基因注释、聚类和网络分析，用于多参数识别 MAGE 对细胞过程和关键下游组件的整体影响。验证将通过 RNAi 选择性抑制途径成分，通过增殖、存活和细胞周期调节分析来测试下游网络成分的作用。为了开始开发和验证抑制 MAGE 效应的小分子，将对 KAP1 结合和基因表达谱的 MAGE 敲低效应与小分子 MAGE 抑制剂的效应进行比较。目标 2：确定 MAGE 对不同基因的差异作用机制。我们发现 MAGE 蛋白可以增强或减少 KZNF 和 KAP1 介导的特定基因抑制。为了确定 MAGE 对基因调控的差异作用机制，我们将检验以下假设：KRAB 结构域的组成差异决定 MAGE 表达是否增加、减少或对 KZNF 介导的基因抑制没有影响。这些实验将通过一系列全面的 KRAB 结构域测试 MAGE 效应：哺乳动物两种杂交测定中的 KAP1-KRAB 结合，b． KAP1 在荧光素酶报告基因测定中介导基因抑制，c．通过免疫印迹 KAP1 介导 KZNF 泛素化。 公共卫生相关性：MAGE 表达与多种肿瘤的侵袭性临床病程、化疗耐药性的获得以及不良临床结果相关。该项目将为 MAGE 效应的机制和模式提供新的见解，从而强调从正常黑色素细胞向侵袭性黑色素瘤的转变。开发基于 MAGE 功能的疗法将挽救生命并产生巨大的经济影响。