Regulatory Variants in HUMAN SKIN DISEASES

人类皮肤病的变异 项目总结 转录因子(TF)与调节DNA结合以控制正常和 病变组织，包括皮肤表皮。我们绘制了人类活跃的调控DNA图谱 并发现它富含单核苷酸多态(eSNPs，表达SNPs)。 通过全基因组关联研究(GWAS)与炎症性皮肤病相关，在这些疾病中 表皮分化有助于发病，包括特应性皮炎(AD)和牛皮癣 普通人(PV)。AD和PV连锁的eSNPs改变人类DNA基序和转录导向活性 角质形成细胞，提示它们是其修饰的TF的结合数量性状基因座(BQTL) 这些位点的活性在病理性地失调靶基因(Egene)。这样的ESNP监管 变异体，它们改变其结合的转录因子，以及它们表达失调的Egene，形成一个 多基因疾病的监管风险框架。这项提案将阐明这种监管的特点 人类组织背景下的风险框架，使用AD和PV作为多基因疾病的原型。 首先，我们将定义人类皮肤组织中与疾病相关的eSNPs的表型影响，重点是 AD和PV的表皮--固有成分。我们将评估疾病ESNP对基因的影响 完整人表皮组织的表达、结构和功能。要做到这一点，我们将生成 其他相同的、同基因的人皮肤异种移植，只是单个调控DNA不同 使用混合基因编辑方法研究核苷酸。ESNPs将单独研究，并在 组合。目的I将定义AD和PV相关的调节变异体在人体组织中的影响。 其次，我们将确定其DNA结合和功能在特定eSNPs处改变的转录因子 将对与疾病相关的调控DNA变体的理解扩展到生化机制的水平。 为了做到这一点，我们将使用新的蛋白质组学和遗传学方法。第一种方法，DNA蛋白质 识别检测(DAPID)使用DNA连接的邻近蛋白质组学来检测和定量转铁蛋白 与活细胞中任何感兴趣的DNA序列结合。第二个，扰动-MPRA，夫妇CRISPR 对大规模平行报告试验(MPRA)的筛选，以确定对TFS至关重要的因子 由任何DNA序列指示的转录活性。AIM II将确定其DNA结合的TF AD和PV中的活性受疾病相关eSNPs的影响。 这个项目将使用新的方法和人体组织环境来定义ESNP、Tf和Egene 常见人类多基因皮肤病的监管风险框架。

VARIANTS IN HUMAN SKIN DISEASE PROJECT SUMMARY Transcription factors (TFs) bind regulatory DNA to control differentiation genes in normal and diseased tissue, including cutaneous epidermis. We mapped regulatory DNA active in human epidermis and found it enriched for single nucleotide polymorphisms (eSNPs, for “expression SNPs”) linked by genome-wide association studies (GWAS) to inflammatory skin diseases in which altered epidermal differentiation contributes to pathogenesis, including atopic dermatitis (AD) and psoriasis vulgaris (PV). AD and PV-linked eSNPs alter DNA motifs and transcription-directing activity in human keratinocytes, suggesting they serve as binding quantitative trait loci (bQTLs) for TFs whose modified activity at these sites pathogenically dysregulates target genes (eGenes). Such eSNP regulatory variants, the TFs whose binding they alter, and the eGenes whose expression they dysregulate, form a regulatory risk framework for polygenic disease. This proposal will elucidate features of such regulatory risk frameworks in the context of human tissue, using AD and PV as polygenic disease prototypes. First, we will define phenotypic impacts of disease-linked eSNPs in human skin tissue, focusing on the epidermis-intrinsic component of AD and PV. We will assess disease eSNP impacts on the gene expression, architecture and function of intact human epidermal tissue. To do this, we will generate otherwise identical, isogenic human skin xenografts that differ only by the single regulatory DNA nucleotides studied using a hybrid gene editing approach. eSNPs will be studied singly and in combination. Aim I will define the impacts of AD and PV-linked regulatory variants in human tissue. Second, we will identify the TFs whose DNA binding and function is changed at specific eSNPs to extend understanding of disease-linked regulatory DNA variants to the level of biochemical mechanism. To do this, we will use new proteomic and genetic methods. The first method, DNA Protein Identification Detection (DAPID) uses DNA-tethered proximity proteomics to detect and quantify TF binding to any DNA sequence of interest in living cells. The second, Perturb-MPRA, couples CRISPR screening to the massively parallel reporter assay (MPRA) to identify the TFs essential for the transcriptional activity directed by any DNA sequence. Aim II will identify the TFs whose DNA binding and activity is affected by disease-linked eSNPs in AD and PV. This project will use new methods and a human tissue context to define the eSNP, TF, and eGene regulatory risk frameworks in common human polygenic skin diseases.