The Cutaneous Biology of MAGE Transcription Factors

描述(申请人提供)：MAGE蛋白通常只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成为肿瘤的特异性靶点，但主导范式认为MAGE表达是细胞转化的一种功能无关的副产品。该项目挑战了这一模式，提出MAGE通过调节KAP1来调节细胞生长和促进肿瘤的发展，KAP1是一种支架蛋白，反过来又通过包含锌指转录因子(KZNF)的KRAB结构域大家族来介导基因调控。长期的目标是：1.阐明MAGE蛋白如何通过作为KZNF的主要调节因子而促进细胞生长和存活；2.开发基于干扰MAGE表达或功能的恶性肿瘤的新疗法。其具体目的是：目的1：鉴定和操纵MAGE对人黑素细胞基因调控的作用。为了确定MAGE在黑素细胞中的作用，我们将使用正常人类黑素细胞的CHIP-SEQ和RNA-SEQ将MAGE的表达与全基因组KAP1结合、染色质压缩和基因抑制相关联，无论是否存在MAGE的异位表达。对照组将使用低传代的人类黑色素瘤细胞系，带有和不带有shRNA介导的MAGE基因敲除，以及出于美容目的从黑色素细胞痣中去除的染色质。分析将包括全面的基因注释、聚类和网络分析，以多参数识别MAGE对细胞过程和关键下游组件的全球影响。验证将通过使用RNAi选择性地抑制途径组件，通过检测增殖、存活和细胞周期调节来测试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，MAGE对KAP1结合和基因表达谱的击倒作用将与小分子MAGE抑制剂的效果进行比较。目的2：探讨MAGE对不同基因的差异作用机制。我们发现MAGE蛋白可以增强或降低KZNF和KAP1介导的特定基因的抑制。为了确定MAGE对基因调控的差异作用机制，我们将检验这样一种假设，即KRAB结构域的组成差异决定了MAGE在KZNF介导的基因抑制中的表达是增加、减少还是没有影响。这些实验将测试一系列完整的KRAB结构域的MAGE效应：a.在哺乳动物双杂交实验中KAP1-KRAb结合，b.KAP1在荧光素酶报告基因实验中介导的基因抑制，以及c.KAP1通过免疫印迹介导的KZNF泛素化。 公共卫生相关性：在许多类型的肿瘤中，MAGE的表达与侵袭性的临床病程、化疗耐药的获得和不良的临床结果相关。这个项目将为MAGE效应的机制和模式提供新的见解，这些效应强调了从正常黑素细胞到侵袭性黑色素瘤的转变。开发基于MAGE功能的疗法将拯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.