The Arabidopsis thaliana checkpoint kinase WEE1 protects against premature vascular differentiation during replication stress

A sessile lifestyle forces plants to respond promptly to factors that affect their genomic integrity. Therefore, plants have developed checkpoint mechanisms to arrest cell cycle progression upon the occurrence of DNA stress, allowing the DNA to be repaired before onset of division. Previously, the WEE1 kinase had been demonstrated to be essential for delaying progression through the cell cycle in the presence of replication-inhibitory drugs, such as hydroxyurea. To understand the severe growth arrest of WEE1-deficient plants treated with hydroxyurea, a transcriptomics analysis was performed, indicating prolonged S-phase duration. A role for WEE1 during S phase was substantiated by its specific accumulation in replicating nuclei that suffered from DNA stress. Besides an extended replication phase, WEE1 knockout plants accumulated dead cells that were associated with premature vascular differentiation. Correspondingly, plants without functional WEE1 ectopically expressed the vascular differentiation marker VND7, and their vascular development was aberrant. We conclude that the growth arrest of WEE1-deficient plants is due to an extended cell cycle duration in combination with a premature onset of vascular cell differentiation. The latter implies that the plant WEE1 kinase acquired an indirect developmental function that is important for meristem maintenance upon replication stress

Een sleutelrol voor NIPP1 in lever stamcel proliferatie en alkylatie-geïntroduceerde leverkanker

Summary The multifunctional scaffold protein NIPP1 interacts with the Ser/Thr phosphatase PP1, but also contains a substrate-interacting domain that binds, amongst others, several ligands when phosphorylated on specific residues, such as protein kinase MELK. Since NIPP1-/- mice proved to be embryonic lethal at the onset of gastrulation, the main goal of my thesis was to further characterize the function of NIPP1 in vivo. This was done by generating a liver-specific NIPP1 knockout (LKO) mouse, whereby NIPP1 is deleted in epithelial liver cells. LKO mice appeared healthy at first. However upon aging they gradually developed a ductular reaction. Upregulation of several Liver Progenitor Cell (LPC) markers suggested an expansion of the LPC compartment. In addition our LKOs also showed an increased sensitivity to a specific DDC-diet that is known to cause a ductular response through LPC proliferation, whereas acute hepatocyte-specific damage evoked a normal response. These data imply that NIPP1 specifically restrains LPC proliferation in the adult liver. We also injected mice with the DNA alkylation agent diethylnitrosamine (DEN), which is well known to cause hepatocellular carcinoma. LKO mice showed much less tumor formation compared to controls. We could link these results to a better repair of specific DEN-induced O6-alkylguanine lesions by the suicide repair enzyme MGMT (O6-Methylguanine-DNA Methyltransferase). Both MGMT expression and activity were upregulated in our LKO mice. Further research in hepatoma cells could link the upregulation of MGMT expression to the two NIPP1 interactors MELK and PP1. Indeed, not only loss of NIPP1, but also a knockdown of MELK or PP1 induced elevated MGMT transcript levels. We hypothesize that PP1-NIPP1 regulates MGMT expression through dephosphorylation of MELK. The second aim of my thesis involved the effect of a mild but stable overexpression of NIPP1 in epithelial HeLa cancer cells. We noticed that a small increase in NIPP1 causes a massive induction of mesenchymal, especially smooth muscle-specific, genes. Transdifferentiation of HeLa cells into smooth-muscle like cells was dependent on a functional PP1-binding and substrate-binding domain of NIPP1. This implies an involvement of the NIPP1:PP1 complex via specific substrate dephosphorylation.status: publishe

Genome-wide promoter binding profiling of protein phosphatase-1 and its major nuclear targeting subunits

Protein phosphatase-1 (PP1) is a key regulator of transcription and is targeted to promoter regions via associated proteins. However, the chromatin binding sites of PP1 have never been studied in a systematic and genome-wide manner. Methylation-based DamID profiling in HeLa cells has enabled us to map hundreds of promoter binding sites of PP1 and three of its major nuclear interactors, i.e. RepoMan, NIPP1 and PNUTS. Our data reveal that the α, β and γ isoforms of PP1 largely bind to distinct subsets of promoters and can also be differentiated by their promoter binding pattern. PP1β emerged as the major promoter-associated isoform and shows an overlapping binding profile with PNUTS at dozens of active promoters. Surprisingly, most promoter binding sites of PP1 are not shared with RepoMan, NIPP1 or PNUTS, hinting at the existence of additional, largely unidentified chromatin-targeting subunits. We also found that PP1 is not required for the global chromatin targeting of RepoMan, NIPP1 and PNUTS, but alters the promoter binding specificity of NIPP1. Our data disclose an unexpected specificity and complexity in the promoter binding of PP1 isoforms and their chromatin-targeting subunits.status: publishe

Enhanced DNA-repair capacity and resistance to chemically induced carcinogenesis upon deletion of the phosphatase regulator NIPP1

Nuclear Inhibitor of PP1 (NIPP1) is a conserved regulatory subunit of protein phosphatase PP1. The selective deletion of NIPP1 in mouse liver parenchymal cells or skin epidermal cells culminates in a late-onset hyperproliferation of a subset of resident progenitor cells. Although a hyperplastic phenotype is usually tumor promoting, we show here that the absence of NIPP1 conferred a strong resistance to chemically induced hepatocellular or skin carcinoma. The ablation of NIPP1 did not affect the metabolism of the administered mutagens (diethylnitrosamine or 7,12-dimethylbenz[a]anthracene), but reduced the conversion of mutagen-induced covalent DNA modifications into cancer-initiating mutations. This reduced sensitivity to mutagens correlated with an enhanced DNA-damage response and an augmented expression of rate-limiting DNA-repair proteins (MGMT in liver, XPD and XPG in skin), hinting at an increased DNA-repair capacity. Our data identify NIPP1 as a repressor of DNA repair and as a promising target for novel cancer prevention and treatment therapies.status: publishe

Protein phosphatase PP1-NIPP1 activates mesenchymal genes in HeLa cells

The deletion of the protein phosphatase-1 (PP1) regulator known as Nuclear Inhibitor of PP1 (NIPP1) is embryonic lethal during gastrulation, hinting at a key role of PP1-NIPP1 in lineage specification. Consistent with this notion we show here that a mild, stable overexpression of NIPP1 in HeLa cells caused a massive induction of genes of the mesenchymal lineage, in particular smooth/cardiac-muscle and matrix markers. This reprogramming was associated with the formation of actin-based stress fibers and retracting filopodia, and a reduced proliferation potential. The NIPP1-induced mesenchymal transition required functional substrate and PP1-binding domains, suggesting that it involves the selective dephosphorylation of substrates of PP1-NIPP1.publisher: Elsevier articletitle: Protein phosphatase PP1-NIPP1 activates mesenchymal genes in HeLa cells journaltitle: FEBS Letters articlelink: http://dx.doi.org/10.1016/j.febslet.2015.04.017 content_type: article copyright: Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.status: publishe

The selective inhibition of protein phosphatase-1 results in mitotic catastrophe and impaired tumor growth

The serine/threonine protein phosphatase-1 (PP1) complex is a key regulator of the cell cycle. However, the redundancy of PP1 isoforms and the lack of specific inhibitors have hampered studies on the global role of PP1 in cell cycle progression in vertebrates. Here, we show that the overexpression of nuclear inhibitor of PP1 (NIPP1; also known as PPP1R8) in HeLa cells culminated in a prometaphase arrest, associated with severe spindle-formation and chromosome-congression defects. In addition, the spindle assembly checkpoint was activated and checkpoint silencing was hampered. Eventually, most cells either died by apoptosis or formed binucleated cells. The NIPP1-induced mitotic arrest could be explained by the inhibition of PP1 that was titrated away from other mitotic PP1 interactors. Consistent with this notion, the mitotic-arrest phenotype could be rescued by the overexpression of PP1 or the inhibition of the Aurora B kinase, which acts antagonistically to PP1. Finally, we demonstrate that the overexpression of NIPP1 also hampered colony formation and tumor growth in xenograft assays in a PP1-dependent manner. Our data show that the selective inhibition of PP1 can be used to induce cancer cell death through mitotic catastrophe.status: publishe

Phosphatase Regulator NIPP1 Restrains Chemokine-Driven Skin Inflammation

Nuclear inhibitor of protein phosphatase 1 (NIPP1) is a ubiquitously expressed nuclear protein that regulates functions of protein serine/threonine phosphatase-1 in cell proliferation and lineage specification. The role of NIPP1 in tissue homeostasis is not fully understood. This study shows that the selective deletion of NIPP1 in mouse epidermis resulted in epidermal hyperproliferation, a reduced adherence of basal keratinocytes, and a gradual decrease in the stemness of hair follicle stem cells, culminating in hair loss. This complex phenotype was associated with chronic sterile skin inflammation and could be partially rescued by dexamethasone treatment. NIPP1-deficient keratinocytes massively expressed proinflammatory chemokines and immunomodulatory proteins in a cell-autonomous manner. Chemokines subsequently induced the recruitment and activation of immune cells, in particular conventional dendritic cells and Langerhans cells, accounting for the chronic inflammation phenotype. The data identifies NIPP1 as a key regulator of epidermal homeostasis and as a potential target for the treatment of inflammatory skin diseases.status: publishe