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

项目概要（项目1） MAPT H1单倍型与多种Tau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），是公认的和复制。 尽管如此，关于与糖尿病相关的疾病风险差异背后的机制知之甚少。 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。然而，没有调查 每个MAPT单倍型的下游功能含义。我们的目标是全面评估 染色质结构、基因表达和H1之间结构差异的功能后果 和H2单倍型，以便更好地了解 Tauopathy的潜在风险机制。主要的MAPT单倍型包括一个970 Kb倒位， 17q21.31基因座，除了MAPT外还包括许多基因，以及广泛遗传和结构基因 变化量我们假设，单倍型之间的总体结构差异赋予了短距离和长距离的遗传变异。 染色质结构的变化，导致细胞内外基因表达的调节改变， 在一些实施方案中，神经元和/或神经胶质中的这些变化有助于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）模型来检查H1/H1之间的多OMIC差异 和H2/H2以及来自H1/H1和H2/H2载体的人脑组织来验证这些变化， 通过基于CRISPR的功能基因组筛选来测试候选致病变体对基因的影响， 表达和功能。我们提出了四个具体目标：1）使用单核苷酸测序，ISOseq和蛋白质组学 表征H1/H1人脑组织中的染色质结构、基因/蛋白质表达和剪接， H2/H2携带者，2）使用RNAseq、HiC、ATACseq、ISOseq和蛋白质组学来表征细胞自主表达。 2D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检查对H1/H1和H2/H2个体中基因表达的影响。 来自H1/H1和H2/H2个体的3D类胡萝卜素; 4）使用基于CRISPRa/i的测定来测试功能性类胡萝卜素。 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蛋白表达的影响 同种型表达和剪接。了解H2保护作用的机制 单倍型有可能揭示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.