[Show abstract][Hide abstract] ABSTRACT: Histone transcription and deposition are tightly regulated with the DNA replication cycle in order to maintain genetic integrity. Ams2 is a GATA containing transcription factor responsible for core histone gene expression and for CENP-A loading at centromeres in fission yeast. Ams2 levels are cell cycle regulated and after S phase it is degraded by the SCFpof3 ubiquitin ligase, however the regulation of Ams2 in G1 or meiosis is poorly understood. Here we show that another ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C) targets Ams2 for destruction in G1. Ubiquitylation and destruction of Ams2 is dependent upon a coactivator Cdh1/Ste9 and the KEN box in the C-terminus of Ams2. We also find that stabilization of Ams2 sensitizes cells to the anti-microtubule drug thiabendazole and a histone deacetylase (HDAC) inhibitor Tricostatin A, when a HDAC gene hst4 is deleted, suggesting that histone acetylation together with Ams2 stability ensures the coupling of mitosis to DNA replication. Furthermore, in meiosis, the failure of the APC/C-mediated destruction of Ams2 is deleterious and pre-meiotic DNA replication is barely completed. These data suggest that Ams2 destruction via both the APC/C and the SCF ubiquitin ligases underlies the coordination of histone expression and DNA replication.
[Show abstract][Hide abstract] ABSTRACT: Targeting mitotic exit has been recently proposed as a relevant therapeutic approach against cancer. By using genetically engineered mice, we show that the APC/C cofactor Cdc20 is essential for anaphase onset in vivo in embryonic or adult cells, including progenitor/stem cells. Ablation of Cdc20 results in efficient regression of aggressive tumors, whereas current mitotic drugs display limited effects. Yet, Cdc20 null cells can exit from mitosis upon inactivation of Cdk1 and the kinase Mastl (Greatwall). This mitotic exit depends on the activity of PP2A phosphatase complexes containing B55α or B55δ regulatory subunits. These data illustrate the relevance of critical players of mitotic exit in mammals and their implications in the balance between cell death and mitotic exit in tumor cells.
Cancer cell 12/2010; 18(6):641-54. DOI:10.1016/j.ccr.2010.10.028 · 23.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Schizosaccharomyces pombe GATA factor Ams2 is responsible for cell cycle-dependent transcriptional activation of all the core histone genes peaking at G1/S phase. Intriguingly, its own protein level also fluctuates concurrently. Here, we show that Ams2 is ubiquitylated and degraded through the SCF (Skp1-Cdc53/Cullin-1-F-box) ubiquitin ligase, in which F box protein Pof3 binds this protein. Ams2 is phosphorylated at multiple sites, which is required for SCF(Pof3)-dependent proteolysis. Hsk1/Cdc7 kinase physically associates with and phosphorylates Ams2. Even mild overexpression of Ams2 induces constitutive histone expression and chromosome instability, and its toxicity is exaggerated when Hsk1 function is compromised. This is partly attributable to abnormal incorporation of canonical H3 into the central CENP-A/Cnp1-rich centromere, thereby reversing specific chromatin structures to apparently normal nucleosomes. We propose that Hsk1 plays a vital role during post S phase in genome stability via SCF(Pof3)-mediated degradation of Ams2, thereby maintaining centromere integrity.
[Show abstract][Hide abstract] ABSTRACT: The anaphase-promoting complex/cyclosome (APC/C), a large (20S) multisubunit E3 ligase, has an essential role to ubiquitylate numerous substrates at specific times during mitosis and G1 phase as well as in meiosis. The deregulation of the APC/C causes cell death or genomic instability, which is a hallmark of cancers. Although 13 years have passed since its discovery, the molecular mechanisms of the APC/C-dependent ubiquitylation and proteolysis are still poorly understood. The development of in vitro systems enables the identification of new substrates and investigation of the molecular mechanisms by which the APC/C recognizes its substrates. This chapter describes in vitro assays reconstituted in Xenopus egg extracts.
[Show abstract][Hide abstract] ABSTRACT: Homologous recombination (HR) is important for maintaining genome integrity and for the process of meiotic chromosome segregation and the generation of variation. HR is regulated throughout the cell cycle, being prevalent in the S and G2 phases and suppressed in the G1 phase. Here we show that the anaphase-promoting complex/cyclosome (APC/C) regulates homologous recombination in the fission yeast Schizosaccharomyces pombe by ubiquitylating Rhp54 (an ortholog of Rad54). We show that Rhp54 is a novel APC/C substrate that is destroyed in G1 phase in a KEN-box- and Ste9/Fizzy-related manner. The biological consequences of failing to temporally regulate HR via Rhp54 degradation are seen in haploid cells only in the absence of antirecombinase Srs2 function and are more extensive in diploid cells, which become sensitive to a range of DNA-damaging agents, including hydroxyurea, methyl methanesulfonate, bleomycin, and UV. During meiosis, expression of nondegradable Rhp54 inhibits interhomolog recombination and stimulates sister chromatid recombination. We thus propose that it is critical to control levels of Rhp54 in G1 to suppress HR repair of double-strand breaks and during meiosis to coordinate interhomolog recombination.
[Show abstract][Hide abstract] ABSTRACT: The anaphase-promoting complex/cyclosome (APC/C) is a cell-cycle-regulated essential E3 ubiquitin ligase; however, very little is known about its meiotic regulation. Here we show that fission yeast Mes1 is a substrate of the APC/C as well as an inhibitor, allowing autoregulation of the APC/C in meiosis. Both traits require a functional destruction box (D box) and KEN box. We show that Mes1 directly binds the WD40 domain of the Fizzy family of APC/C activators. Intriguingly, expression of nonubiquitylatable Mes1 blocks cells in metaphase I with high levels of APC/C substrates, suggesting that ubiquitylation of Mes1 is required for partial degradation of cyclin B in meiosis I by alleviating Mes1 inhibitory function. Consistently, a ternary complex, APC/C-Fizzy/Cdc20-Mes1, is stabilized by inhibiting Mes1 ubiquitylation. These results demonstrate that the fine-tuning of the APC/C activity, by a substrate that is also an inhibitor, is required for the precise coordination and transition through meiosis.
[Show abstract][Hide abstract] ABSTRACT: Ubiquitination of proliferating cell nuclear antigen (PCNA) plays a crucial role in regulating replication past DNA damage in eukaryotes, but the detailed mechanisms appear to vary in different organisms. We have examined the modification of PCNA in Schizosaccharomyces pombe. We find that, in response to UV irradiation, PCNA is mono- and poly-ubiquitinated in a manner similar to that in Saccharomyces cerevisiae. However in undamaged Schizosaccharomyces pombe cells, PCNA is ubiquitinated in S phase, whereas in S. cerevisiae it is sumoylated. Furthermore we find that, unlike in S. cerevisiae, mutants defective in ubiquitination of PCNA are also sensitive to ionizing radiation, and PCNA is ubiquitinated after exposure of cells to ionizing radiation, in a manner similar to the response to UV-irradiation. We show that PCNA modification and cell cycle checkpoints represent two independent signals in response to DNA damage. Finally, we unexpectedly find that PCNA is ubiquitinated in response to DNA damage when cells are arrested in G2.
Molecular Biology of the Cell 08/2006; 17(7):2976-85. DOI:10.1091/mbc.E05-11-1008 · 4.47 Impact Factor