Mediator kinases as interferon antagonists in Down Syndrome

介导激酶作为唐氏综合症干扰素拮抗剂

• 批准号: 10677733
• 负责人: Dylan J Taatjes
• 金额: $ 46.5万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-22 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677733
• 关键词: Acute; Address; Affect; Age; Area; Autoimmune Diseases; Automobile Driving; B lymphocyte immortalization; Basic Science; Biochemical; Biological Assay; Cells; Chromosome 21; Chronic; Classification; Clinical; Code; Complement; Complex; DNA Binding; Defect; Dependence; Development; Down Syndrome; Ensure; Enzymes; Equilibrium; Foundations; Gene Expression; General Population; Genes; Genetic Transcription; Goals; Human; Human Genome; Hyperactivity; Hypersensitivity; Immune response; Immunity; In Vitro; Individual; Infection; Inflammation; Inflammatory; Interferon Activation; Interferon Receptor; Interferons; Link; Mediator; Metabolism; Methods; Molecular; Nuclear; Pathway interactions; Phosphotransferases; Polymerase; Proteins; RNA Polymerase II; RNA Splicing; Reagent; Regulation; Repression; Research Personnel; Role; Runaway; STAT1 gene; Sampling; Siblings; Signal Transduction; Solid; Spliced Genes; Stimulus; System; Testing; Therapeutic; Transcription Initiation; Transcriptional Regulation; United States National Institutes of Health; Untranslated RNA; antagonist; biobank; comorbidity; cortistatin; cytokine; druggable target; experience; experimental study; genome-wide; high reward; high risk; immune function; inhibitor; insight; interferon antagonist; metabolomics; new therapeutic target; novel therapeutic intervention; paralogous gene; programs; promoter; public health relevance; response; scaffold; sex; therapeutic target; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

SUMMARY This collaborative project seeks to gain a deep mechanistic understanding of the Mediator kinases (CDK8 and CDK19) and their roles in driving hyper-active interferon (IFN) signaling in Down syndrome (DS). A chronic inflammatory state underlies the DS condition and contributes to its co-morbidities; consequently, a means to dampen IFN activation could have broad therapeutic benefits. CDK8 and CDK19 each represent druggable targets and, as potent activators of IFN responses, inhibition of their function could effectively antagonize runaway IFN signaling in DS. CDK8 and its paralog, CDK19, are considered “Mediator kinases” because of their stable, but reversible, association with the 26-subunit Mediator complex. Mediator is a global regulator of RNA polymerase II (pol II) activity, which transcribes all protein-coding and many non-coding RNAs in the human genome. CDK8 and CDK19 have each been shown to help drive transcriptional responses to IFN cytokines, which are universal regulators of inflammation and immune function. Here, we seek to define their essential— yet poorly understood—cellular and mechanistic roles in the context of DS (Biobank-matched D21 and T21 cells) and IFN signaling. We will combine sophisticated cell-based methods (e.g. transcriptomics and metabolomics) with detailed, cutting-edge biochemical approaches to allow rigorous and reliable assessment of the mechanisms by which CDK8 vs. CDK19 control IFN responses in DS individuals. In Aim 1, we will probe the role of Mediator kinase activity, whereas in Aim 2 we will focus on the CDK19 protein, which serves key structural/scaffolding roles to enable robust transcriptional responses upon IFN stimulation. Transcriptomic (RNA-seq and PRO-seq) and metabolomics approaches will be applied in Aims 1 and 2, to assess T21-specific signatures (including potential splicing defects) and to identify how CDK8 and/or CDK19 impact cellular responses to IFN. These experiments will also uncover how sequence-specific, DNA-binding transcription factors (TFs) are differentially mobilized in T21 vs. D21 cells, including during IFN stimulation. As part of this, we will establish how CDK8 and CDK19 independently control TF activation or repression, through kinase-dependent or -independent mechanisms. In Aim 3, we will perform detailed in vitro studies to define the molecular mechanisms by which Mediator kinases regulate pol II transcription initiation, promoter-proximal pausing, or elongation. Mechanistic findings will then be probed further in cells, to better characterize their significance in DS and related IFN signaling cascades. Collectively, these experiments (Aims 1-3) may help establish Mediator kinases (CDK8 & CDK19) as viable therapeutic targets to antagonize hyper-active IFN signaling in DS. Moreover, the balance of targeted/mechanistic studies with discovery-based approaches may yield new and unanticipated therapeutic strategies with relevance for DS and its associated co-morbidities, which also affect the general population.

总结 这个合作项目旨在获得对介体激酶（CDK 8和 CDK 19）及其在唐氏综合症（DS）中驱动过度活跃的干扰素（IFN）信号传导中的作用。一种慢性 炎症状态是DS病症的基础，并导致其合并症;因此， 抑制IFN的活化可能具有广泛的治疗益处。CDK 8和CDK 19各自代表可药用的 作为IFN应答的有效激活剂，抑制它们的功能可以有效地拮抗 在DS中失控的IFN信号传导。CDK 8和它的副产物CDK 19被认为是“介体激酶”，因为它们的 与26-亚基介体复合物稳定但可逆的结合。介体是RNA的全局调节因子 聚合酶II（pol II）活性，其转录人类中的所有蛋白质编码和许多非编码RNA。 基因组CDK 8和CDK 19均已显示有助于驱动对IFN细胞因子的转录应答， 它们是炎症和免疫功能的通用调节剂。在这里，我们试图定义其基本- 然而，在DS（生物库匹配的D21和T21细胞）背景下，对细胞和机制作用的理解不足 和IFN信号。我们将结合联合收割机复杂的基于细胞的方法（例如转录组学和代谢组学） 详细的，尖端的生化方法，以允许严格和可靠的评估机制， CDK 8与CDK 19控制DS个体中的IFN应答。在目标1中，我们将探讨中介人的作用 激酶活性，而在目标2中，我们将重点关注CDK 19蛋白，它提供关键的结构/支架 在IFN刺激后能够产生稳健的转录应答。转录组学（RNA-seq和PRO-seq） 和代谢组学方法将应用于目标1和2，以评估T21特异性特征（包括 潜在的剪接缺陷）和鉴定CDK 8和/或CDK 19如何影响细胞对IFN的应答。这些 实验还将揭示序列特异性的DNA结合转录因子（TF）是如何与其他转录因子的差异。 在T21与D21细胞中动员，包括在IFN刺激期间。作为其中的一部分，我们将确定CDK 8和 CDK 19通过激酶依赖性或非依赖性途径独立控制TF的激活或抑制， 机制等在目标3中，我们将进行详细的体外研究，以确定 介体激酶调节pol II转录起始、启动子近端暂停或延伸。机械论 这些发现将在细胞中进一步探索，以更好地表征它们在DS和相关IFN中的意义 信号级联。总的来说，这些实验（目的1-3）可以帮助建立介体激酶（CDK 8 & CDK 19）作为可行的治疗靶标来拮抗DS中的过度活跃的IFN信号传导。此外， 采用基于发现的方法进行的靶向/机制研究可能会产生新的和非预期的治疗方法， 与DS及其相关合并症相关的策略，也影响一般人群。

项目成果

Transcription dosage compensation does not occur in Down syndrome.

• DOI: 10.1186/s12915-023-01700-4
• 发表时间: 2023-11-10
• 期刊: BMC BIOLOGY
• 影响因子: 5.4
• 作者: Hunter, Samuel;Hendrix, Jo;Freeman, Justin;Dowell, Robin D.;Allen, Mary A.
• 通讯作者: Allen, Mary A.

Metabolic and Transcriptomic Effects of Mediator Kinase Inhibition on the Interferon Response in Down Syndrome.

介导激酶抑制对唐氏综合症干扰素反应的代谢和转录组学影响。

• 发表时间: 2022
• 期刊: FASEB journal : official publication of the Federation of American Societies for Experimental Biology
• 作者: Cozzolino,KiraA;Jones,Taylor;Ajit,Deepa;Dowell,Robin;Taatjes,Dylan
• 通讯作者: Taatjes,Dylan