IPMK function in chromatin

IPMK 在染色质中的功能

• 批准号: 10350670
• 负责人: Raymond Daniel Blind
• 金额: $ 37.4万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-15 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10350670
• 关键词: Acetylation; Address; Astrocytes; Astrocytoma; Binding Proteins; Biological; Biological Models; Cell Line; Cell model; Cells; Chromatin; Chronic; Complement; Cues; Data; Detection; Development; Elements; Enzymes; Etiology; Eukaryota; Event; Experimental Models; FDA approved; Feeds; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Epistasis; Genetic Transcription; Genomic approach; Genomics; HDAC1 gene; Histone Acetylation; Histone Deacetylase; Histones; Human; Hydrophobicity; In Vitro; Individual; Inositol; Inositol Phosphates; Lipid Binding; Lipids; Location; Mediating; Modeling; Mus; Nuclear; Nuclear Receptors; Nucleosomes; Paper; Pathway interactions; Pharmaceutical Preparations; Phenotype; Phospholipids; Phosphotransferases; Physiological; Plants; Production; Regulation; Second Messenger Systems; Series; Signal Transduction; Signaling Molecule; Site; Slide; Testing; Transcript; Transcription Initiation Site; Transcription Repressor; Transcriptional Regulation; Work; Yeasts; animal tissue; base; chemical genetics; chromatin remodeling; enzyme activity; in vitro activity; inhibitor; kinase inhibitor; mutant; novel; promoter; recruit; response; structural biology; transcription factor; tumor microenvironment; tumor xenograft; yeast genetics

Abstract: For the past two decades, several labs including John York, Susan Wente, Steve Shears, Adolfo Saiardi and Solomon Synder have tried to elucidate how higher-order inositol phosphate 2nd messenger signaling molecules (inositols) regulate transcription, mainly examining single transcriptional units in yeast by genetic complementation/epistasis analyses. These studies focused on the completely conserved and ubiquitous inositol phosphate multikinase (IPMK, ipk2), as this kinase sits at the nexus of several pathways required for production of all higher inositols. IPMK activity is clearly required to rescue yeast phenotypes and transcripts from individual elements, but how inositols achieved this regulation was undescribed, as the chromatin effectors of inositols were unknown. In 2003, Erin O'Shea showed the kinase activity of IPMK regulates nucleosome sliding in yeast, Carl Wu and another group showed inositols regulate ATP-dependent chromatin remodelers Ino80 and Swi/Snf in vitro. However, inositol regulation of ATP-remodelers has not been built upon in any cellular studies since, despite availability of genomic approaches to examine open chromatin. We discovered a completely different way IPMK could regulate transcription, by directly phosphorylating a phospholipid while the lipid is bound in the hydrophobic cleft of a nuclear receptor. This model threatened to explain why the chromatin targets of IPMK were difficult to identify - they might be lipid- binding proteins, not inositol-binding proteins. This led us to attempt to identify other transcription factors regulated similarly by IPMK using genomics, presented in this proposal. In our human cell models we see IPMK is recruited to hundreds of transcriptional start sites, controlling transcript accumulation at those promoters in a kinase-dependent manner. But to our great surprise, GSEA immediately suggested IPMK primarily (but certainly not exclusively) regulates gene expression through histone deacetylases (HDACs). HDACs are transcriptional repressors shown in a series of structural biology papers by John Schwabe's group to require inositols, not lipids, for full activity in vitro. Indeed, histone acetylation increases upon IPMK loss, occurring at specific subsets of transcriptional start sites that recruit IPMK. All these aspects of IPMK functions in chromatin and at transcriptional start sites are novel. This proposal more deeply interrogates the new chromatin functions of IPMK described in our preliminary data, taking advantage of new chemical-genetics and other mutants of IPMK we have developed. Aim 1 identifies which of the new chromatin events are mediated most directly by IPMK, so mechanism can be studied. Aim 2 determines which IPMK-mediated chromatin events are shared between physiologically relevant model systems. Aim 3 resolves the mechanism of IPMK gene regulation. This proposal addresses long standing questions of how IPMK regulates gene expression while introducing a new chromatin-based 2nd messenger signaling paradigm that controls histone marks and transcription.

抽象的： 在过去的二十年里，包括 John York、Susan Wente、Steve Shears、Adolfo 在内的多个实验室 Saiardi 和 Solomon Synder 试图阐明高阶肌醇磷酸盐第二信使的作用 信号分子（肌醇）调节转录，主要通过以下方式检查酵母中的单个转录单位： 遗传互补/上位分析。这些研究集中于完全保守和 普遍存在的肌醇磷酸多激酶（IPMK、ipk2），因为该激酶位于多个途径的连接处 生产所有高级肌醇所需的。 IPMK 活性显然是拯救酵母表型所必需的 单个元件的转录本，但肌醇如何实现这种调节尚未描述，因为 肌醇的染色质效应子尚不清楚。 2003年，Erin O'Shea展示了IPMK的激酶活性 调节酵母中的核小体滑动，Carl Wu 和另一组表明肌醇调节 ATP 依赖性 体外染色质重塑剂 Ino80 和 Swi/Snf。然而，肌醇对 ATP 重塑剂的调节尚未得到证实。 尽管可以使用基因组方法来检查开放染色质，但此后的任何细胞研究都建立在这一基础上。 我们发现了一种完全不同的 IPMK 调节转录的方式，通过直接 磷酸化磷脂，同时脂质结合在核受体的疏水裂口中。这 模型可能会解释为什么 IPMK 的染色质目标难以识别——它们可能是脂质—— 结合蛋白，而不是肌醇结合蛋白。这导致我们尝试识别其他转录因子 本提案中提出的 IPMK 使用基因组学进行类似的监管。在我们的人体细胞模型中我们看到 IPMK 被招募到数百个转录起始位点，控制这些转录起始位点的积累 以激酶依赖性方式启动子。但令我们大吃一惊的是，GSEA 立即建议了 IPMK 主要（但肯定不是唯一）通过组蛋白脱乙酰酶 (HDAC) 调节基因表达。 John Schwabe 小组在一系列结构生物学论文中展示了 HDAC 是转录抑制因子 需要肌醇，而不是脂质，才能在体外发挥全部活性。事实上，IPMK 丢失后组蛋白乙酰化会增加， 发生在招募 IPMK 的转录起始位点的特定子集上。 IPMK 功能的所有这些方面 染色质和转录起始位点的变化是新颖的。 该提案更深入地探讨了我们的文章中描述的 IPMK 新染色质功能。 初步数据，利用新的化学遗传学和我们开发的 IPMK 的其他突变体。 目标 1 确定哪些新染色质事件最直接由 IPMK 介导，因此机制可以是 研究过。目标 2 确定生理学之间共享哪些 IPMK 介导的染色质事件 相关模型系统。目标3解决IPMK基因调控机制。该提案解决了 长期存在的问题是 IPMK 如何在引入新的基于染色质的第二个基因的同时调节基因表达 控制组蛋白标记和转录的信使信号范式。