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Regulatory Mechanism for Cellular Dedifferentiation in C. elegans Germline

线虫种系细胞去分化的调控机制

基本信息

批准号：
2132286
负责人：
Myon Hee Lee
金额：
$ 60万
依托单位：
East Carolina University
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2025-01-31
项目状态：
未结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=2132286&HistoricalAwards=false
关键词：
Regulatory Mechanism Cellular Dedifferentiation C.

项目摘要

Throughout their lifespan, stem cells make several critical fate decisions: the initial decision between self-renewal (remaining pluripotent) and differentiation, and then the subsequent decision between whether to continue in the differentiation path or to return to an undifferentiated state, i.e., to engage in a form of dedifferentiation. Defects in cellular machineries governing differentiation decisions can result in loss of a specific cell type, overpopulation of undifferentiated cells, or overt growth of cancer cells. Over the past decade, the molecular mechanisms related to the initial decision have been described in major model organisms. However, an understanding of the underlying determinants of the subsequent decisions is vague at best. The long-term research objectives are to elucidate the mechanisms that cause differentiating cells to return to an undifferentiated state, and to develop an animal model to interrogate these mechanisms in the living organism. A molecular understanding of this cellular event holds promise for the development of powerful tools in stem cell biology, regenerative medicine, and oncotherapy. The project also includes a component in which traditionally underserved populations of students in North Carolina (minority high school students, community college and undergraduate students) will be actively involved in the basic research.This project addresses three key questions concerning the fundamental molecular controls of a known dedifferentiation mechanism: (1) How do differentiating cells maintain their state or return to undifferentiated cells? (2) What regulatory network is involved in this cellular process in vivo? (3) Is aberrant regulation of this network associated with oncogenesis? Using the nematode C. elegans germline as a model system, the investigators have previously reported that PUF-8 (an RNA-binding protein) and LIP-1 (a dual-specificity phosphatase) are required to (a) maintain the meiosis (differentiating state) of spermatocytes, and (b) prevent their dedifferentiation into mitotic cells (undifferentiating state) by inhibiting MPK-1 (an ERK homolog) signaling. Further studies found that the differentiation/dedifferentiation decision is regulated in part by cell cycle regulators (CYB-1 and X-ray) and mRNA decay factors (CGH-1 and CAR-1). In this project, the researchers will investigate two hypotheses: In the absence of PUF-8 and LIP-1, (1) spermatocyte dedifferentiation requires cell cycle arrest and the activation of cell survival pathways (Objective 1), and (2) inactivation of mRNA decay factors induces the ectopic translation of mRNAs that initiate spermatocyte dedifferentiation (Objective 2). To accomplish this goal, the researchers will take multidisciplinary approaches, including RNAi screening, genetic/functional analysis, CRISPR/Cas9-based genome editing, X-ray-based biophysical analysis, and high-resolution polysome profiling. Findings from the study will not only elucidate the fundamental mechanisms of the differentiation/dedifferentiation decision in vivo, but they will also provide a novel working platform for the identification of therapeutic targets for dedifferentiation-mediated cellular regeneration and tumorigenesis.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

在干细胞的整个生命周期中，干细胞会做出几个关键的命运决定：自我更新（保持多能性）和分化之间的最初决定，然后是继续分化路径还是返回未分化状态（即，进行某种形式的去分化。控制分化决定的细胞机制的缺陷可导致特定细胞类型的丧失、未分化细胞的过度增殖或癌细胞的明显生长。在过去的十年中，与初始决定相关的分子机制已经在主要的模式生物中描述。然而，对随后决定的基本决定因素的理解充其量是模糊的。长期的研究目标是阐明导致分化细胞恢复到未分化状态的机制，并开发动物模型来询问活生物体中的这些机制。对这种细胞事件的分子理解有望为干细胞生物学、再生医学和肿瘤治疗提供强大的工具。该项目还包括一个组成部分，其中传统上缺乏服务的人口在北卡罗来纳州的学生（少数民族高中生，社区学院和本科生）将积极参与基础研究。该项目解决了三个关键问题，有关的基本分子控制的一个已知的去分化机制：（1）分化细胞如何保持其状态或返回到未分化的细胞？(2)在体内这个细胞过程中涉及到什么样的调控网络？(3)这个网络的异常调节与肿瘤发生有关吗？利用线虫C.以线虫种系为模型系统，研究者先前报道了PUF-8（一种RNA结合蛋白）和LIP-1（一种双特异性磷酸酶）是（a）维持精母细胞减数分裂（分化状态）和（B）通过抑制MPK-1（一种ERK同源物）信号传导防止它们去分化成有丝分裂细胞（未分化状态）所必需的。进一步的研究发现，分化/去分化决定部分受细胞周期调节因子（CYB-1和X射线）和mRNA衰变因子（CGH-1和CAR-1）的调节。在这个项目中，研究人员将研究两个假设：在没有PUF-8和LIP-1的情况下，（1）精母细胞去分化需要细胞周期停滞和细胞存活途径的激活（目标1），（2）mRNA衰变因子的失活诱导启动精母细胞去分化的mRNA的异位翻译（目标2）。为了实现这一目标，研究人员将采取多学科方法，包括RNAi筛选，遗传/功能分析，基于CRISPR/Cas9的基因组编辑，基于X射线的生物物理分析和高分辨率多核糖体分析。这项研究的发现不仅将阐明体内分化/去分化决定的基本机制，而且还将为去分化介导的细胞再生和肿瘤发生的治疗靶点的鉴定提供一个新的工作平台。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。