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.

威胁生命的疾病的遗传学提供了对病理生理学的基本见解。 这可以改变诊断和管理。全基因组遗传关联研究(GWASs) 十年来已经确定了DNA序列与疾病表型的基因类型关系，通常没有任何 伴随着对在基因和疾病风险之间运行的令人难以置信的复杂生物学的洞察。 我们的DVA优秀奖的工作重点是系统性红斑狼疮(SLE)的遗传学。确实有 现在&gt；50已经建立并发表了遗传关联，我们最近的结果将把这一风险提高到&gt；100 SLE风险 精神错乱。没有机制的协会的实际效用非常有限。我们目前的重点已经变成了 阐明了这些机制，我们在IRF5和ETS1狼疮风险基因座上已经取得了很大的进展。vbl.使用 频率统计量和贝叶斯统计量以及与之相关的变种的异同 主要的人类祖先，我们产生的祖先信息可信的集合(AICS)看似因果的变异。 随后对这些变体的等位基因特定功能结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物种。vbl.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 将重点放在STAT4基因与SLE的重要关联上，这一次是通过其 表达，再次显得很重要。我们已经将看似合理的因果变量从56个减少到只有4个 STAT4基因第2和第4内含子的变异。我们的研究结果表明，有一种惊人的可能性 HMGA1以不同的等位基因特异性结合到STAT4基因座AICS的4个变异体中的3个。HMGA1作用 作为影响DNA环和染色质构象的染色质支架。 我们(和其他人)已经证明，STAT4的表达会因风险单倍型而改变。我们有最新的数据 表明STAT1的表达也与STAT4等位基因相关。此外，我们还展示了 STAT1DNA结合位点与已发表的53个系统性红斑狼疮风险基因座(p≤10-10)。与STAT4的强大关联性 在所有人类祖先都患有系统性红斑狼疮(OR)1.8的情况下，STAT4等位基因依赖的表达 STAT4和STAT1，STAT1与ETS1等位基因特异性结合的证明，STAT1与ETS1的关系 SLE危险基因，以及STAT4与其他炎症性疾病(类风湿关节炎(RA)、 干燥综合征(SS)、原发性胆汁性肝硬变(PBC)、进行性系统性硬化症(PSS)和1型 糖尿病(T1D)，我们得出结论，STAT1-STAT4基因座为我们赢得了集中的努力。 我们将通过实验评估AICS变体(AIM 1)与STAT4和STAT1之间的关系 在HMGA1与AICS结合的背景下表达，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，我们还将使用染色质编辑策略(CRISPR 技术)来确定4个看似合理的因果变异上的等位基因差异对 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)使用染色质免疫沉淀和测序的标准方案(CHIP-SEQ)。 实验将在转化的B细胞系和分离的B细胞中进行，这两种细胞都来自患者和 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)，并用于探讨其他STAT1-STAT4相关的机制 疾病：RA、SS、PBC、PSS和T1D，强调了解机制的重要性(S) 了解人类自身免疫力。这个项目将帮助照亮目前存在的黑匣子的内部。 STAT4和SLE疾病表达的DNA变异之间的关系，并在此过程中提供洞察力、数据和 有可能影响管理和治疗学发展的新工具。