Lupus Association with Signal Transducer and Activator of Transcription 4 (STAT4)

狼疮与信号转导器和转录激活剂 4 (STAT4) 的关联

• 批准号: 9898284
• 负责人: John Barker Harley
• 金额: --
• 依托单位: CINCINNATI VA MEDICAL CENTER RESEARCH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9898284
• 关键词: Affect; Alleles; Autoimmunity; Award; B-Lymphocytes; Bayesian Analysis; Binding; Binding Sites; Biological Assay; Biology; CRISPR/Cas technology; Cell Line; Cells; ChIP-seq; Chromatin; Chromatin Loop; Complex; DNA; DNA Binding; DNA Sequence; Data; Data Set; Databases; Dendritic Cells; Development; Diagnosis; Disease; ETS1 gene; Etiology; Evaluation; Functional disorder; Funding; Future; Gene Expression; Genes; Genetic; Genetic Diseases; Genetic Predisposition to Disease; Genetic Risk; Genetic Transcription; Genome; Genotype; Goals; HMGA1 gene; Haplotypes; Human; Immunologics; Inflammatory; Informatics; Infrastructure; Insulin-Dependent Diabetes Mellitus; Interferon Type I; Interferon Type II; Interferons; Interleukin-12; Introns; Laboratories; Lead; Life; Ligands; Luciferases; Lupus; Mathematics; Mediating; Methodology; Methods; Molecular; Molecular Conformation; Monitor; Nucleic Acid Regulatory Sequences; Odds Ratio; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Peripheral Blood Mononuclear Cell; Positioning Attribute; Prevention strategy; Primary biliary cirrhosis; Process; Production; Property; Proteins; Protocols documentation; Publishing; RNA; Receptor Signaling; Receptors, Antigen, B-Cell; Reporter; Rheumatoid Arthritis; Risk; Role; STAT1 gene; STAT4 protein; Signal Transduction; Signaling Molecule; Single Nucleotide Polymorphism; Sjogren' s Syndrome; Specificity; Systemic Lupus Erythematosus; Systemic Scleroderma; T-Lymphocyte; TLR7 gene; Testing; Therapeutic; Time; Toll-like receptors; Variant; Veterans; Work; causal variant; cell type; chromatin immunoprecipitation; cytokine; design; disease phenotype; disorder risk; experimental study; expression vector; genetic architecture; genetic association; genetic element; genetic variant; genome editing; genome-wide; genomic locus; immune function; immunoregulation; insight; monocyte; novel; novel diagnostics; novel therapeutics; peripheral blood; programs; receptor internalization; response; risk variant; scaffold; therapeutic development; tool; transcription factor

The genetics of life threatening diseases offer the possibility of fundamental insights into pathophysiology that can transform diagnosis and management. Genome wide genetic association studies (GWASs) of the past decade have identified DNA sequence genotypic relationships to disease phenotypes, usually without any accompanying insight into the incredibly complex biology that operates between genotype and disease risk. Our DVA Merit Award work has focused on the genetics of Systemic Lupus Erythematosus (SLE). There are now >50 established and published genetic associations and our recent results will raise this to >100 SLE risk loci. Associations without mechanisms are of very limited practical utility. Our present focus has become elucidating these mechanisms and we have made much progress at the IRF5 and ETS1 lupus risk loci. Using frequentist and Bayesian statistics along with the differences and similarities of the associated variants in the major human ancestries we generate Ancestry Informed Credible Sets (AICSs) of plausibly causal variants. The subsequent search for allele specific functional consequences for these variants is enormously aided by all of the work now underway characterizing the protein and RNA species that interact with chromatin. Using the dataset infrastructure now available and methods that identify DNA ligands, we have identified ZBTB3 and STAT1 as relatively specific AICS risk allele transcription factors for IRF5 and ETS1, respectively. We propose to focus on the important association in the STAT4 gene with SLE where STAT1, this time through its expression, again appears to be important. We have reduced the plausibly causal variants from 56 to only 4 variants in the 2nd and 4th introns of STAT4. We have results suggesting the astonishing possibility that HMGA1 binds with varying allele specificity to 3 of the 4 variants in the AICS for the STAT4 locus. HMGA1 acts as a chromatin scaffold influencing DNA looping and chromatin conformation. We (and others) have shown that STAT4 expression is altered by the risk haplotype. We have recent data showing that STAT1 expression is also associated with STAT4 alleles. In addition, we show association of the DNA binding sites of STAT1 with the 53 published SLE risk loci (p≤10-10). With the strong association at STAT4 with SLE across all human ancestries (1.2<odds ratio (OR)<1.8), the STAT4 allele dependent expression of STAT4 and STAT1, the demonstration of allele specific binding of STAT1 at ETS1, the relationship of STAT1 to SLE risk loci, and the association of STAT4 with other inflammatory diseases (rheumatoid arthritis (RA), Sjögren's syndrome (SS), primary biliary cirrhosis (PBC), progressive systemic sclerosis (PSS), and type 1 diabetes (T1D)), we conclude that the STAT1-STAT4 locus has earned our concentrated effort. We will experimentally evaluate the relationship between the AICS variants (Aim 1) and STAT4 and STAT1 expression in the context of HMGA1 binding to the AICS, especially using luciferase expression vectors and with chromatin editing of the AICS variants. In addition, we will use chromatin editing strategies (CRISPR technologies) to establish the influence of allelic differences at the 4 plausibly causal variants on the expression of STAT1 and STAT4. We will identify the allele specific binding of STAT1 and STAT4 at SLE loci (Aim 2) using a standard protocol for chromatin immunoprecipitation followed by sequencing (ChIP-seq). Experiments will be performed in transformed B cell lines and in isolated B cells, both from patients and controls. These experiments will set the stage for future experiments in other cell types (various T cells, monocytes, dendritic cells) and for the exploration of mechanism in the other STAT1-STAT4 associated diseases: RA, SS, PBC, PSS, and T1D, emphasizing how important understanding the mechanism(s) will be for understanding human autoimmunity. This project will help illuminate the inside of a black box now existent between DNA variants in STAT4 and SLE disease expression and in the process provide insights, data, and new tools that have the potential to influence management and the development of therapeutics.

危及生命的疾病的遗传学为深入了解病理生理学提供了可能性 可以改变诊断和管理。过去的全基因组遗传关联研究（GWAS） 十年来已经确定了 DNA 序列基因型与疾病表型的关系，通常没有任何关系 伴随着对基因型和疾病风险之间极其复杂的生物学的深入了解。 我们的 DVA 优异奖工作重点是系统性红斑狼疮 (SLE) 的遗传学。有 现在超过 50 个已建立并公布的遗传关联，我们最近的结果将把系统性红斑狼疮风险提高到 >100 基因座。没有机制的协会的实际用途非常有限。我们现在的重点已经变成 通过阐明这些机制，我们在 IRF5 和 ETS1 狼疮风险位点方面取得了很大进展。使用 频率论和贝叶斯统计以及相关变体的差异和相似之处 我们根据主要人类祖先生成合理因果变异的祖先知情可信集（AICS）。 随后对这些变异的等位基因特异性功能后果的搜索得到了极大的帮助 目前正在进行的所有工作都在表征与染色质相互作用的蛋白质和 RNA 种类。使用 现在可用的数据集基础设施和识别 DNA 配体的方法，我们已经识别了 ZBTB3 和 STAT1 分别作为 IRF5 和 ETS1 相对特异的 AICS 风险等位基因转录因子。我们建议 重点关注 STAT4 基因与 SLE 的重要关联，其中 STAT1，这次是通过其 表达，再次显得很重要。我们将看似合理的因果变异从 56 个减少到只有 4 个 STAT4 第二个和第四个内含子的变异。我们的研究结果表明，有一种令人惊讶的可能性： HMGA1 以不同的等位基因特异性与 AICS 中 STAT4 位点的 4 个变体中的 3 个结合。 HMGA1作用 作为影响 DNA 循环和染色质构象的染色质支架。 我们（和其他人）已经证明 STAT4 表达会因风险单倍型而改变。我们有最新数据 显示 STAT1 表达也与 STAT4 等位基因相关。另外，表示协会 STAT1 与 53 个已发表的 SLE 风险位点的 DNA 结合位点 (p≤10-10)。与 STAT4 的强关联 所有人类祖先的 SLE 患者（1.2<优势比 (OR)<1.8），STAT4 等位基因依赖性表达 STAT4和STAT1，STAT1在ETS1上等位基因特异性结合的演示，STAT1的关系 STAT4 与 SLE 风险位点的关系，以及 STAT4 与其他炎症性疾病（类风湿性关节炎 (RA)、 干燥综合征 (SS)、原发性胆汁性肝硬化 (PBC)、进行性系统性硬化症 (PSS) 和 1 型 糖尿病（T1D）），我们的结论是 STAT1-STAT4 位点赢得了我们的集中努力。 我们将通过实验评估 AICS 变体（目标 1）与 STAT4 和 STAT1 之间的关系 HMGA1 与 AICS 结合的背景下的表达，特别是使用荧光素酶表达载体和 对 AICS 变体进行染色质编辑。此外，我们将使用染色质编辑策略（CRISPR 技术）来确定 4 个可能因果变异的等位基因差异对 STAT1 和 STAT4 的表达。我们将鉴定 STAT1 和 STAT4 在 SLE 基因座上的等位基因特异性结合 （目标 2）使用标准方案进行染色质免疫沉淀，然后进行测序 (ChIP-seq)。 实验将在转化的 B 细胞系和分离的 B 细胞中进行，这些细胞均来自患者和 控制。这些实验将为未来在其他细胞类型（各种 T 细胞、 单核细胞、树突状细胞）以及其他 STAT1-STAT4 相关机制的探索 疾病：RA、SS、PBC、PSS 和 T1D，强调了解其机制的重要性 用于了解人类自身免疫。该项目将有助于照亮现有黑匣子的内部 STAT4 和 SLE 疾病表达中的 DNA 变异之间的关系，并在此过程中提供见解、数据和 有潜力影响管理和治疗方法开发的新工具。