The molecular roles of RFX3 in neurodevelopment and Autism Spectrum disorder

项目摘要 自闭症谱系障碍（ASD）是最常见的神经发育障碍，然而， ASD发病机制的神经生物学机制仍不清楚。大规模 ASD患者的外显子组测序研究已经鉴定了100多个基因， 与ASD风险有关。ASD风险基因的功能表征可以提供对ASD风险基因的深入了解。 ASD发病机制我们和其他研究人员最近发现了一种新的功能丧失变异， 转录因子RFX 3作为ASD的一个相对常见的单基因病因，这意味着 RFX 3在人类神经发育中的重要作用。我们已经发现RFX 3可能 是II/III层神经元发育和功能的关键转录调节因子：其 表达在皮质层II/III兴奋性神经元中显著富集，并且RFX 3结合 基序特异性地富集在人胎儿生殖带的可接近的染色质区域中 和II/III层兴奋性神经元。在这项拟议的研究中，我将解决的假设， RFX 3调节II/III层兴奋性神经元的关键神经发育过程， 其他ASD风险基因的表达影响神经元的形成和功能。在目标1中，我将 通过分析人类皮层神经元中RFX 3的表达， RFX 3的全基因组结合位点和RFX 3缺失引起的转录变化 RFX 3单倍不足的人iPSC衍生的神经元中的占据。在目标2中，我将评估 RFX 3单倍不足对人皮层神经元形成和突触功能影响 iPSC衍生的前脑类器官。我将使用单细胞RNA测序来鉴定细胞中的变化， 类型组成和推断RFX 3缺陷类器官发育轨迹的改变， 和多电极阵列评估RFX 3缺陷类器官中的突触可塑性平衡 与同基因对照相比。总的来说，这一建议将使人们了解 人类神经元中RFX 3调控的转录程序，以及RFX 3单倍不足如何影响神经元的转录程序。 破坏神经元的发育和功能。这将有助于更好地了解ASD 神经生物学和ASD的新型靶向治疗的发展。

Project Abstract Autism Spectrum Disorder (ASD) is the most common neurodevelopmental disorder, yet the neurobiological mechanisms underlying ASD pathogenesis remain largely unknown. Large-scale exome sequencing studies of individuals with ASD have identified over 100 genes significantly associated with ASD risk. Functional characterization of ASD risk genes can provide insight to ASD pathogenesis. We and others have recently identified de novo loss-of-function variants in the transcription factor RFX3 as a relatively common monogenic cause of ASD, implying an important role for RFX3 in human neurodevelopment. We have found evidence that RFX3 may be a critical transcriptional regulator of the development and function of layer II/III neurons: its expression is significantly enriched in cortical layer II/III excitatory neurons, and the RFX3 binding motif is specifically enriched in accessible chromatin regions of the human fetal germinal zone and layer II/III excitatory neurons. In this proposed research, I will address the hypothesis that RFX3 regulates key neurodevelopmental processes in layer II/III excitatory neurons and the expression of other ASD risk genes that affect neuronal formation and function. In Aim 1, I will identify the genes and pathways regulated by RFX3 in human cortical neurons by profiling the genome-wide binding sites of RFX3 and the transcriptional changes induced by loss of RFX3 occupancy in RFX3 haploinsufficient human iPSC-derived neurons. In Aim 2, I will evaluate the effect of RFX3 haploinsufficiency on cortical neuron formation and synaptic function in human iPSC-derived forebrain organoids. I will use single-cell RNA-sequencing to identify changes in cell type composition and infer alterations in developmental trajectories in RFX3 deficient organoids, and multielectrode array to assess synaptic plasticity balance in RFX3 deficient organoids compared to isogenic controls. Taken together, this proposal will yield insight on the transcriptional programs regulated by RFX3 in human neurons, and how RFX3 haploinsufficiency disrupts neuronal development and function. This will allow for improved understanding of ASD neurobiology, and the development of novel targeted therapies for ASD.