Epigenetic Control of Retinal Development

摘要：我的最终目标是阐明控制视网膜发育的表观遗传学机制。 为了寻找新的靶点来再生在视网膜退行性疾病中丢失的组织。这些令人眼花缭乱 疾病几乎没有治疗方法，作为一名视网膜医生，我照顾的大多数人都受到这种疾病的困扰 外科医生。例如，老年性黄斑变性(AMD)，其中光感受器退化是主要的 失明，是一种影响1000万美国人的疾病。AMD的一种再生策略是多能性 干细胞来源的光感受器移植。然而，PSC来源的视网膜前体细胞的来源 细胞(RPC)-光感受器的组织特异性前体-仍然效率低下，依赖于供体，而且效果很差 明白了。为了更好地理解RPC是如何产生的，并实现我职业目标的初始步骤，我有 我计划了一个为期三年、有指导的职业发展计划，旨在促进我向 独立调查员。我设计了授课和实践训练目标，将干细胞和 染色质动力学与最先进的表观遗传技术。这项工作将在芝加哥大学进行。 密歇根大学(密歇根大学)，一个出色的环境，在培养年轻教师走向独立方面有着良好的记录 从事研究工作。我的主要导师Dou是国际公认的表观遗传学领域的专家 转录调控。萨莉·坦普尔，共同导师，是神经干细胞的先驱，并在定义它们的 发育转录组。托马斯·加德纳，U-M K12首席研究员兼高级临床医生兼科学家， 其工作重点是糖尿病视网膜病变，将担任职业导师。这是多种多样和有成就的 团队将促进我的培训目标、职业目标和我解决中心研究主题的努力：如何 表观遗传酶M111协调PSCs形成RPC。这项提议的理由是，通过 确定mll1在视网膜分化中的作用，将获得有关未知表观遗传学的知识 控制视网膜形成的机制。我们最近发现了M111-Rx视网膜发育 Axis，并发现M111调节视网膜Meis1。Mll1、Meis1或Rx的调节失调 哺乳动物的视网膜发育，但这是如何发生的仍不清楚。为了解决这一知识鸿沟， 拟议的研究旨在：1)确定MLI1缺陷是否损害通过MEIS1和 Rx抑制；以及2)在视网膜Mll1-Rx中定义依赖于Mll1的转录组和增强子网络 轴心。为了实现这些目标，我将学习和应用创新技术，如基因编辑、RNA 干扰、芯片和RNA测序以及增强子基序分析。这条集成的管道将允许 视网膜发育中有针对性的和临床相关的表观遗传途径的全基因组询问，以及 最终可能被应用于视网膜疾病。总而言之，从 建议的学习、职业发展计划和指导团队将促进我从被指导的人转变为 临床医生研究员至独立的、R01支持的翻译科学家。

ABSTRACT: My ultimate goal is to elucidate the epigenetic mechanisms that control retinal development in order to identify novel targets to regenerate tissues lost in the retinal degenerative diseases. These blinding disorders have few treatments and afflict the majority of individuals I care for as a retinal physician and surgeon. For example, age-related macular degeneration (AMD), in which photoreceptors degenerate leading to vision loss, is a disease that affects 10 million Americans. One regenerative strategy for AMD is pluripotent stem cell (PSC)-derived photoreceptor transplantation. However, derivation of PSC-derived retinal progenitor cells (RPCs)—tissue-specific precursors to photoreceptors—remains inefficient, donor-dependent, and poorly understood. To better understand how RPCs arise, and to achieve the initial steps of my career goals, I have planned a three-year, mentored career development program designed to foster my transition to an independent investigator. I have devised didactic and hands-on training aims that integrate stem cells and chromatin dynamics with state-of-the-art epigenetic techniques. This work will take place at University of Michigan (U-M), an outstanding environment with a track record of nurturing young faculty toward independent research careers. My primary mentor, Yali Dou, is an internationally recognized expert in the field of epigenetic regulation of transcription. Sally Temple, co-mentor, is a pioneer in neural stem cells and in defining their developmental transcriptome. Thomas Gardner, U-M K12 Principal Investigator and senior clinician-scientist, whose work focuses on diabetic retinopathy, will serve as a career mentor. This diverse and accomplished team will foster my training aims, career goals, and my efforts to address the central research theme: how an epigenetic enzyme, Mll1, orchestrates formation of RPCs from PSCs. The rationale for this proposal is that by determining the role of Mll1 in retinal differentiation, knowledge will be gained about unknown epigenetic mechanisms that govern retinal formation. We have recently discovered the Mll1-Rx retinal developmental axis, and have also found that Mll1 regulates retinal Meis1. Dysregulation of Mll1, Meis1, or Rx disrupts mammalian retinal development, but how this occurs remains unknown. To address this knowledge gap, the proposed research aims to: 1) determine whether Mll1 deficiency impairs generation of RPCs via Meis1 and Rx repression; and 2) define Mll1-dependent transcriptome and enhancer networks within the retinal Mll1-Rx axis. To accomplish these aims, I will learn and apply innovative technologies such as gene editing, RNA interference, ChIP- and RNA-sequencing, and enhancer motif analysis. This integrated pipeline will allow genome-wide interrogation of targetable and clinically relevant epigenetic pathways in retinal development, and could ultimately be applied to retinal disease. Together, the insights, skills, and guidance gained from the proposed studies, career development plan, and mentorship team will facilitate my transition from a mentored clinician investigator to an independent, R01-supported, translational scientist.