The Cutaneous Biology of MAGE Transcription Factors

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，但在一些痣、大多数黑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成为肿瘤特异性靶点，但主导范式将法师表达视为细胞转化的功能无关副产物。该项目通过提出法师调节细胞生长并通过调节KAP 1促进肿瘤发展来挑战这种范式，KAP 1是一种支架蛋白，其反过来介导含有锌指转录因子（KZNF）的KRAB结构域的大家族的基因调节。长期目标是：1。显示法师蛋白如何通过作为KZNF的主要调节剂而促进细胞生长和存活，以及2.开发基于干扰法师表达或功能的恶性肿瘤新疗法。具体目的是：目的1：鉴定和操纵法师对人黑素细胞基因调控的作用。为了确定法师在黑素细胞中的作用，我们将使用正常人黑素细胞的ChIP-seq和RNA-seq将法师表达与全基因组KAP 1结合、染色质压实和基因阻遏相关联，有和没有异位法师表达。对照将使用低传代人黑色素瘤细胞系，具有和不具有shRNA介导的法师敲低，并且出于美容目的去除来自黑色素细胞痣的染色质。分析将包括全面的基因注释、聚类和网络分析，用于法师对细胞过程和关键下游组分的全局影响的多参数鉴定。验证将通过RNAi选择性抑制途径组分，用增殖、存活和细胞周期调节试验测试下游网络组分的作用。为了开始开发和验证抑制法师效应的小分子，将对KAP 1结合和基因表达谱的法师敲低效应与小分子法师抑制剂的效应进行比较。目的2：研究法师对不同基因的不同作用机制。我们已经发现法师蛋白可以增强或减少KZNF和KAP 1介导的特定基因的抑制。为了确定差异法师对基因调控的作用机制，我们将检验KRAB结构域组成的差异决定法师表达是否增加、减少或对KZNF介导的基因抑制没有影响的假设。这些实验将用一系列全面的KRAB结构域测试法师效应：哺乳动物双杂交测定中的KAP 1-KRA B结合，B.在荧光素酶报告基因测定中KAP 1介导的基因阻遏，和c.免疫印迹法检测KAP 1介导的KZNF泛素化。 公共卫生相关性：在许多类型的肿瘤中，法师表达与侵袭性临床过程、获得对化疗的抗性和不良临床结果相关。这个项目将提供新的见解机制和模式的法师的影响，强调从正常的黑色素细胞的过渡到侵略性黑色素瘤。开发基于法师功能的疗法将挽救生命并产生巨大的经济影响。

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.