3/3 Chromatin regulation during brain development and in ASD

3/3 大脑发育和自闭症谱系障碍中的染色质调节

• 批准号: 9727072
• 负责人: GREGORY E CRAWFORD
• 金额: $ 35.59万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9727072
• 关键词: ATAC-seq; Adult; Autistic Disorder; Autopsy; Biological Assay; Brain; CRISPR/Cas technology; Cell Line; Cell model; Cells; ChIP-seq; Chromatin; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Data; Data Set; Databases; Development; Disease; Distal; Elements; Engineering; Enhancers; Epigenetic Process; Event; Family; Fathers; Female; Functional disorder; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Enhancer Element; Genomic Segment; Goals; Histones; Human; Individual; Link; Maps; Mutation; Neurons; Nucleic Acid Regulatory Sequences; Organoids; Pathogenesis; Patients; Phenotype; Population; Prosencephalon; Regulation; Regulatory Element; Site; Specimen; Stem cells; Synapses; Time; Tissue-Specific Gene Expression; Tissues; Variant; autism onset; autism spectrum disorder; base; brain tissue; cell type; chromosome conformation capture; developmental disease; epigenome; experience; experimental study; fetal; functional genomics; genetic variant; induced pluripotent stem cell; male; molecular sequence database; neurodevelopment; pluripotency; prenatal; proband; progenitor; promoter; sex; transcription factor; transcriptome sequencing; treatment strategy; whole genome; wiki

Abstract Autism spectrum disorder (ASD) is a developmental disorder that emerges in the prenatal period, likely during the first weeks of brain development. Chromatin regulatory events in early brain development have been repeatedly implicated in ASD. Chromatin regulation in prenatal development differs in fundamental ways from chromatin regulation in adulthood, which has been an obstacle to understand ASD pathogenesis. Here, we will use telencephalic organoids derived from human iPSCs to assess the functional activity of regulatory elements we identified through the PsychENCODE project to begin to unravel chromatin and gene regulation during early stages of cortical development, including stages that are not commonly accessible using postmortem brain tissue. We will longitudinally map the activity of these elements at critical developmental transitions in both normal organoids and ASD organoids, fractioned in different cell types (progenitors and neurons), examine their functional disruption in ASD by assessing their enrichment in disease-associated variants and determine their target genes from chromatin conformation capture experiments. In Aim 1, we will use STARR- seq to map the activity of H3K27ac histone-associated putative enhancers in organoids mimicking early cortical development and will compare the STARR-seq enhancers with histone-based enhancers active in stem cells, prenatal and adult postmortem brain identified through PsychENCODE and Epigenome Road map projects. In Aim 2, we will use ATAC-seq and STARR-seq to identify and compare enhancer activity in organoids from ASD patients and controls across early development and in different cell types. For this, we will use a collection of iPSC lines we generated from families with ASD. In Aim 3, we will use capture Hi-C and RNA-seq to study the 3D chromatin organization and promoter-enhancer interactions and their effect on gene expression in ASD neural cells. We will then explore whether ASD-implicated enhancers harbor disease- associated mutations by intersection with Simons and MSSNG whole genome public databases sequence variants. Finally, in Aim 4, we will carry out detailed functional analyses on ASD-associated mutations found in the implicated enhancers. We will engineer mutations in control iPSC lines, compare pairs of isogeneic organoids with or without the mutations, and perform capture Hi-C to identify their target genes and RNA-seq to confirm their effect on gene expression. These studies will chart gene regulation in human prenatal forebrain, across stages and cell types, map enhancers that are differentially active in early neural development in autism and identify mutations that are putatively responsible for these alterations. The end results will be the identification of a network of interacting genes involved in the pathophysiology of ASD, and the genetic/epigenetic mechanism responsible for their altered function in the disorder.

摘要 自闭症谱系障碍(ASD)是一种发育障碍，出现在产前，很可能是在 大脑发育的头几周。大脑发育早期的染色质调节事件 多次与ASD有牵连。产前发育中的染色质调节在根本上不同于 成年期的染色质调节一直是理解ASD发病机制的障碍。在这里，我们将 使用来源于人IPSCs的端脑有机体来评估调节元件的功能活性 我们通过心理密码项目确定开始解开染色质和基因调控在 皮质发育的早期阶段，包括通常不能通过尸检获得的阶段 脑组织。我们将纵向绘制出这些元素在关键发育转变时期的活动 正常类器官和自闭症类器官，分成不同类型的细胞(祖细胞和神经元)， 通过评估它们在疾病相关变异体中的丰富和 通过染色质构象捕捉实验确定其靶基因。在目标1中，我们将使用STAR- SEQ定位H3K27ac组蛋白相关组蛋白增强子在早期模拟有机体中的活性 并将比较STARR-SEQ增强剂和活性在 心理编码和表观基因组路线图鉴定干细胞、出生前和成人死后脑 项目。在目标2中，我们将使用atac-seq和starr-seq来鉴定和比较 来自ASD患者和对照组的不同细胞类型的早期发育的有机化合物。为此，我们将 使用我们从患有ASD的家庭中生成的IPSC行的集合。在目标3中，我们将使用Capture Hi-C和 RNA-SEQ研究3D染色质结构和启动子-增强子相互作用及其对基因的影响 在ASD神经细胞中表达。然后我们将探索与ASD相关的增强子是否存在疾病- 与SIMONS和MSSNG全基因组公共数据库序列交叉的关联突变 变种。最后，在目标4中，我们将对发现的ASD相关突变进行详细的功能分析 隐含的增强剂。我们将在控制IPSC系中设计突变，比较成对的同源基因 有或没有突变的有机化合物，并进行捕获Hi-C以确定其目标基因和RNA-seq 以确认它们对基因表达的影响。这些研究将绘制人类产前的基因调控图。 跨阶段和不同细胞类型的前脑，在早期神经中具有不同活性的MAP增强剂 自闭症的发展，并确定可能导致这些变化的突变。结束 结果将是识别参与ASD病理生理学的相互作用的基因网络，以及 导致它们在疾病中功能改变的遗传/表观遗传机制。