Pax6, CtBP and the regulation of transcription in Drosophilia eye specification

Pax6、CtBP 和果蝇眼睛规范中的转录调控

• 批准号: 8180550
• 负责人: JENNIFER R CURTISS
• 金额: $ 20.97万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8180550
• 关键词: Alleles; Aniridia; Blindness; Cells; Chromatin; Co-Immunoprecipitations; Computational Technique; Coupled; Data; Defect; Development; Distal; Drosophila genus; Eye; Eye Development; Eye diseases; Foundations; Future; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Goals; Histone H3; Human; Immunofluorescence Immunologic; In Vitro; Lead; Link; Mediating; Methods; Mining; Molecular; Molecular Genetics; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Organism; Pathway interactions; Phase; Proliferating; Proliferation Marker; Proteins; RNA Interference; Reporter Genes; Research; Retinal; Role; Stem cells; Techniques; Testing; Time; Transcriptional Regulation; Vertebrates; Work; chromatin modification; cofactor; fly; gene function; homeodomain; induced pluripotent stem cell; mutant; notch protein; novel; retinal neuron; retinal progenitor cell; tool; transcription factor; transcriptomics

DESCRIPTION (provided by applicant): The Paired Homeodomain transcription factor Pax6 is an important regulator of cell fate in the human eye, and mutations cause congenital eye defects, including aniridia, leading to blindness. Recent studies in both vertebrates and in Drosophila show that Pax6 has a role both in proliferation of retinal progenitor cells, and in regulating their transition to differentiation, in part through activation of proneural genes. Accordingly, Pax6 is currently the topic of intense work aimed towards converting e.g. induced pluripotent stem cells to retinal neurons. Such work holds spectacular promise for treating neurodegenerative eye disease. However, for this type of approach to be successful as well as safe, it is imperative that we understand the mechanisms by which Pax6 regulates gene expression during the transition between proliferation and differentiation. The long- term goal of this application is to understand the mechanisms by which Pax6 regulates gene expression during proliferative phases of retinal development as well as during the transition to differentiation. Our previous work has demonstrated that Drosophila Pax6, Eyeless (Ey), is physically and functionally linked during eye development with the transcriptional regulator CtBP, which is known to link sequence-specific transcription factors to chromatin modification factors. Additional data suggests that Ey and CtBP have antagonistic functions in regulating proliferation as well as expression of the proneural gene atonal. This data provides an exceptional opportunity to probe the mechanisms by which Pax6 regulates gene expression during the transition from proliferation to differentiation. The proposed research will test the hypothesis that CtBP interacts with Drosophila Ey/Pax6 and other eye specification factors to regulate gene expression important for proliferation as well as differentiation. We will integrate the powerful genetics available in Drosophila with molecular and transcriptomic approaches to determine (1) whether CtBP antagonizes Ey's function in regulating proliferation and ato expression, (2) which ato enhancer elements respond to CtBP and other repressive factors linked to CtBP [e.g. the bHLH factor Hairy and the Notch pathway transcription factor Su(H)], (3) the mechanisms by which Ey and CtBP associate and whether the switch in target gene expression between proliferating and differentiating cells depends on the presence of different cofactors, and (4) what genes besides ato are co-regulated by Ey and CtBP, which will provide abundant future opportunities for understanding the mechanisms by which Pax6 functions. In summary, the proposed research will provide a novel mechanistic approach to the study of Pax6 function in eye specification and proliferation, and lay the foundation for development of new treatments for blindness. PUBLIC HEALTH RELEVANCE: The Pax6 gene is a critical regulator of cell fate in the human eye and mutations cause congenital eye defects, including aniridia, that lead to blindness. Furthermore, Pax6 is at the center of recent efforts to use stem cells to treat neurodegenerative disease. A detailed understanding of how Pax6 works is therefore important because it will facilitate the development of novel therapies for blindness.

描述（由申请人提供）：配对同源结构域转录因子Pax 6是人眼细胞命运的重要调节因子，突变会导致先天性眼缺陷，包括导致失明的无虹膜。最近在脊椎动物和果蝇中的研究表明，Pax 6在视网膜祖细胞的增殖和调节其向分化的转变中均具有作用，部分通过激活前神经基因。因此，Pax 6目前是旨在将例如诱导多能干细胞转化为视网膜神经元的密集工作的主题。这项工作为治疗神经退行性眼病带来了巨大的希望。然而，为了使这种方法成功且安全，我们必须了解Pax 6在增殖和分化之间的过渡期间调节基因表达的机制。本申请的长期目标是了解Pax 6在视网膜发育的增殖阶段以及向分化过渡期间调节基因表达的机制。我们以前的工作已经证明，果蝇Pax 6，Eyeless（Ey），在眼睛发育过程中与转录调节因子CtBP，这是已知的连接序列特异性转录因子染色质修饰因子的物理和功能上的联系。其他数据表明，Ey和CtBP在调节增殖以及前神经基因无调表达方面具有拮抗作用。这些数据为探索Pax 6在从增殖到分化的过渡过程中调节基因表达的机制提供了一个难得的机会。这项研究将检验CtBP与果蝇Ey/Pax 6和其他眼睛特异性因子相互作用以调节对增殖和分化重要的基因表达的假设。我们将整合果蝇中可用的强大遗传学与分子和转录组学方法，以确定（1）CtBP是否拮抗Ey在调节增殖和ato表达中的功能，（2）哪些ato增强子元件响应CtBP和与CtBP相关的其他抑制因子[例如bHLH因子Hairy和Notch途径转录因子Su（H）]，（3）Ey和CtBP相关的机制以及增殖和分化细胞之间靶基因表达的转换是否取决于不同辅因子的存在，以及（4）除了ato之外还有哪些基因受到Ey和CtBP的共调节，这将为理解Pax 6的功能机制提供丰富的未来机会。综上所述，本研究为研究Pax 6在眼特化和增殖中的作用提供了一种新的机制，并为开发新的失明治疗方法奠定了基础。 公共卫生关系：Pax 6基因是人眼细胞命运的关键调节因子，突变会导致先天性眼部缺陷，包括导致失明的无虹膜。此外，Pax 6是最近使用干细胞治疗神经退行性疾病的努力的中心。因此，详细了解Pax 6的工作原理非常重要，因为它将有助于开发新的失明疗法。