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

摘要 运动神经元(MN)功能或存活缺陷会导致严重的人类病理，如肌营养不良 侧索硬化症(ALS)和脊髓性肌萎缩症(SMA)，不同的MN亚型在其 对疾病易感性。目前还没有有效的治疗MN疾病的方法，部分原因是缺乏 理解允许不同的MN亚型获得和保持其 定义功能的属性。因此，对线虫、苍蝇和老鼠等模式生物的基础研究是 需要揭示这样的机制。 MN亚型功能的基础是MN亚型特有的差异表达 末端分化基因，编码蛋白质(如离子通道、神经递质受体)，从而定义 给定的MN亚型在整个生命周期中的功能特性。在这里，我们提出了一种新的方法， 特别关注MN亚型特异性末端分化基因的转录调控。我们的 目标是发现在发育过程中建立并贯穿始终的基因调控机制 LIFE表达MN亚型特异的末端分化基因。这样的知识将促进我们的 了解MN亚型特定的功能属性是如何建立和维护的，从而 为MN疾病的病因和诊断提供新的见解。 线虫MN亚型特异性末端分化标记的存在使我们能够 鉴定三种高度保守的基因调控因子，转录因子UNC-3和CFI-1以及 染色质重塑因子PBRM-1通过调节MN亚型的表达决定MN亚型的功能 末端分化基因。有趣的是，我们的结果表明，虽然UNC-3激活了MN的表达 亚型特异的末端分化基因，调节因子CFI-1和PBRM-1，抵消了这一点 激活器通过压制特定MN亚型中的UNC-3目标来起作用。这些观察结果很重要 因为它们可以指示获得和维持MN亚型的一般机制- 广泛作用的激活物(UNC-3)的转录靶标在 MN亚型特定方式(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 采用以UNC-3和CFI-1为重点的创新方法。首先，我们制定了一项新战略，以 分离不同的线虫MN亚型，从而能够鉴定UNC-3和CFI-1转录 通过RNA测序打靶(目标1)。其次，我们将利用强大的生长素诱导降解 (辅助)系统，以检验UNC-3和CFI-1除了发育功能外，还表现出 通过持续控制MN亚型特异性末端分化的表达而发挥维持作用 生命中的基因(目标2)。我们的发现可能为未来的基因调控研究提供蓝图。 神经系统中神经元亚型发育和功能的机制。

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.