Regulation of Nuclear Receptor Function during Drosophila Development

果蝇发育过程中核受体功能的调控

• 批准号: 1457145
• 负责人: Leslie Pick
• 金额: $ 74.5万
• 依托单位: University of Maryland, College Park
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-15 至 2019-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1457145&HistoricalAwards=false
• 关键词: Regulation; Nuclear; Receptor; Function; during

The formation of a complex organism from a single cell, the fertilized egg, requires the expression of different genes at different times and in different cells as the embryo develops. Many of the gene products controlling these early steps in development determine cell fate through their function as regulatory genes, encoding transcription factors or signaling proteins that regulate the expression of downstream cascades of genes encoding products directly involved in growth and differentiation of different cell types. If these regulatory genes are active in the wrong cells at the wrong time, it is catastrophic for the embryo, which will likely die as a result of this mis-expression. The goal of this project is to unravel the mechanisms controlling the activity of embryonic regulatory genes. It will use a well-established animal model, the fruit fly Drosophila melanogaster, for these studies. The focus is on one family of embryonic transcription factors, a nuclear receptor, which is regulated by interactions with protein partners. Findings will provide a fundamental model for studies of other members of the nuclear receptor family, in Drosophila and other species, by contributing to an understanding of cell type specification during embryonic development and elucidating different levels at which the function of transcription factors is regulated to produce unique cell types. This proposal will train students from diverse backgrounds in the laboratory in bench science, critical thinking and scientific presentation. In addition, it will support formal collaborations with a majority minority middle school in Maryland(Wilde Lake Middle School)to bolster the genetics curriculum for 7th graders and with the MARC program at Northeastern Illinois University that will send up to two students/year for summer internships to Northwestern. These efforts are part of ongoing efforts on the part of both the PI and Co-PI to educate the public about science. Nuclear Receptors (NRs) are a large family of transcription factors that control a broad range of biological processes. Although the activity of many NRs is regulated by ligand, a large number of these receptors are orphan NRs that share a common fold with NRs with known ligands. This likely reflects the ancestral state of this protein family. How is the activity of orphan NRs regulated? Drosophila Ftz-F1 is the founding member of the NR5A nuclear receptor family. In previous work, the Principle Investigator and colleagues showed that Drosophila Ftz-F1 interacts with the homeodomain protein Ftz, a classic pair-rule protein expressed in seven stripes in embryos in the primordia of body regions missing in ftz mutant animals. Ftz-F1 and Ftz form a stable complex in vivo and bind to composite DNA sites, synergistically activating target gene expression. Thus, although Ftz-F1 is a strong transcriptional activator alone in vitro, and it is present in the nuclei of all somatic cells of the embryo, its activity is limited to the cells in which Ftz is also expressed (Ftz+ cells). These studies demonstrate that protein-protein interactions modulate Ftz-F1 activity and provide an in vivo model system to dissect the mechanisms underlying this regulation. Here we will test whether Ftz-F1 is regulated: (1) at the level of DNA binding; (2) by a corepressor/coactivator exchange, and/or (3) by Ftz-induced conformational change. This work will combine biochemical and structural methods with molecular genetic approaches in Drosophila embryos to evaluate the contributions of each of these mechanisms to the regulation of NR activity in vivo. It will train postdoctoral fellows, graduate students and undergraduate students in molecular biology, genetics, biochemistry, biophysics and, most importantly, critical thinking: generating, testing and evaluating hypotheses.

从一个单细胞，即受精卵，形成一个复杂的有机体，需要在胚胎发育的不同时间和不同细胞中表达不同的基因。控制这些发育早期步骤的许多基因产物通过其作为调控基因的功能决定细胞命运，编码转录因子或信号蛋白，这些基因调节直接参与不同细胞类型生长和分化的下游级联基因编码产物的表达。如果这些调控基因在错误的时间在错误的细胞中活跃，对胚胎来说是灾难性的，胚胎可能会因为这种错误表达而死亡。本项目的目标是揭示控制胚胎调控基因活性的机制。它将使用一种成熟的动物模型——果蝇来进行这些研究。重点是胚胎转录因子的一个家族，一个核受体，它是通过与蛋白质伴侣的相互作用来调节的。这一发现将为研究核受体家族的其他成员、果蝇和其他物种提供一个基础模型，有助于理解胚胎发育过程中的细胞类型规范，并阐明转录因子的功能在不同水平上被调节以产生独特的细胞类型。本计划将培养来自不同背景的学生在实验室的实验科学、批判性思维和科学陈述。此外，它还将支持与马里兰州一所以少数民族为主的中学（王尔德湖中学）的正式合作，以加强七年级学生的遗传学课程，并与东北伊利诺伊大学的MARC项目合作，该项目每年将向西北大学输送最多两名学生进行暑期实习。这些努力是PI和Co-PI正在进行的对公众进行科学教育的努力的一部分。核受体（NRs）是一个控制广泛生物过程的转录因子大家庭。尽管许多rna的活性受配体调节，但这些受体中有大量是孤儿rna，它们与已知配体的rna有共同的折叠。这可能反映了这种蛋白质家族的祖先状态。孤儿nr的活性是如何调控的？果蝇Ftz-F1是NR5A核受体家族的创始成员。在之前的工作中，首席研究员和同事们发现果蝇Ftz- f1与同位结构域蛋白Ftz相互作用，Ftz是一种经典的配对规则蛋白，在Ftz突变动物缺失的身体区域原基中，在胚胎中以七条条纹表达。Ftz- f1和Ftz在体内形成稳定的复合物，结合复合DNA位点，协同激活靶基因表达。因此，尽管Ftz- f1在体外是一个强大的转录激活因子，并且存在于胚胎所有体细胞的细胞核中，但其活性仅限于Ftz也表达的细胞（Ftz+细胞）。这些研究表明，蛋白质-蛋白质相互作用调节Ftz-F1活性，并提供了一个体内模型系统来剖析这种调节的机制。在这里，我们将测试Ftz-F1是否受到调节：(1)在DNA结合水平上；(2)通过辅抑制因子/辅激活因子交换，和/或(3)通过自贸区诱导的构象变化。这项工作将结合生物化学和结构方法以及分子遗传学方法在果蝇胚胎中评估这些机制对体内NR活性调节的贡献。它将培养分子生物学、遗传学、生物化学、生物物理学方面的博士后、研究生和本科生，最重要的是培养批判性思维：生成、测试和评估假设。