Regulation of Replicative Stress Signaling by Deacetylation and Dephosphorylation

点击翻译按钮获取中文摘要

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.