R-loops as Mechanisms Governing Neural Differentiation and Cell Type-Specific Transcription

项目总结 神经精神障碍、精神分裂症和自闭症共同影响着超过15%的儿童 美国，这是一个重大的公共卫生问题。开发有效的治疗方法和 这些疾病的诊断工具由于我们对其复杂性的不完全理解而受到阻碍 神经发育病因学。基因表达变异是这些疾病的共同属性，被认为是 产生于神经发育过程中发育阶段和组织特异性的RNA调节。新的基因组- 广泛的作图策略已经确定了R环(一种三链核酸结构)之间的联系 包含DNA/RNA杂交体)和转录调控，这表明R-环和 基因表达变异。然而，R-环以前从未在全基因组范围内被表征过 人类大脑，排除了研究它们在神经发育疾病中的作用。我们的研究已经 显示R-环可能平衡发育基因在神经分化过程中的转录，而我们的 目前的目标是通过利用基于人类诱导多能干细胞(HiPSC)来验证这一假设 从功能上操作R环路电平的模型。具体地说，我们的目标是降低HiPSC衍生的R-loop水平 神经前体细胞，并测量这对神经细胞转录、分化和能力的影响 形成功能性突触。我们的目标是确定这种R-环介导的转录的原因机制 通过探索R-环使RNA聚合酶II停滞的假设保持镇定，因为人们认为停滞的RNA聚合酶II 保持发育基因在转录上不活跃，但准备好在发育提示下表达。 重要的是，我们将首次在发育中的人脑中绘制全基因组范围的R-环，并将R-环 人类神经发生过程中的分布变化与神经发育疾病相关基因的表达 签名。在这里，我们将使用尖端技术，包括DNA/RNA免疫沉淀，然后是Next- 世代排序，以实现这些目标。了解神经细胞中R环功能的第一步 细胞将有助于我们的长期目标，以揭示异常的RNA调控的表观遗传机制 为了改进诊断和确定针对这些疾病的新的治疗目标，我们将继续开展神经精神疾病方面的研究。

PROJECT SUMMARY The neuropsychiatric disorders schizophrenia and autism together affect over 15% of children in the United States, representing a significant public health concern. Development of effective therapeutics and diagnostic tools for these disorders has been hindered by our incomplete understanding of their complex neurodevelopmental etiologies. Gene expression variation is a common attribute of these disorders, thought to arise from developmental stage- and tissue-specific RNA regulation during neurodevelopment. New genome- wide mapping strategies have identified a connection between R-loops (a three-stranded nucleic acid structure containing a DNA/RNA hybrid) and transcriptional regulation, suggesting a possible link between R-loops and gene expression variation. However, R-loops have never before been characterized on a genome-wide scale in the human brain, precluding research studying their role in neurodevelopmental illnesses. Our research has showed that R-loops may poise developmental genes for transcription during neural differentiation, and our immediate goal is to test this hypothesis by leveraging human induced pluripotent stem cell (hiPSC)-based models to functionally manipulate R-loop levels. Specifically, we aim to reduce R-loop levels in hiPSC-derived neural progenitor cells and measure the effect this has on transcription, differentiation, and ability of neural cells to form functional synapses. We aim to identify a causal mechanism of this R-loop-mediated transcriptional poising by exploring the hypothesis that R-loops stall RNA polymerase II, as stalled RNA polymerase II is thought to keep developmental genes transcriptionally inactive but ready for expression upon developmental cues. Importantly, we will map R-loops genome-wide for the first time in the developing human brain, and link R-loop distributional shifts during human neurogenesis with neurodevelopmental disease-associated gene expression signatures. Here, we will use cutting-edge techniques, including DNA/RNA immunoprecipitation followed by next- generation sequencing, to achieve these goals. These first steps toward understanding R-loop function in neural cells will facilitate our long-term objective to uncover the epigenetic mechanisms of aberrant RNA regulation in neuropsychiatric illness, in order to improve diagnostics and identify novel therapeutic targets for these illnesses.