Regulation of Nuclear Receptor Function during Drosophila Development

从单个细胞，即受精卵形成复杂的有机体，需要随着胚胎的发育在不同的时间和不同的细胞中表达不同的基因。许多控制这些发育早期步骤的基因产物通过它们作为调节基因、编码转录因子或信号蛋白的功能来决定细胞的命运，这些转录因子或信号蛋白调节下游级联基因的表达，这些基因编码的产物直接涉及不同类型细胞的生长和分化。如果这些调控基因在错误的时间在错误的细胞中激活，对胚胎来说是灾难性的，胚胎很可能会因为这种错误表达而死亡。该项目的目标是解开控制胚胎调控基因活性的机制。它将使用一个成熟的动物模型--果蝇黑腹果蝇--来进行这些研究。重点放在一个胚胎转录因子家族上，它是一种核受体，由与蛋白质伙伴的相互作用来调节。这些发现将为研究果蝇和其他物种中核受体家族的其他成员提供一个基本的模型，有助于理解胚胎发育过程中细胞类型的指定，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。这项提议将在实验室培训来自不同背景的学生，包括板凳科学、批判性思维和科学陈述。此外，它还将支持与马里兰州一所少数民族占多数的中学(王德湖中学)的正式合作，以支持7年级学生的遗传学课程，以及东北伊利诺伊大学的MARC项目，该项目将每年向西北大学派遣最多两名学生进行暑期实习。这些努力是国际和平协会和联合国际正在进行的教育公众科学知识的努力的一部分。核受体(NRs)是一个转录因子大家族，控制着广泛的生物过程。虽然许多NRs的活性受配体的调节，但这些受体中有很大一部分是孤立的NRs，与已知配体的NRs有一个共同的折叠。这可能反映了这个蛋白质家族的祖先状态。孤儿NRS的活动是如何监管的？果蝇FTZ-F1是NR5A核受体家族的创始成员。在之前的工作中，Prof Investigator和他的同事们证明了果蝇FTZ-F1与同源结构域蛋白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的原基中，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体，结合到复合DNA位点，协同激活靶基因的表达。因此，尽管FTZ-F1在体外是一种强大的转录激活剂，并且存在于胚胎的所有体细胞的细胞核中，但其活性仅限于表达FTZ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是否受到以下调控：(1)在DNA结合水平上；(2)通过辅阻遏子/辅活化子交换，和/或(3)通过FTZ诱导的构象变化。这项工作将结合生化和结构方法与分子遗传学方法在果蝇胚胎中评估这些机制中的每一个对体内NR活性调节的贡献。它将培训博士后研究员、研究生和本科生分子生物学、遗传学、生物化学、生物物理学，以及最重要的批判性思维：生成、测试和评估假设。

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.