Regulation of Replicative Stress Signaling by Deacetylation and Dephosphorylation

通过脱乙酰化和去磷酸化调节复制应激信号

• 批准号: 325554574
• 负责人: Professor Dr. Oliver Holger Krämer
• 金额: --
• 依托单位: Institut für Toxikologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/325554574?language=en
• 关键词: Regulation; Replicative; Stress; Signaling; Deacetylation

Over 3 billion base pairs of a mammalian cell are replicated with every cell division. Limitations in the supply of nucleotides and DNA lesions slow down replication forks and trigger complex stress responses. The checkpoint kinases ataxia telangiectasia mutated (ATM), ATM/Rad3-related (ATR), checkpoint kinase-1 (CHK1) and -2 (CHK2) are at the heart of such responses. These kinases catalyze processes that slow down the cell cycle as well as mechanisms that stabilize replication forks and initiate DNA repair. Such mechanisms ensure the faithful transmission of DNA without epigenetic alterations and oncogenic mutations. As expected for such a pivotal mechanism, checkpoint kinases are highly regulated. Posttranslational modifications including phosphorylation and acetylation regulate checkpoint kinases. Recent data accordingly show that the class I histone deacetylases (HDACs) HDAC1 and HDAC2 have an impact on genomic stability. However, it is unknown whether these HDACs affect checkpoint kinases and their activities. Moreover, phosphorylation activates checkpoint kinases and the trimeric phosphatase PP2A attenuates checkpoint kinase phosphorylation, but it is unknown how this activity of PP2A is modulated. Active PP2A consists of the subunits A (structural component, PPP2R1A/B), C (catalytic activity, PPP2CA/B), and B (discriminates between substrates to allow specificity of the PP2A holoenzyme). To better understand how checkpoint kinase signaling becomes terminated, it should be defined which PP2A B subunit(s) are responsible for the recognition and the subsequent dephosphorylation of these kinases. Such knowledge will give deeper insights into molecular mechanisms that terminate the signaling pathways of the replicative stress cascade. Our new data illustrate that class I HDACs are required to sustain checkpoint kinase phosphorylation in human and murine cells. We show that HDAC1 and HDAC2 suppress the expression of the PP2A B subunit PR130 and that HDACi and PR130 target ATM and CHK1, but not ATR, for dephosphorylation by the PP2A holoenzyme. We further show that PR130 controls cell cycle progression of cells under replicative stress and HDAC inhibition. We now want to define precisely how HDAC1/HDAC2, PR130, CHK1, and ATM interact and how they affect cell cycle control, replication fork speed, DNA integrity and stability, and cellular fate. Our novel CRISPR-Cas9 HCT116 cells devoid of PR130 are an invaluable tool for these analyses. We use genetic and biochemical approaches, including RNAi, CRISPR-Cas9, DNA fiber assays, i-POND, confocal microscopy, phospho-proteomics, and DNA and protein analyses. In order to reveal common and specific pathways that are regulated by HDAC1/HDAC2, PR130, and checkpoint kinases, we want to clarify whether and how these molecules affect replicative stress and DNA damage signaling upon the addition of chemotherapeutics and after oncogene activation.

哺乳动物细胞的每次细胞分裂都会复制超过 30 亿个碱基对。核苷酸供应的限制和 DNA 损伤会减慢复制叉并引发复杂的应激反应。检查点激酶共济失调毛细血管扩张突变 (ATM)、ATM/Rad3 相关 (ATR)、检查点激酶-1 (CHK1) 和 -2 (CHK2) 是此类反应的核心。这些激酶催化减慢细胞周期的过程以及稳定复制叉和启动 DNA 修复的机制。这种机制确保 DNA 的忠实传递，而不会发生表观遗传改变和致癌突变。正如对这样一个关键机制的预期，检查点激酶受到高度监管。包括磷酸化和乙酰化在内的翻译后修饰调节检查点激酶。因此，最近的数据表明，I 类组蛋白脱乙酰酶 (HDAC) HDAC1 和 HDAC2 对基因组稳定性有影响。然而，尚不清楚这些 HDAC 是否影响检查点激酶及其活性。此外，磷酸化会激活检查点激酶，而三聚磷酸酶 PP2A 会减弱检查点激酶磷酸化，但尚不清楚 PP2A 的这种活性是如何调节的。活性 PP2A 由亚基 A（结构成分，PPP2R1A/B）、C（催化活性，PPP2CA/B）和 B（区分底物以实现 PP2A 全酶的特异性）组成。为了更好地理解检查点激酶信号传导如何终止，应该定义哪些 PP2A B 亚基负责这些激酶的识别和随后的去磷酸化。这些知识将使我们更深入地了解终止复制应激级联信号通路的分子机制。我们的新数据表明，I 类 HDAC 是维持人和鼠细胞中检查点激酶磷酸化所必需的。我们发现 HDAC1 和 HDAC2 抑制 PP2A B 亚基 PR130 的表达，并且 HDACi 和 PR130 靶向 ATM 和 CHK1，但不靶向 ATR，以实现 PP2A 全酶的去磷酸化。我们进一步表明，PR130 在复制应激和 HDAC 抑制下控制细胞的细胞周期进程。我们现在想要精确定义 HDAC1/HDAC2、PR130、CHK1 和 ATM 如何相互作用，以及它们如何影响细胞周期控制、复制叉速度、DNA 完整性和稳定性以及细胞命运。我们不含 PR130 的新型 CRISPR-Cas9 HCT116 细胞是这些分析的宝贵工具。我们使用遗传和生化方法，包括 RNAi、CRISPR-Cas9、DNA 纤维测定、i-POND、共焦显微镜、磷酸蛋白质组学以及 DNA 和蛋白质分析。为了揭示受 HDAC1/HDAC2、PR130 和检查点激酶调节的常见和特定途径，我们希望阐明这些分子在添加化疗药物和癌基因激活后是否以及如何影响复制应激和 DNA 损伤信号传导。