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

项目总结(项目1) MAPT H1单倍型与多发性肌萎缩侧索硬化症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)是公认的和可复制的疾病。 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。到目前为止，这些单倍型的比较仅限于对 MAPT剪接，以及与临床表型的关联。然而，目前还没有对 每种MAPT单倍型的下游功能暗示。我们的目标是全面评估 染色质结构、基因表达和H1之间结构差异的功能后果 以及来自欧洲和非洲血统的个体的H2单倍型，以便更好地了解 牵张症潜在风险的机制。主要的MAPT单倍型包括一个970Kb的倒位 17q21.31位点除MAPT外，还包括许多基因，以及广泛的遗传和结构 变种。我们假设，单倍型之间的总体结构差异提供了短期和长期 染色质结构的变化，导致细胞内外基因表达调控的改变 倒置，神经元和/或神经胶质细胞的这些变化有助于紧张症的风险/保护。我们会 使用2D和3D诱导多能干细胞(IPSC)模型研究H1/H1间的多组学差异 而H2/H2和来自H1/H1和H2/H2载体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 表情和功能。我们提出了四个具体的目标：1)使用单个nuc测序、isseq和蛋白质组学 研究人脑组织中h1/h1和h1/h1的染色质结构、基因/蛋白表达和剪接。 H_2/H_2载体，2)用RNAseq、HIC、ATACseq、ISOseq和蛋白质组学来表征细胞自主性 2D神经元、星形胶质细胞和小胶质细胞培养中染色质结构、基因表达和剪接的变化 来自H1/H1和H2/H2的个体；3)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集合体；4)使用基于CRISPRa/I的分析来测试功能 改变单倍型和细胞特异性基因增强子区域对全球基因表达和Tau的影响 异构体表达和剪接。理解H2保护作用的潜在机制 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。

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.