Project 1: Determination of molecular differences caused by tauopathy-associated H1 and H2 haplotypes

项目 1：确定 tau 蛋白病相关 H1 和 H2 单倍型引起的分子差异

基本信息

批准号：
10295517
负责人：
ALISON M GOATE
金额：
$ 77.65万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10295517
关键词：
17q21 3-Dimensional ATAC-seq Address African Astrocytes Biological Assay CRISPR screen Cell Nucleus Cells Chromatin Chromatin Structure Chromosomes Clinical Clustered Regularly Interspaced Short Palindromic Repeats Complex Dementia Disease Enhancers Epigenetic Process European Frontotemporal Dementia Gene Expression Gene Expression Regulation Gene Proteins Genes Genetic Genetic Structures Genomics Goals Haplotypes Hi-C Human Individual Induced pluripotent stem cell derived neurons Investigation Linkage Disequilibrium Methods Microglia Modernization Molecular Mutation Nerve Degeneration Neurodegenerative Disorders Neuroglia Neurons Pathology Pathway interactions Patients Population Progressive Supranuclear Palsy Protein Isoforms Protein Splicing Proteomics RNA Splicing Regulator Genes Regulatory Element Risk Risk Factors Signal Transduction Spliced Genes Structure Tauopathies Testing Variant Work XCL1 gene base brain tissue causal variant cell type clinical phenotype differential expression disorder risk functional genomics gene function genetic association genome wide association study induced pluripotent stem cell multiple omics novel therapeutic intervention prevent protective effect protein expression risk variant stem cell model targeted treatment tau Proteins transcriptome transcriptome sequencing transcriptomics

项目摘要

PROJECT SUMMARY (PROJECT 1) Genetic association between the MAPT H1 haplotype and increased risk for multiple Tauopathies, including Frontotemporal Dementia (FTD) and Progressive Supranuclear Palsy (PSP), is well-established and replicated. Despite this, very little is known regarding the mechanisms behind the differences in disease risk associated with the H1 and H2 haplotypes. To date, comparison of these haplotypes has been restricted to studies of MAPT splicing, as well as associations with clinical phenotypes. However, there has been no investigation of the downstream functional implications of each MAPT haplotype. Our goal is to comprehensively assess the chromatin structure, gene expression and functional consequences of structural differences between the H1 and H2 haplotypes in cells from individuals of European and African ancestry, in order to better understand the mechanisms underlying risk for Tauopathy. The major MAPT haplotypes encompass a 970Kb inversion within the 17q21.31 locus and include many genes in addition to MAPT, as well as extensive genetic and structural variation. We hypothesize that the gross structural differences between haplotypes confer short- and long-range changes in chromatin structure, leading to altered regulation of gene expression within and outside the inversion, and that these changes in neurons and/or glia contribute to risk/protection for tauopathies. We will use 2D and 3D induced pluripotent stem cell (iPSC) models to examine multi-OMIC differences between H1/H1 and H2/H2 and human brain tissue derived from H1/H1 and H2/H2 carriers to validate these changes, followed by CRISPR-based functional genomic screens to test the impact of candidate causal variants on gene expression and function. We propose four specific aims: 1) Use single nuc sequencing, ISOseq and proteomics to characterize chromatin structure, gene/protein expression and splicing in human brain tissue from H1/H1 and H2/H2 carriers, 2) Use RNAseq, HiC, ATACseq, ISOseq and proteomics to characterize cell-autonomous changes in chromatin structure, gene expression and splicing in 2D neuron, astrocyte and microglial cultures from H1/H1 and H2/H2 individuals; 3) Use single cell ATAC/RNAseq to examine effects on gene expression in 3D assembloids from H1/H1 and H2/H2 individuals; 4) Use CRISPRa/i-based assays to test the functional impact of altering haplotype- and cell-specific gene enhancer regions on global gene expression and Tau- isoform expression and splicing. Understanding the mechanisms underlying the protective effects of the H2 haplotype has the potential to uncover new therapeutic strategies for FTD and other neurodegenerative diseases.

项目概要（项目 1） MAPT H1 单倍型与多种 Tau蛋白病风险增加之间的遗传关联，包括额颞叶痴呆 (FTD) 和进行性核上性麻痹 (PSP) 已得到充分证实和复制。尽管如此，人们对相关疾病风险差异背后的机制知之甚少。具有 H1 和 H2 单倍型。迄今为止，这些单倍型的比较仅限于以下研究 MAPT 剪接以及与临床表型的关联。但目前还没有对此进行调查每个 MAPT 单倍型的下游功能含义。我们的目标是全面评估 H1 之间结构差异的染色质结构、基因表达和功能后果和 H2 单倍型来自欧洲和非洲血统的个体的细胞，以便更好地了解 Tau 病的潜在风险机制。主要的 MAPT 单倍型包含 970Kb 的倒转 17q21.31 位点，包括除 MAPT 之外的许多基因，以及广泛的遗传和结构变化。我们假设单倍型之间的总体结构差异赋予短期和长期染色质结构的变化，导致细胞内外基因表达调控的改变反转，并且神经元和/或神经胶质细胞的这些变化有助于 tau蛋白病的风险/保护。我们将使用 2D 和 3D 诱导多能干细胞 (iPSC) 模型检查 H1/H1 之间的多组学差异和 H2/H2 以及源自 H1/H1 和 H2/H2 携带者的人脑组织来验证这些变化，随后通过基于 CRISPR 的功能基因组筛选来测试候选因果变异对基因的影响表达和功能。我们提出四个具体目标：1）使用单核测序、ISOseq 和蛋白质组学表征 H1/H1 和人脑组织中的染色质结构、基因/蛋白质表达和剪接 H2/H2 载体，2) 使用 RNAseq、HiC、ATACseq、ISOseq 和蛋白质组学来表征细胞自主性二维神经元、星形胶质细胞和小胶质细胞培养物中染色质结构、基因表达和剪接的变化来自 H1/H1 和 H2/H2 个体； 3) 使用单细胞 ATAC/RNAseq 检测对基因表达的影响来自 H1/H1 和 H2/H2 个体的 3D 组装体； 4) 使用基于 CRISPRa/i 的检测来测试功能改变单倍型和细胞特异性基因增强子区域对整体基因表达和 Tau 的影响同种型表达和剪接。了解 H2 保护作用的机制单倍型有可能发现 FTD 和其他神经退行性疾病的新治疗策略疾病。