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，出现在产前时期，可能在 大脑发育的最初几周早期大脑发育中的染色质调节事件已经被发现， 多次与自闭症有牵连产前发育中的染色质调节在根本上不同于 成人期的染色质调控，这一直是理解ASD发病机制的障碍。在这里，我们将 使用来源于人iPSC的端脑类器官来评估调节元件的功能活性 我们通过PsychENCODE项目确定，开始解开染色质和基因调控， 皮质发育的早期阶段，包括通常无法使用死后解剖的阶段。 脑组织我们将纵向绘制这些元素在关键发育过渡期的活动， 正常类器官和ASD类器官，在不同的细胞类型（祖细胞和神经元）中分级， 通过评估它们在疾病相关变体中的富集来检查它们在ASD中的功能破坏， 从染色质构象捕获实验中确定它们的靶基因。在目标1中，我们将使用STARR- seq以绘制类器官中H3 K27 ac组蛋白相关的推定增强子的活性， 皮质发育，并将比较STARR-seq增强子与基于组蛋白的增强子， 通过PsychENCODE和表观基因组路线图识别干细胞，产前和成人死后大脑 项目在目的2中，我们将使用ATAC-seq和STARR-seq来鉴定和比较在人结肠癌中的增强子活性。 来自ASD患者和对照的类器官在早期发育和不同细胞类型中。为此，我们 使用我们从ASD家庭中产生的iPSC系的集合。在目标3中，我们将使用捕获Hi-C， RNA-seq研究3D染色质组织和启动子-增强子相互作用及其对基因表达的影响 在ASD神经细胞中的表达。然后我们将探索ASD相关的增强子是否携带疾病- 相关突变与Simons和MSSNG全基因组公共数据库序列交叉 变体。最后，在目标4中，我们将对在以下中发现的ASD相关突变进行详细的功能分析： 相关的增强子我们将在对照iPSC细胞系中设计突变，比较同基因突变对， 具有或不具有突变的类器官，并进行捕获Hi-C以鉴定其靶基因和RNA-seq 来证实它们对基因表达的影响。这些研究将绘制人类产前基因调控图 前脑，跨阶段和细胞类型，地图增强，是差异活跃的早期神经 自闭症的发展，并确定突变，是puronuncially负责这些变化。年底 结果将是鉴定出ASD病理生理学中涉及的相互作用基因网络， 遗传/表观遗传机制导致其在疾病中的功能改变。