Identification of the transcriptional targets of three conserved regulatory factors necessary for motor neuron subtype function - Resubmission 01

运动神经元亚型功能所需的三种保守调节因子的转录靶标的鉴定 - 重新提交 01

• 批准号: 9757517
• 负责人: Paschalis Kratsios
• 金额: $ 44.22万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-15 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9757517
• 关键词: Activator Appliances; Adult; Amyotrophic Lateral Sclerosis; Animal Model; Auxins; Basic Science; Biological Models; Brain Stem; Caenorhabditis elegans; Cells; Chromatin; Chromatin Remodeling Factor; Clinical; Code; Defect; Development; Developmental Delay Disorders; Diagnosis; Differentiated Gene; Differentiation Antigens; Disease; Etiology; Exhibits; Fluorescence-Activated Cell Sorting; Future; Genes; Genetic; Genetic Screening; Genetic Transcription; Glutamate Receptor; Goals; Human; Human Pathology; Investigation; Ion Channel; Knowledge; Label; Life; Locomotion; Maintenance; Molecular; Morphology; Motor; Motor Neurons; Mus; Mutation; Nematoda; Nerve; Nervous system structure; Neurons; Neuropeptides; Neurotransmitter Receptor; Orthologous Gene; Outcome; Phylogenetic Analysis; Predisposition; Property; Proteins; Public Health; Regulator Genes; Research; Role; Spinal Cord; Spinal Muscular Atrophy; Syndrome; System; Technology; Testing; Transcriptional Regulation; cholinergic; differential expression; effective therapy; experimental study; fly; gene repression; innovation; insight; intersectionality; motor neuron function; novel; novel strategies; prevent; tool; transcription factor; transcriptome sequencing

Summary Defects in motor neuron (MN) function or survival result in severe human pathologies, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), with distinct MN subtypes differing in their susceptibility to disease. There is currently no effective treatment for MN disorders in part due to a lack of understanding of the molecular mechanisms that allow distinct MN subtypes to acquire and maintain their function-defining properties. Thus, basic research in model organisms such as nematodes, flies, and mice is needed to reveal such mechanisms. The underlying basis of MN subtype function is the differential expression of MN subtype-specific terminal differentiation genes, which encode proteins (e.g. ion channels, neurotransmitter receptors) that define the functional properties of a given MN subtype throughout life. Here, we propose a novel approach that specifically focuses on the transcriptional regulation of MN subtype-specific terminal differentiation genes. Our goal is to uncover the gene regulatory mechanisms that establish during development and maintain throughout life the expression of MN subtype-specific terminal differentiation genes. Such knowledge will advance our understanding of how MN subtype-specific functional properties are established and maintained, thereby providing new insights into the etiology and diagnosis of MN disorders. The availability of MN subtype-specific terminal differentiation markers in C. elegans has enabled us to identify three highly conserved gene regulatory factors, the transcription factors UNC-3 and CFI-1 and the chromatin remodeling factor PBRM-1, that determine MN subtype function by regulating the expression of terminal differentiation genes. Intriguingly, our results suggest that while UNC-3 activates expression of MN subtype-specific terminal differentiation genes, the regulatory factors CFI-1 and PBRM-1, counteract this activator function by repressing UNC-3 targets in specific MN subtypes. These observations are important because they could be indicative of a general mechanism for the acquisition and maintenance of MN subtype- specific features in which the transcriptional targets of a broadly acting activator (UNC-3) are repressed in a MN subtype-specific fashion (by CFI-1 and PBRM-1). To test this idea within the 2-year R21 timeframe, we will employ an innovative approach that focuses on UNC-3 and CFI-1. First, we have devised a new strategy to isolate distinct C. elegans MN subtypes, thereby enabling the identification of UNC-3 and CFI-1 transcriptional targets through RNA-sequencing (Aim 1). Second, we will employ the powerful auxin-inducible degradation (AID) system to test the hypothesis that UNC-3 and CFI-1, apart from their developmental function, also exhibit a maintenance role by continuously controlling the expression of MN subtype-specific terminal differentiation genes throughout life (Aim 2). Our findings may serve as a blueprint for future investigations of gene regulatory mechanisms for neuronal subtype development and function throughout the nervous system.

总结 运动神经元（MN）功能或存活的缺陷导致严重的人类病理学，例如肌萎缩性神经营养不良。 脊髓侧索硬化症（ALS）和脊髓性肌萎缩症（SMA），具有不同的MN亚型， 对疾病的易感性。目前对于MN病症没有有效的治疗，部分原因是缺乏有效的治疗方法。 了解允许不同MN亚型获得和维持其 功能定义属性。因此，对线虫、苍蝇和小鼠等模式生物的基础研究是必要的。 需要揭示这种机制。 MN亚型功能的潜在基础是MN亚型特异性的差异表达， 终末分化基因，其编码蛋白质（例如离子通道、神经递质受体）， 特定MN亚型在整个生命过程中的功能特性。在这里，我们提出了一种新的方法， 特别关注MN亚型特异性终末分化基因的转录调控。我们 我们的目标是揭示在发育过程中建立并在整个发育过程中维持的基因调控机制。 生活MN亚型特异性终末分化基因的表达。这些知识将促进我们的 了解MN亚型特异性功能特性是如何建立和维持的， 为MN疾病的病因学和诊断提供了新的见解。 MN亚型特异性终末分化标记在C. elegans使我们能够 鉴定了三个高度保守的基因调控因子，转录因子β 1 -3和CFI-1， 染色质重塑因子PBRM-1，通过调节 终末分化基因有趣的是，我们的研究结果表明，虽然ESTA-3激活MN的表达， 亚型特异性终末分化基因，调节因子CFI-1和PBRM-1，抵消了这一点 激活剂通过抑制特定MN亚型中的β-3靶点发挥作用。这些观察结果很重要 因为它们可能指示MN亚型获得和维持的一般机制- 广泛作用的激活因子（β-3）的转录靶点在细胞中被抑制的特异性特征， MN亚型特异性方式（通过CFI-1和PBRM-1）。为了在2年的R21时间框架内测试这一想法，我们将 采用创新的方法，重点是ESTA-3和CFI-1。首先，我们制定了一项新战略， 分离出不同的C. elegans MN亚型，从而能够鉴定转录因子β 3和CFI-1， 通过RNA测序（Aim 1）。其次，我们将采用强大的生长素诱导降解， (AID)系统来测试的假设，除了他们的发育功能，ESTA-3和CFI-1也表现出 通过持续控制MN亚型特异性终末分化的表达来维持作用 基因在整个生命（目标2）。我们的研究结果可以作为未来基因调控研究的蓝图 神经元亚型的发育机制和整个神经系统的功能。