Determining mechanisms of PRC2-mediated disease using Drosophila

项目摘要/摘要 正常发育需要严格控制基因表达，以精确调节组织模式和 如果不能适当地控制表达谱，可能会导致疾病。的翻译后修改 包装DNA基因组的组蛋白可以通过使 DNA或多或少可用于转录机器。多梳抑制复合体2(PRC2)，一种高度的 保守的组蛋白甲基转移酶，通过促进染色质在发育过程中维持基因抑制 压实。PrC2催化组蛋白H3(H3K27me3)上赖氨酸27的甲基化及其表观遗传缺失 MARK与许多发育疾病有关。弥漫性固有桥脑胶质瘤(DIPG)，一种致命的 儿童脑癌，是由组蛋白H3(H3K27M)上的赖氨酸到蛋氨酸突变引起的，它抑制了PRC2， 导致H3K27me3几乎全部损失。同样，EZHIP基因的异常表达对PRC2的抑制作用 结果另1例儿童脑胶质瘤为A型后颅窝室管膜瘤。相比之下，功能丧失 两个核心PRC2亚基的突变导致Weaver综合征，这是一种以躯体症状为特征的先天性疾病 过度生长和认知迟缓，但不是癌症。由这些疾病引起的不同的临床表型 等位基因可能是不同的PRC2抑制模式和组织特定的发育背景的结果 它们是被表达出来的。这项建议寻求利用遗传和基因组方法的组合。 明确不同的PRC2抑制模式和发育背景对临床表型的重要性。 由于PRC2在物种间高度保守，因此将用黑腹果蝇来测试其亲缘关系 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 疾病等位基因在组织构型、PRC2功能和基因调控方面的动态评估， 发展背景。调节疾病等位基因影响的基因，包括潜在的治疗作用 目标，将进行系统的测试。这些方法为哈里森实验室(基因)的专业知识搭建了桥梁 表达和发育)和Lewis实验室(染色质调节和癌症)以解决基本问题 关于这些疾病等位基因的机械问题。威斯康星大学丰富的资源 医学与公共卫生学院和威斯康星州癌症中心提供了一个理想的开展地点 建议的工作。这项工作将一起定义PRC2疾病等位基因和PRC2疾病等位基因之间的机制差异 将揭示潜在的差异，这些差异可能导致它们不同的、毁灭性的临床表型。完成 这项研究将确保发展实验、指导、临床和沟通技能， 能够成功地过渡到医生-科学家的职业生涯。

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.