IPMK function in chromatin

摘要： 在过去的二十年里，几个实验室，包括约翰约克，苏珊温特，史蒂夫希尔斯，阿道夫 Saiardi和所罗门辛德试图阐明高级肌醇磷酸第二信使 信号分子（肌醇）调节转录，主要是检查酵母中的单个转录单位， 遗传互补/上位性分析。这些研究的重点是完全保守且 普遍存在的肌醇磷酸多激酶（IPMK，ipk 2），因为这种激酶位于几种途径的连接处 用于生产所有高级肌醇。IPMK活性显然是拯救酵母表型所需的， 转录本从个别元素，但如何肌醇实现这一调节是没有描述的，因为 肌醇的染色质效应物是未知的。2003年，Erin O 'Shea展示了IPMK的激酶活性， 调节酵母核小体滑动，Carl Wu和另一个小组表明肌醇调节ATP依赖性 体外染色质重塑Ino 80和Swi/Snf。然而，肌醇调节ATP-重塑还没有被证实。 尽管有基因组方法来检查开放的染色质，但这是建立在任何细胞研究之上的。 我们发现了一种完全不同的IPMK调节转录的方式， 当脂质结合在核受体的疏水裂缝中时，使磷脂磷酸化。这 该模型可能解释了为什么IPMK的染色质靶点难以识别-它们可能是脂质- 而不是肌醇结合蛋白。这促使我们尝试鉴定其他转录因子 IPMK使用基因组学进行类似的监管，在本提案中提出。在我们的人类细胞模型中， IPMK被募集到数百个转录起始位点，控制这些位点的转录物积累。 以激酶依赖的方式启动子。但令我们惊讶的是，GSEA立即建议IPMK 主要（但肯定不是排他地）通过组蛋白脱乙酰酶（HDAC）调节基因表达。 HDAC是转录抑制因子，在John施瓦贝的研究小组的一系列结构生物学论文中得到了证实 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损失后增加， 发生在募集IPMK的转录起始位点的特定子集上。IPMK功能的所有这些方面 在染色质中和在转录起始位点是新的。 这个建议更深入地询问了我们在论文中描述的IPMK的新染色质功能。 初步数据，利用新的化学遗传学和其他突变体的IPMK，我们已经开发。 目的1确定哪些新的染色质事件是由IPMK最直接介导的，因此机制可以是 研究了目的2确定哪些IPMK介导的染色质事件在生理学上是共享的。 相关模型系统。目的3解决IPMK基因调控机制。该提案涉及 长期存在的问题是IPMK如何调节基因表达，同时引入新的基于染色质的第二代 控制组蛋白标记和转录的信使信号传导模式。

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.