Mechanisms of trans-repression by Myc in Drosophila

描述（由申请人提供）：该项目的长期目标是了解分化过程如何防止胚胎发生过程中的过度增殖。更具体地说，转录因子和癌蛋白Myc是胚胎发生过程中增殖所必需的，然而Myc蛋白的过丰度诱导肿瘤；胚胎细胞如何实现Myc功能的正确平衡？Myc是一种转录调节剂，具有激活和抑制不同靶标转录的能力。Myc的转录激活需要其与DNA结合，尽管Myc的抑制不一定是由其与DNA的结合介导的，但无法结合DNA的Myc突变体无法抑制。最早发现的Myc抑制靶点之一是c-myc基因本身，所有测试的人类肿瘤来源细胞系都存在Myc自我抑制的缺陷。为了了解myc自动抑制的机制，我和普林斯顿大学的Eric Wieschaus和Michael Cole一起，用果蝇开发了一种新的基因筛选方法。我们发现，维持细胞命运所需的染色质结合抑制因子Polycomb的突变消除了果蝇myc基因的自身抑制，也消除了胚胎中由异位myc介导的大部分抑制。Polycomb的突变也导致许多Myc激活靶点的表达增加，这表明Polycomb、Myc的抑制和Myc的反激活之间存在联系。虽然在生物学上很重要，但Polycomb和Myc之间的联系尚不清楚是直接的还是间接的。事实上，在果蝇胚胎中，Myc诱导了Polycomb功能相关基因产物的表达：Su(z)2、Esc和Pho（分别是哺乳动物bmi-1、eed和YY1的同源物）。在这个提议中，我假设Polycomb Group基因产物Su(z)2， Esc和Pho的Myc激活导致Myc对靶标的下游抑制，并且这些抑制因子在整个胚胎发生过程中的相对丰度影响Myc对靶标的反式抑制。为了验证这一假设，我的具体目标是：(1)确定Myc是否直接与Su(z)2、pho和/或esc基因结合，导致它们的激活；(2)确定是否需要Su(z)2和/或Esc来抑制Myc；(3)确定在胚胎发生过程中与Myc相关的蛋白质。导致Myc表达增加的基因改变导致不受控制的增殖和肿瘤发生。然而，在胚胎中，Myc水平很高，增殖受到控制。本提案中所描述的研究将深入了解胚胎细胞维持正常增殖和防止肿瘤发生的机制。在果蝇中可用的遗传和分子生物学工具将迅速提供一个难以（如果不是不可能的话）利用哺乳动物系统获得的深度理解。公共卫生相关性：生物体在正常过程中需要细胞增殖，如发育过程中的生长和血液产生。一种名为Myc的蛋白质是正常增殖所必需的，尽管Myc过多会导致癌症。本研究旨在探讨Myc在具有天然高水平Myc的组织中的活性机制。

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand how the process of differentiation prevents over-proliferation during embryogenesis. More specifically, the transcription factor and onco-protein Myc is required for proliferation during embryogenesis, and yet an over-abundance of Myc protein induces tumors; how do embryonic cells achieve the correct balance of Myc function? Myc is a transcriptional regulator with the ability to activate and repress transcription of different targets. Transcriptional activation by Myc requires its binding to DNA, and though repression by Myc is not necessarily mediated by its binding to DNA, a Myc mutant that is unable to bind DNA is unable to repress. One of the first identified targets of Myc repression was the c-myc gene itself, and all human tumor-derived cell lines tested are defective for this auto-repression of myc. In an effort to understand the mechanism of myc auto-repression, along with Eric Wieschaus and Michael Cole at Princeton University, I developed a novel genetic screen using Drosophila melanogaster. We found that mutations in Polycomb, a chromatin binding repressor required for maintenance of cell fates, eliminate auto-repression of the Drosophila myc gene, and also the majority of repression mediated by ectopic Myc in the embryo. Mutations in Polycomb also led to increased expression of many of Myc's targets of activation, suggesting a link between Polycomb, repression by Myc and transactivation by Myc. Though biologically important, the link between Polycomb and Myc is not known to be direct or indirect. In fact, in the Drosophila embryo, Myc induces the expression of gene products functionally related to Polycomb: Su(z)2, Esc and Pho (homologs of mammalian bmi-1, eed and YY1, respectively). In this proposal, I hypothesize that Myc activation of Polycomb Group gene products Su(z)2, Esc and Pho causes Myc's downstream repression of targets, and that the relative abundance of these repressors throughout embryogenesis affects Myc trans-repression of targets. To test this hypothesis, my specific aims are: (1) to determine whether Myc binds directly to Su(z)2, pho and/or esc genes, causing their activation; (2) to determine whether Su(z)2 and/or Esc are required for repression by Myc; and (3) to determine what proteins associate with Myc throughout embryogenesis. Genetic alterations that lead to increased Myc expression cause uncontrolled proliferation and tumorigenesis. In an embryo, however, Myc levels are high and proliferation is controlled. The research described in this proposal will provide insight into the mechanisms operating in embryonic cells that maintain normal proliferation and prevent tumorigenesis. The genetic and molecular biological tools available in Drosophila will rapidly provide a depth of understanding that is difficult, if not impossible, to obtain using mammalian systems. PUBLIC HEALTH RELEVANCE: Organisms require cellular proliferation for normal processes such as growth during development and blood production. A protein called Myc is required for normal proliferation, although Myc causes cancer when over-abundant. This proposal investigates mechanisms of Myc activity in a tissue with naturally high levels of Myc.