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提供了深入了解病理生理学的可能性 可以改变诊断和管理。过去的全基因组遗传关联研究（GWAS） 几十年来，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 伴随着对基因型和疾病风险之间难以置信的复杂生物学的深入了解。 我们的DVA优异奖工作集中在系统性红斑狼疮（SLE）的遗传学上。有 现在有超过50个已建立和发表的遗传关联，我们最近的结果将把这个风险提高到超过100 SLE风险 的位点没有机制的协会实际效用非常有限。我们目前的重点是 阐明这些机制，我们已经在IRF 5和ETS 1狼疮风险位点取得了很大进展。使用 频率论和贝叶斯统计沿着的差异和相似性的相关变量中， 主要的人类祖先，我们生成可能的因果变体的遗传学信息可信集（AICS）。 随后对这些变体的等位基因特异性功能结果的搜索极大地帮助了 目前正在进行的所有工作都是描述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鉴定DNA配体的方法，我们已经鉴定了ZBTB 3和 STAT 1分别是IRF 5和ETS 1相对特异的AICS风险等位基因转录因子。我们提出 重点关注STAT 4基因与SLE的重要关联，而STAT 1，这次是通过其 这一点，再次显得很重要。我们已经将可能的因果变异从56个减少到只有4个 STAT 4的第2和第4内含子中的变体。我们的研究结果显示了一种惊人的可能性， HMGA 1以不同的等位基因特异性结合AICS中STAT 4基因座的4种变体中的3种。HMGA 1作用 作为染色质支架影响DNA成环和染色质构象。 我们（和其他人）已经表明，STAT 4的表达是由风险单倍型改变。我们有最近的数据 表明STAT 1表达也与STAT 4等位基因相关。此外，我们还显示了 STAT 1与已发表的53个SLE风险位点的DNA结合位点（p≤10-10）。在STAT 4的强关联下， 在所有人类祖先的SLE患者中（1.2<比值比（OR）<1.8）， STAT 1与ETS 1等位基因特异性结合的证明，STAT 1与ETS 1的关系， SLE风险基因座，以及STAT 4与其他炎性疾病（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（T1 D）），我们得出结论，STAT 1-STAT 4基因座值得我们集中精力。 我们将通过实验评估AICS变体（Aim 1）与STAT 4和STAT 1之间的关系。 在HMGA 1结合AICS的情况下表达，特别是使用荧光素酶表达载体， 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），以确定在4个可能的因果变异体上的等位基因差异对 STAT 1和STAT 4的表达。我们将确定SLE基因座上STAT 1和STAT 4的等位基因特异性结合 (Aim 2）使用染色质免疫沉淀随后测序的标准方案（ChIP-seq）。 实验将在转化的B细胞系和分离的B细胞中进行，这两种细胞均来自患者， 对照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），并探索其他STAT 1-STAT 4相关的机制。 疾病：RA，SS，PBC，PSS和T1 D，强调了解机制的重要性 来理解人类自身免疫。这个项目将有助于照亮一个黑匣子的内部， STAT 4和SLE疾病表达中的DNA变异之间的关系，并在此过程中提供了见解，数据， 新的工具，有可能影响管理和治疗的发展。