Molecular and neurodevelopmental consequences of ADNP mutation

项目总结 自闭症谱系障碍(ASD)患者染色质调节蛋白的大量突变突出了 染色质结构在正常脑发育中的意义。一种相对较新且鲜为人知的染色质 与几种神经发育障碍有关的结构是R环。R-环有三个- 含有DNA：RNA杂合体和移位的单链DNA的链状核酸结构，即 并不断地下决心。然而，当R-环积累时，它们会阻碍基因转录，促进DNA 损害，并导致疾病。R环与几种神经退行性疾病有关，例如 额颞叶痴呆和肌萎缩侧索硬化症，以及神经发育障碍，如脆性 X综合征。目前尚不清楚R环的松弛是否与ASD的病因有关。核心挑战 在将R环放松与ASD联系起来的过程中，人们一直在找出在ASD中发生突变的因素，这些因素也是R环 监管者。我们已经使用邻近标记蛋白质组学方法确定了定位于R环的因子。 我们的方法产生了染色质重构体、同源盒转录因子和解旋酶。许多这样的 在ASD中也有蛋白质发生突变的报道，如ADNP、POGZ、CHD2和DHX30。这些 初步结果首次表明，R环功能障碍可能与ASD有关。我们专注于我们的研究 对ADNP的分析表明，ADNP是一种在ASD中频繁突变的R环调节因子，是ADNP综合征的病因。 我们的初步研究表明，小鼠胚胎干细胞(MESCs)中ADNP的丢失导致R环 特异地在ADNP结合部位积累。重要的是，我们的数据显示ADNP的同源结构域是 对R环抑制至关重要，而同源结构域的缺失损害了mESCs的能力 分化为神经前体细胞(NPC)。我们发现人类诱导的多能干细胞(HiPSCs) ADNP酪氨酸719*突变(ADNP Tyr719*)也表现出R环去调节。根据这些数据，我们 假设R-环在特定基因组区域的积累改变了正常的基因表达 导致神经元分化不正确的程序，这最终可能导致ASD。在这里，我们将 确定ADNP Tyr719*hiPSCs中R-环的积累如何影响多能状态下的基因表达 在神经元分化的基础上。我们将评估ADNP Tyr719*的神经分化潜能 建立脑器官模型并确定R环衰减是否能改善神经分化缺陷 在ADNP突变体中观察到。

PROJECT SUMMARY The abundance of mutations in chromatin regulatory proteins in autism spectrum disorders (ASD) highlights the significance of chromatin structure in normal brain development. A relatively new and little understood chromatin structure that has been implicated in several neurodevelopmental disorders is the R-loop. R-loops are three- stranded, nucleic acid structures containing a DNA:RNA hybrid and a displaced single stranded DNA, that form and resolve continually. However, when R-loops accumulate, they can impede gene transcription, promote DNA damage, and result in disease. R-loops are implicated in several neurodegenerative disorders, such as frontotemporal dementia and amyotrophic lateral sclerosis, and in neurodevelopmental disorders such as Fragile X Syndrome. Whether R-loop deregulation contributes to the etiology of ASD is not clear. The central challenge in linking R-loop deregulation to ASDs has been pinpointing factors that are mutated in ASD that are also R-loop regulators. We have identified factors that localize to R-loops using a proximity labeling proteomic approach. Our approach yielded chromatin remodelers, homeobox transcription factors, and helicases. A number of these proteins had also been reported to be mutated in ASD, e.g. ADNP, POGZ, CHD2, and DHX30. These preliminary results provide the first indication that R-loop dysfunction may contribute to ASD. We focus our study on the analysis of ADNP, an R-loop regulator that is frequently mutated in ASD and is causal in ADNP syndrome. Our preliminary studies show that ADNP loss in mouse embryonic stem cells (mESCs) results in R-loop accumulation specifically at ADNP binding sites. Importantly, our data show that the homeodomain of ADNP is critical for R-loop suppression and that deletion of the homeodomain compromises the ability of mESCs to differentiate into neural progenitor cells (NPCs). We found that human induced pluripotent stem cells (hiPSCs) with an ADNP tyrosine 719* mutation (ADNP Tyr719*) also show R-loop deregulation. Based on these data, we hypothesize that accumulation of R-loops at specific genomic regions changes normal gene expression programs leading to improper neuronal differentiation, which can ultimately contribute to ASD. Here we will determine how R-loop accumulation in ADNP Tyr719* hiPSCs affects gene expression in the pluripotent state and upon neuronal differentiation. We will evaluate the neurodifferentiation potential of ADNP Tyr719* using a cerebral organoid model and determine if R-loop attenuation can ameliorate the neurodifferentiation defects observed in ADNP mutants.