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.

摘要 这个合作项目寻求获得对介体蛋白激酶(CDK8和CDK8)的深入机械性理解 CDK19)及其在唐氏综合征(DS)中驱动高活性干扰素信号转导中的作用。一种慢性病 炎症状态是DS状况的基础，并导致其共病；因此，一种手段 抑制干扰素的激活可能具有广泛的治疗益处。CDK8和CDK19分别代表可用药 作为干扰素反应的有效激活剂，抑制它们的功能可以有效地拮抗 DS中的干扰素信号失控。CDK8及其类似物CDK19被认为是“介体蛋白激酶”，因为它们 稳定的，但可逆的，与26亚单位介体复合体的联系。介体是RNA的全球调节者 聚合酶II(PolII)活性，转录人类所有编码蛋白质和许多非编码RNA 基因组。CDK8和CDK19各自被证明有助于推动对干扰素细胞因子的转录反应， 它们是炎症和免疫功能的通用调节器。在这里，我们试图定义它们的本质- 然而知之甚少-在DS(生物库匹配的D21和T21细胞)背景下的细胞和机械作用 和干扰素信号。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) 具有详细、尖端的生化方法，以允许对机制进行严格和可靠的评估 CDK8与CDK19控制DS患者干扰素应答的比较。在目标1中，我们将探讨调解人的角色 而在目标2中，我们将重点关注CDK19蛋白，它为关键的结构/支架提供服务 在干扰素刺激下实现强大的转录反应的作用。转录(RNA-seq和proseq) 代谢组学方法将在AIMS 1和2中应用，以评估T21特异性签名(包括 潜在的剪接缺陷)，并确定CDK8和/或CDK19如何影响细胞对干扰素的反应。这些 实验还将揭示序列特异性的、DNA结合的转录因子(TF)是如何不同的 在T21和D21细胞中动员，包括在干扰素刺激期间。作为这项工作的一部分，我们将确定CDK8和 CDK19通过依赖或非依赖的方式独立控制转铁蛋白的激活或抑制 机制。在目标3中，我们将进行详细的体外研究，以确定通过 介体蛋白激酶调节PolII转录的启动、启动子近端的暂停或延伸。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和相关干扰素中的意义 信号级联。总的来说，这些实验(目标1-3)可能有助于建立介体蛋白激酶(CDK8& CDK19)可作为抗DS高活性干扰素信号转导的有效治疗靶点。此外，这一平衡 以发现为基础的针对性/机械性研究可能会产生新的和意想不到的治疗方法 与DS及其相关的共病相关的战略，这也影响到普通人群。

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