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.

概括 该合作项目旨在深入了解介导激酶（CDK8 和 CDK19）及其在唐氏综合症（DS）中驱动高活性干扰素（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，有一种手段 抑制干扰素激活可能具有广泛的治疗益处。 CDK8和CDK19各自代表可成药 作为 IFN 反应的有效激活剂，抑制其功能可以有效拮抗 DS 中失控的 IFN 信号传导。 CDK8 及其旁系同源物 CDK19 被认为是“介导激酶”，因为它们 与 26 个亚基介体复合体的关联稳定但可逆。 Mediator 是 RNA 的全局调节因子 聚合酶 II (pol II) 活性，可转录人类体内所有蛋白质编码和许多非编码 RNA 基因组。 CDK8 和 CDK19 均已被证明有助于驱动对 IFN 细胞因子的转录反应， 它们是炎症和免疫功能的通用调节剂。在这里，我们试图定义他们的本质—— 但知之甚少——DS 背景下的细胞和机制作用（生物库匹配的 D21 和 T21 细胞） 和干扰素信号传导。我们将结合复杂的基于细胞的方法（例如转录组学和代谢组学） 采用详细、尖端的生化方法，可以对机制进行严格和可靠的评估 CDK8 与 CDK19 控制 DS 个体中的 IFN 反应。在目标 1 中，我们将探讨调解员的角色 激酶活性，而在目标 2 中，我们将重点关注 CDK19 蛋白，它充当关键的结构/支架 在 IFN 刺激下实现强有力的转录反应。转录组学（RNA-seq 和 PRO-seq） 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）并确定 CDK8 和/或 CDK19 如何影响细胞对 IFN 的反应。这些 实验还将揭示序列特异性、DNA 结合转录因子 (TF) 之间的差异 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 CDK8 和 CDK19 通过激酶依赖性或独立性独立控制 TF 激活或抑制 机制。在目标 3 中，我们将进行详细的体外研究，以确定其分子机制 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 然后将在细胞中进一步探讨这些发现，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（目标 1-3）可能有助于建立介导激酶（CDK8 和 CDK19）作为拮抗 DS 中过度活跃的 IFN 信号传导的可行治疗靶点。此外，平衡 采用基于发现的方法进行靶向/机制研究可能会产生新的和意想不到的治疗方法 与 DS 及其相关并发症相关的策略，这些疾病也影响普通人群。

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