Molecular and neurodevelopmental consequences of ADNP mutation

ADNP 突变的分子和神经发育后果

• 批准号: 10372679
• 负责人: Kavitha Sarma
• 金额: $ 29.51万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10372679
• 关键词: Affect; Binding Sites; Brain; Cerebrum; Chromatin; Chromatin Structure; DNA; DNA Damage; Data; Data Set; Defect; Deletion Mutation; Development; Disease; Etiology; Exhibits; Fragile X Syndrome; Functional disorder; Future; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Genome; Genomic Segment; Homeobox; Human; Hybrids; Label; Link; Malignant Neoplasms; Mediating; Modeling; Molecular; Morphology; Mus; Mutate; Mutation; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neuronal Differentiation; Neurons; Oncogene Deregulation; Organoids; Population; Proteins; Proteomics; RNA; Reporting; Research; Ribonuclease H; Single-Stranded DNA; Site; Structure; Syndrome; Therapeutic Intervention; Tyrosine; Undifferentiated; attenuation; autism spectrum disorder; base; chromatin remodeling; embryonic stem cell; frontotemporal lobar dementia-amyotrophic lateral sclerosis; genetic regulatory protein; genome-wide; helicase; homeodomain; induced pluripotent stem cell; insight; mutant; nerve stem cell; new therapeutic target; nucleic acid structure; progenitor; programs; single-cell RNA sequencing; transcription factor

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.

项目摘要 自闭症谱系中染色质调节蛋白的突变丰度（ASD）突出了 染色质结构在正常脑发育中的重要性。一个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环是三 滞留的含有DNA的核酸结构：RNA杂交和一个位移的单链DNA，该形式 并不断解决。但是，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 额颞痴呆和肌萎缩性侧面硬化症，以及在神经发育障碍中，例如脆弱 X综合征。 R环的放松管制是否有助于ASD的病因，尚不清楚。中心挑战 在将R-loop放松管制与ASD的连接时，已经指出了在ASD中突变的因素，这些因素也是R-loop 监管机构。我们已经确定了使用接近标记蛋白质组学方法将其定位为R-loops的因素。 我们的方法产生了染色质重塑，同型转录因子和解旋酶。其中一些 据报道，蛋白质在ASD中被突变，例如ADNP，POGZ，CHD2和DHX30。这些 初步结果提供了第一个迹象，表明R环功能障碍可能有助于ASD。我们集中研究 关于ADNP的分析，ADNP是一种经常在ASD中突变而在ADNP综合征因果的R环调节剂。 我们的初步研究表明，小鼠胚胎干细胞（MESC）中的ADNP损失导致R环 专门在ADNP结合位点积累。重要的是，我们的数据表明ADNP的同源域是 对于R环抑制至关重要，并且删除同源域损害了MESC的能力 分化为神经祖细胞（NPC）。我们发现人类诱导多能干细胞（HIPSC） 使用ADNP酪氨酸719*突变（ADNP Tyr719*）也显示R环放松管制。基于这些数据，我们 假设R环在特定基因组区域的积累改变了正常基因表达 导致神经元分化不当的程序，最终可能导致ASD。我们会在这里 确定ADNP Tyr719中的R环积累如何影响多能状态的基因表达 以及神经元分化。我们将使用A评估ADNP Tyr719*的神经畸形潜力 大脑器官模型，并确定R环衰减是否可以改善神经差异缺陷 在ADNP突变体中观察到。