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

描述（由申请人提供）：配对同源结构域转录因子Pax6是人眼细胞命运的重要调节因子，突变可导致先天性眼睛缺陷，包括无虹膜，导致失明。最近对脊椎动物和果蝇的研究表明，Pax6在视网膜祖细胞的增殖和调节其向分化的转变中都有作用，部分是通过激活祖细胞基因来实现的。因此，Pax6目前是旨在将诱导多能干细胞转化为视网膜神经元的紧张工作的主题。这一研究为治疗神经退行性眼病带来了巨大的希望。然而，为了使这种方法既成功又安全，我们必须了解Pax6在增殖和分化之间转变过程中调节基因表达的机制。该应用程序的长期目标是了解Pax6在视网膜发育增殖阶段以及向分化过渡期间调节基因表达的机制。我们之前的工作已经证明，果蝇Pax6， Eyeless (Ey)，在眼睛发育过程中与转录调节因子CtBP在物理和功能上存在联系，CtBP已知将序列特异性转录因子与染色质修饰因子联系起来。另外的数据表明，Ey和CtBP在调节增殖和前膜基因的表达方面具有拮抗作用。这些数据为探索Pax6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绝佳的机会。这项拟议的研究将验证CtBP与果蝇Ey/Pax6和其他眼睛规范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。我们将整合果蝇中强大的遗传学与分子和转录组学方法，以确定(1)CtBP是否拮抗Ey调节增殖和ato表达的功能，(2)ato增强元件是否对CtBP和其他与CtBP相关的抑制因子（例如bHLH因子Hairy和Notch通路转录因子Su(H)）做出反应。(3) Ey和CtBP的关联机制，以及增殖和分化细胞之间靶基因表达的转换是否依赖于不同辅因子的存在；(4)Ey和CtBP除了共同调控哪些基因外，还共同调控哪些基因，这些将为进一步了解Pax6的作用机制提供丰富的机会。综上所述，本研究将为研究Pax6蛋白在眼睛功能规范和增殖中的作用提供新的机制途径，并为开发新的失明治疗方法奠定基础。

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.