Determining mechanisms of PRC2-mediated disease using Drosophila

项目摘要/总结 正常发育需要严格控制基因表达，以精确调节组织模式， 不能正确控制表达谱可导致疾病。翻译后修饰 包装DNA基因组的组蛋白，可以通过使基因组中的蛋白质与DNA结合来调节潜在基因的表达。 DNA或多或少可用于转录机器。Polycomb repressive complex 2（PRC 2）是一种高度抑制性的蛋白质。 保守的组蛋白甲基转移酶，在发育过程中通过促进染色质 压实PRC 2催化组蛋白H3上赖氨酸27的甲基化（H3 K27 me 3），并使这种表观遗传的甲基化丧失。 标记与许多发育性疾病有关。弥漫性内在脑桥胶质瘤（DIPG），一种致命的 儿科脑癌，是由组蛋白H3（H3 K27 M）上的赖氨酸-甲硫氨酸突变引起的，该突变抑制PRC 2， 导致H3 K27 me 3几乎全部损失。类似地，通过EZHIP的异常表达抑制PRC 2， 导致另一种小儿神经胶质瘤，后颅窝室管膜瘤A型（PFA）。相比之下，功能丧失 两个核心PRC 2亚基的突变导致Weaver综合征，这是一种先天性疾病， 发育过度和认知迟缓，但不是癌症这些疾病引起的不同临床表型 等位基因可能是不同模式的PRC 2抑制和组织特异性发育背景的结果， 他们所表达的。该提案寻求利用遗传学和基因组学方法的组合 以确定PRC 2抑制的不同模式和发育背景对临床表型的重要性。 由于PRC 2在物种间高度保守，因此将使用黑腹果蝇来测试PRC 2的相对保守性。 使用一系列实验方法来研究这些特征中的每一个对疾病的贡献。的影响 疾病等位基因对组织模式、PRC 2功能和基因调控的影响将在动态， 发展背景。调节疾病等位基因效应的基因，包括潜在的治疗基因， 目标，将进行系统的测试。这些方法将哈里森实验室的专业知识（基因） 表达和发展）和刘易斯实验室（染色质调控和癌症），以解决基本的 关于这些疾病等位基因的机制问题。威斯康星州大学资源丰富 医学和公共卫生学院和华盛顿大学Carbone癌症中心提供了一个理想的位置， 建议的工作。总之，这项工作将定义PRC 2疾病等位基因之间的机制差异， 将揭示潜在的差异，可能导致其独特的，毁灭性的临床表型。完成 这项研究将确保实验，指导，临床和沟通技能的发展， 使成功过渡到职业生涯作为一个物理学家，科学家。

PROJECT ABSTRACT/SUMMARY Normal development requires tightly controlled gene expression to precisely regulate tissue patterning and failure to properly control the expression profiles can lead to disease. Post-translational modifications to the histone proteins, which package the DNA genome, can regulate expression of the underlying genes by making DNA more or less available to transcriptional machinery. Polycomb repressive complex 2 (PRC2), a highly conserved histone methyltransferase, maintains gene repression during development by facilitating chromatin compaction. PRC2 catalyzes the methylation of lysine 27 on histone H3 (H3K27me3) and loss of this epigenetic mark is associated with a number of developmental diseases. Diffuse intrinsic pontine glioma (DIPG), a deadly pediatric brain cancer, is caused by a lysine-to-methionine mutation on histone H3 (H3 K27M) that inhibits PRC2, resulting in a near-total loss of H3K27me3. Similarly, inhibition of PRC2 by the aberrant expression of EZHIP results in another pediatric glioma, posterior fossa ependymoma type A (PFA). By contrast, loss-of-function mutations in two core PRC2 subunits result in Weaver syndrome, a congenital disorder marked by somatic overgrowth and cognitive delays, but not cancer. The disparate clinical phenotypes caused by these disease alleles may be the result of different modes of PRC2 inhibition and the tissue-specific developmental context in which they are expressed. This proposal seeks to leverage a combination of genetic and genomic approaches to define the importance of distinct modes of PRC2 inhibition and developmental context to clinical phenotypes. Because PRC2 is highly conserved across species, Drosophila melanogaster will be used to test the relative contributions of each of these features to disease using an array of experimental approaches. The effects of disease alleles on tissue patterning, PRC2 function, and gene regulation will be assessed in a dynamic, developmental context. Genes that modulate the effects of disease alleles, including potential therapeutic targets, will be systematically tested. These approaches bridge the expertise of the Harrison lab (gene expression and development) and the Lewis lab (chromatin regulation and cancer) to address fundamental mechanistic questions about these disease alleles. The abundant resources at the University of Wisconsin School of Medicine and Public Health and the UW Carbone Cancer Center provide an ideal location to carry out the proposed work. Together this work will define mechanistic differences between PRC2-disease alleles and will reveal underlying differences that may lead to their distinct, devastating clinical phenotypes. Completion of this research will ensure development of experimental, mentorship, clinical, and communication skills that will enable successful transition to a career as a physician-scientist.