Regulatory Variants in HUMAN SKIN DISEASES

人类皮肤疾病的监管变异

• 批准号: 10396026
• 负责人: PAUL KHAVARI
• 金额: $ 47.18万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10396026
• 关键词: Affect; Architecture; Atopic Dermatitis; Bar Codes; Binding; Biochemical; Biological Assay; CRISPR screen; Cell Line; Cells; ChIP-seq; Chromatin; Chromosomes; Chronic small plaque psoriasis; Code; Complement; Couples; Cutaneous; DNA; DNA Binding; DNA Sequence; Dependovirus; Detection; Development; Differentiated Gene; Diploidy; Disease; Dissection; Enhancers; Epidermis; Gene Expression; Genes; Genetic; Genetic Transcription; Genomics; Histones; Human; Hybrids; Impairment; Inflammatory; Inherited; Knock-out; Lasers; Link; Maps; Mediating; Methods; Molecular Conformation; Natural regeneration; Nature; Nucleotides; Pathogenesis; Pathogenicity; Permeability; Phenotype; Proteins; Proteomics; Quantitative Trait Loci; RNA; Reporter; Risk; Single Nucleotide Polymorphism; Site; Skin; Skin Tissue; Tissues; Validation; Variant; combinatorial; design; genetic approach; genetic variant; genome wide association study; human disease; human tissue; hybrid gene; interest; keratinocyte; promoter; prototype; single-cell RNA sequencing; skin disorder; skin xenograft; transcription factor

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.

人类皮肤病的变异 项目摘要 转录因子（TF）结合调节DNA以控制正常和非正常细胞中的分化基因。 病变组织，包括皮肤表皮。我们绘制了人类中活跃的调节DNA， 表皮，并发现它富含单核苷酸多态性（eSNPs，“表达SNPs”） 全基因组关联研究（GWAS）将其与炎症性皮肤病联系起来， 表皮分化有助于发病机制，包括特应性皮炎（AD）和银屑病 寻常型（PV）。AD和PV连锁的eSNPs改变人类DNA基序和转录指导活性 角质形成细胞，表明它们作为TF的结合数量性状位点（bQTL），这些TF的修饰 这些位点的活性致病性地失调靶基因（eGenes）。这种eSNP调控 它们改变了转录因子的结合，并使eGene的表达失调，形成了一个新的基因组。 多基因疾病的监管风险框架。本建议将阐明这种监管的特点， 在人体组织的背景下，使用AD和PV作为多基因疾病原型的风险框架。 首先，我们将定义人类皮肤组织中疾病相关eSNPs的表型影响，重点是 AD和PV的表皮内源性成分。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。为此，我们将生成 在其他方面相同的、同基因的人类皮肤异种移植物，其差异仅在于单个调控DNA 使用混合基因编辑方法研究核苷酸。eSNPs将被单独研究， 组合.目的我将定义AD和PV相关的调节变体在人体组织中的影响。 其次，我们将鉴定其DNA结合和功能在特定eSNPs处改变的TF， 将对疾病相关调控DNA变异的理解扩展到生化机制的水平。 为此，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 鉴定检测（DAPID）使用DNA连接的邻近蛋白质组学来检测和定量TF 与活细胞中任何感兴趣的DNA序列结合。第二个是Perturb-MPRA， 筛选大规模平行报告基因分析（MPRA），以确定转录因子对转录的重要性。 任何DNA序列指导的转录活性。目的II将鉴定其DNA结合 并且活性受到AD和PV中疾病相关的eSNP的影响。 该项目将使用新的方法和人体组织背景来定义eSNP，TF和eGene 常见人类多基因皮肤病的监管风险框架。