Characterizing the CHD8 Complex to Determine its Role in Autism Spectrum Disorder

项目摘要/摘要 自闭症谱系障碍(ASD)是一种以言语障碍为特征的神经发育状况 以及非语言交流，据估计全世界有2170万人受到影响。它由来已久。 ASD有很强的遗传成分，有等位基因和基因座异质性的证据。然而，潜在的 在至少70%的病例中，ASD的遗传原因未知。最近，大规模的外显子组测序研究 导致了基于从头功能丧失突变的候选基因的发现 具有巨大的生物效应和ASD的高风险。目前，几种染色质调节剂的突变已经 与人类神经发育和精神障碍有因果关系。一种这样的染色质重构体， 染色域解旋酶DNA结合蛋白8(CHD8)与12个独立的从头丢失相关。 功能突变，这是在ASD患者中发现的单个基因中最大数量的突变。 CHD8是一种依赖于ATP的染色质重塑酶，调节转录、复制、修复和 真核生物基因组的重组。大多数依赖于ATP的染色质重塑因子在 更大的多亚单位复合体，以及我们实验室以前的研究表明，单体CHD8(~290 KDA)参与了一个约1个丙二醛的更大的复合体。这项建议的长期目标是 确定复合体的构成成员，并分析CHD8的紧密联系 与其他多肽的结合可能有助于该复合体在ASD分子病理中的作用。 我们对内源性CHD8进行了亲和纯化/质谱学分析 在成年小鼠皮质中发现了一个复合体，并确定了几个候选亚基。最有希望的热门作品之一是 α地中海贫血/智力低下综合征X连锁(ATRX)，一种先前被证明参与 智力残疾。在这项提案中，我们假设CHD8之间的关系 而ATRX通过生化研究和功能分析将阐明该蛋白的作用 ASD病理中的复杂性。这里提出的目标代表了对ASD的新的和重大的贡献 通过将重点从CHD8的瞬时伙伴转移到CHD8复合体的组成成员进行研究，如 以ATRX的身份。通过生化和结构表征实验，我们期待ATRX成为一种专门的 该复合体的亚基与CHD8稳定地相互作用。我们期望了解以下功能角色 CHD8和ATRX通过使用神经前体细胞在复合体中发挥作用。通过质谱分析 在胚胎CHD8复合体中，我们希望发现专门的亚基，并确定 从胚胎发育阶段到成年发育阶段的复杂变化。之间的联系被发现 CHD8和其他与神经发育疾病有关的蛋白质有望提供更深层次的 了解ASD患者的病理机制和确定治疗靶点。

Project Summary/Abstract Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by impaired verbal and non-verbal communication and is estimated to affect 21.7 million people worldwide. It is well established that ASD has a strong genetic component with evidence of allelic and locus heterogeneity. Yet, the underlying genetic cause of ASD is unknown for at least 70% of cases. Recently, large-scale exome sequencing studies of ASD patients have led to the discovery of candidate genes based on de novo loss-of-function mutations that carry large biological effects and high risk for ASD. Currently, mutations in several chromatin regulators have been causally linked to human neurodevelopmental and psychiatric disorders. One such chromatin remodeler, chromodomain helicase DNA binding protein 8 (CHD8), is associated with 12 independent de novo loss-of- function mutations, which is the largest number of mutations in a single gene identified in individuals with ASD. CHD8 is an ATP-dependent chromatin-remodeling enzyme that regulates transcription, replication, repair, and recombination of the eukaryotic genome. Most ATP-dependent chromatin remodeling factors function within larger multi-subunit complexes, and previous studies from our lab have indicated that monomeric CHD8 (~290 kDa) participates in a larger complex of approximately 1 MDa. The long-term objective of this proposal is to determine the constitutive members of the complex and analyze how the tight association of CHD8 with other polypeptides may contribute to the role of the complex in the molecular pathology of ASD. We performed an affinity purification/mass spectrometry-based analysis of the endogenous CHD8 complex in the adult mouse cortex and identified several subunit candidates. One of the most promising hits is alpha thalassemia/mental retardation syndrome X-linked (ATRX), a protein previously shown to be involved in intellectual disability. In this proposal, we hypothesize that characterizing the relationship between CHD8 and ATRX through biochemical investigations and functional analyses will elucidate the role of the complex in ASD pathology. The aims proposed here represent a novel and significant contribution to ASD research by shifting focus from transient partners of CHD8 to constituent members of the CHD8 complex, such as ATRX. From biochemical and structural characterization experiments, we expect ATRX to be a dedicated subunit of the complex and stably interact with CHD8. We anticipate understanding the functional roles that CHD8 and ATRX play in the complex by using neural progenitor cells. Through a mass spectrometric analysis of the embryonic CHD8 complex, we expect to discover the dedicated subunits and determine how the complex changes from the embryonic to adult stages of development. The connection uncovered between CHD8 and other proteins with contributions to neurodevelopmental diseases promises to provide a deeper understanding of pathological mechanisms and the identification of treatment targets for ASD individuals.