IPMK function in chromatin

IPMK 在染色质中的功能

• 批准号: 9973484
• 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/9973484
• 关键词: Acetylation; Address; Animals; Astrocytes; Astrocytoma; Binding Proteins; Biological; Biological Models; Cell Line; Cell model; Cells; Chromatin; Chronic; Complement; Data; Detection; Elements; Enzymes; Etiology; Event; Experimental Models; FDA approved; Feeds; Future; Gap Junctions; 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; 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; base; chemical genetics; chromatin remodeling; enzyme activity; in vitro activity; inhibitor/antagonist; 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.

摘要： 在过去的二十年里，包括约翰·约克、苏珊·温特、史蒂夫·希尔斯、阿道夫在内的几个实验室 Saiardi和所罗门·辛德试图解释高阶肌醇磷酸二号信使是如何 信号分子(肌醇)调节转录，主要通过以下方式检测酵母中的单个转录单位 遗传互补/上位性分析。这些研究集中在完全保守和 无处不在的磷酸肌醇多激酶(IPMK，IPK2)，因为该激酶位于几个通路的连接处 是生产所有高级肌醇所必需的。IPMK活性显然是拯救酵母菌表型和 来自个别元素的转录本，但肌醇是如何实现这一调控的还没有描述，因为 肌醇的染色质效应尚不清楚。2003年，Erin O‘Shea显示了IPMK的激酶活性 在酵母中调节核小体滑动，Carl Wu和另一个小组发现肌醇调节ATP依赖 染色质重构体INO80和Swi/Snf的体外实验。然而，肌醇对ATP重构体的调节尚未得到 建立在此后的任何细胞研究中，尽管有基因组方法来检查开放染色质。 我们发现了一种完全不同的IPMK调控转录的方式，通过直接 当磷脂结合在核受体的疏水裂隙中时，使磷脂磷酸化。这 模型威胁要解释为什么IPMK的染色质靶标很难识别-它们可能是脂质- 结合蛋白，而不是肌醇结合蛋白。这导致我们试图鉴定其他转录因子 由IPMK使用基因组学进行类似的调控，如本提案所示。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点，控制这些位置的转录积累 启动子以一种依赖于激酶的方式。但令我们非常惊讶的是，GSEA立即建议IPMK 主要(但当然不是唯一的)通过组蛋白脱乙酰酶(HDAC)调节基因表达。 HDAC是由John Schwabe的团队在一系列结构生物学论文中展示的转录抑制因子 需要肌醇，而不是脂质，才能在体外发挥全部活性。事实上，组蛋白乙酰化在IPMK丢失时增加， 发生在招募IPMK的转录起始点的特定亚群。IPMK函数的所有这些方面 在染色质和转录起始处都是新的。 这一提议更深入地询问了我们在我们的 初步数据，利用我们开发的IPMK的新的化学遗传学和其他突变体。 目标1确定哪些新的染色质事件是由IPMK最直接地介导的，因此机制可以是 学习。目标2确定哪些IPMK介导的染色质事件在生理上共享 相关模式体系。目的3解决IPMK基因调控的机制。这项提案涉及 IPMK在引入一种新的以染色质为基础的第二种蛋白时如何调节基因表达的长期问题 信使信号模式，控制组蛋白标记和转录。