Regulation of Replicative Stress Signaling by Deacetylation and Dephosphorylation

哺乳动物细胞的每一次分裂都要复制超过30亿个碱基对。核苷酸供应的限制和DNA损伤减缓了复制分叉并引发了复杂的应激反应。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）是这些反应的核心。这些激酶催化减缓细胞周期的过程，以及稳定复制分叉和启动DNA修复的机制。这种机制确保了DNA的忠实传递，没有表观遗传改变和致癌突变。正如对这种关键机制的预期，检查点激酶受到高度调节。翻译后修饰包括磷酸化和乙酰化调节检查点激酶。最近的数据表明，I类组蛋白去乙酰化酶（HDACs） HDAC1和HDAC2对基因组稳定性有影响。然而，尚不清楚这些hdac是否会影响检查点激酶及其活性。此外，磷酸化激活了检查点激酶，三聚体磷酸酶PP2A减弱了检查点激酶的磷酸化，但尚不清楚PP2A的活性是如何被调节的。活性PP2A由亚基A（结构成分，PPP2R1A/B）， C（催化活性，PPP2CA/B）和B（区分底物以允许PP2A全酶的特异性）组成。为了更好地理解检查点激酶信号是如何终止的，应该定义哪些PP2A B亚基负责识别和随后的这些激酶的去磷酸化。这些知识将为终止复制应激级联信号通路的分子机制提供更深入的见解。我们的新数据表明，I类hdac是维持人类和小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，而不是ATR，通过PP2A全酶进行去磷酸化。我们进一步表明，PR130在复制应激和HDAC抑制下控制细胞周期进程。我们现在想要精确地定义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。我们的新型CRISPR-Cas9 HCT116细胞缺乏PR130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，共聚焦显微镜，磷酸化蛋白质组学以及DNA和蛋白质分析。为了揭示由HDAC1/HDAC2、PR130和检查点激酶调控的常见和特定途径，我们想要阐明这些分子是否以及如何在添加化疗药物和癌基因激活后影响复制应激和DNA损伤信号。

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.