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

项目总结/摘要 自闭症谱系障碍（ASD）是一种神经发育疾病，其特征是言语功能受损， 和非语言交流，估计影响全球2170万人。公认的情况是 自闭症谱系障碍具有很强的遗传成分，并有等位基因和基因座异质性的证据。然而， ASD的遗传原因至少有70%是未知的。最近，大规模外显子组测序研究 ASD患者的研究发现了基于从头功能丧失突变的候选基因， 具有很大的生物学效应和ASD的高风险。目前，几种染色质调节因子的突变 与人类神经发育和精神疾病有因果关系。一种这样的染色质重塑剂， chromodomain helicase DNA binding protein 8（CHD 8），与12个独立的 功能突变，这是在ASD个体中鉴定的单个基因中的最大数量的突变。 CHD 8是一种ATP依赖性染色质重塑酶，其调节转录、复制、修复和代谢。 真核基因组的重组。大多数ATP依赖性染色质重塑因子在 更大的多亚基复合物，我们实验室以前的研究表明，单体CHD 8（~290 kDa）参与约1 MDa的更大复合物。这项建议的长远目标是 确定复合物的组成成员，并分析CHD 8的紧密结合如何 与其他多肽的结合可能有助于该复合物在ASD的分子病理学中的作用。 我们对内源性CHD 8进行了基于亲和纯化/质谱的分析。 复合物中的成年小鼠皮层，并确定了几个亚基候选人。其中最有希望的一首歌是 α地中海贫血/精神发育迟滞综合征X连锁（ATRX），一种蛋白质，以前显示参与 智力残疾。在这个建议中，我们假设，表征CHD 8之间的关系， 和ATRX通过生化调查和功能分析将阐明的作用， 复杂的ASD病理学这里提出的目标是对ASD的一个新的和重要的贡献 通过将重点从CHD 8的瞬时伴侣转移到CHD 8复合物的组成成员， 作为ATRX。从生化和结构表征实验，我们预计ATRX将是一个专门的 亚基的复合物和稳定的相互作用CHD 8。我们期望了解的职能作用， CHD 8和ATRX通过使用神经祖细胞在复合物中发挥作用。通过质谱分析 胚胎CHD 8复合物的，我们希望发现专用的亚基，并确定如何 从胚胎到成体的复杂变化。发现的联系 CHD 8和其他与神经发育疾病有关的蛋白质有望提供更深入的研究。 了解ASD个体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.