Control and Implementation of a Morphogenetic Program

项目摘要/摘要 细胞形态发生对动物发育、伤口愈合和癌症都是必不可少的。在.期间 在这个过程中，细胞协调地改变形状、迁移，并可能融合。细胞形态发生是由 被称为“效应器”的分子参与连接、细胞骨架、细胞极性、囊泡运输等。 细胞学机器的模块/过程。性别、组织、位置和时间的特异性 形态发生是由转录因子等“调节器”决定的。有两个主要的知识 差距：(1)形态发生事件是如何在时间上安排的，以及(2)转录调控输入是如何 链接到效应器。在这里，总体目标是阐明计时机制中一个有趣的部分 这涉及到一个细胞质中的长非编码RNA在翻译后的调节，并描绘出 调节器和效应器之间的链接，形成了一种实验上可以接触到的结构，线虫的尾尖。 尾尖由4个细胞组成，仅在雄性中，这些细胞在幼年到成年的转变过程中改变形状并迁移。 (J/A)，这一过程称为尾尖形态发生(TTM)。先前的研究表明：(1)一个长的非编码RNA (2)转录因子DMD-3是转录因子DMD-3所必需的， 对TTM来说足够了，并协调细胞学机器的几个过程。总体方法是 (目标1)测试lep-5lncRNA自主地调度TTM细胞的假设--通过构建 马可林线虫对异时通路中心保守节点LIN-28的泛素化 蛋白质，LEP-2(另一种高度保守但特性不足的蛋白质)，以及(目标2)确定 DMD-3的直接靶点及其在TTM中的作用。AIM 1将主要使用免疫共沉淀 (CoIP)和泛素化试验测试lep-5 lncRNA的结合伙伴。AIM 2将使用染色质 免疫沉淀(CHIP-SEQ)识别DMD-3的候选靶点；这些候选靶点在TTM中的作用 然后通过标记它们或在带有亚细胞标志的菌株中对它们进行基因干扰来进行测试 不同的荧光蛋白。预期的结果包括：(1)对小说的机械理解 IncRNA在关键保守基因调控中的指导性作用和翻译后作用 (2)形态发生联系的系统网络模型的建立 DMD-3(一种与人DMRT同源的保守转录因子)对 参与形态发生细胞行为的细胞学机制。这些结果预计将有更多的 通过以下方式对生物学和医学产生积极影响：(A)通过将性别联系起来确定如何发生性二型性 确定形态发生效应因子；(B)确定控制形态发生的关键保守基因和 它的时机，从而潜在地确定了治疗青春期时机缺陷、性反转或癌症的新靶点， 或帮助伤口愈合；以及(C)提供可能构成进化多样性基础的候选基因。

PROJECT SUMMARY / ABSTRACT Cellular morphogenesis is essential to animal development as well as wound healing and cancer. During this process, cells coordinately change shape, migrate, and may fuse. Cellular morphogenesis is executed by molecules called "effectors" involved in junctions, cytoskeleton, cell polarity, vesicular trafficking, and other modules/processes of the cytological machinery. The sex-, tissue-, position-, and time-specificity of morphogenesis is determined by "regulators", such as transcription factors. There are two main knowledge gaps: (1) how morphogenetic events are temporally scheduled, and (2) how the transcriptional regulatory input is linked to the effectors. Here, the overall objective is to elucidate an intriguing part of the timing mechanism that involves a cytoplasmic long-noncoding RNA in post-translational regulation, and to delineate the "missing links" between regulators and effectors for an experimentally accessible structure, the tail tip of C. elegans. The tail tip is made of 4 cells which, in males only, change shape and migrate at the juvenile-to-adult transition (J/A), a process called Tail Tip Morphogenesis (TTM). Prior studies showed that (1) a long non-coding RNA (lep-5) is an instructive switch for TTM onset at the J/A, and (2) transcription factor DMD-3 is required and sufficient for TTM and coordinates several processes of the cytological machinery. The overall approach is to (Aim 1) test the hypothesis that the lep-5 lncRNA acts to schedule TTM cell-autonomously by scaffolding the ubiquitination of a central, conserved node in the heterochronic pathway, LIN-28, by the C. elegans Makorin protein, LEP-2 (another highly conserved but under-characterized protein), and (Aim 2) to determine what are the direct targets of DMD-3 and how they function in TTM. Aim 1 will use primarily co-immunoprecipitation (coIP) and ubiquitination assays to test binding partners of lep-5 lncRNA. Aim 2 will use chromatin immunoprecipitation (ChIP-seq) to identify candidate targets of DMD-3; the roles of these candidates in TTM will then be tested by labeling them or genetically perturbing them in a strain with subcellular landmarks tagged with different fluorescent proteins. The expected outcomes include (1) a mechanistic understanding for a novel paradigmatic role for a lncRNA acting instructively and post-translationally in the regulation of a key conserved temporal regulator, and (2) the foundation of a systems network model for morphogenesis connecting transcriptional control by DMD-3 (a conserved transcription factor homologous to human DMRTs) to the cytological machinery involved in morphogenetic cell behaviors. These results are expected to have additional positive impacts on biology and medicine by (a) determining how sexual dimorphism occurs by linking sex determination to morphogenetic effectors; (b) identifying key conserved genes controlling morphogenesis and its timing, thus potentially identifying new targets for treating defects in pubertal timing, sex-reversal or cancer, or to aid wound healing; and (c) providing candidate genes that may underlie evolutionary diversity.