Recent Submissions

  • The transcription factor Atf1 binds and activates the APC/C ubiquitin ligase in fission yeast.

    Ors, Aslihan; Grimaldi, Margaret; Kimata, Yuu; Wilkinson, Caroline R M; Jones, Nic; Yamano, Hiroyuki; Cell Cycle Control Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, United Kingdom. (2009-09-04)
    Fission yeast Atf1 is a member of the ATF/CREB basic leucine zipper (bZIP) family of transcription factors with strong homology to mammalian ATF2. Atf1 regulates transcription in response to stress stimuli and also plays a role in controlling heterochromatin formation and recombination. However, its DNA binding independent role is poorly studied. Here, we report that Atf1 has a distinct role in regulating the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. We have identified atf1(+) as a dose-dependent suppressor of apc5-1, a mutation causing mitotic arrest. Remarkably, the suppression is not dependent upon the bZIP domain and is therefore independent of the ability of Atf1 to bind DNA. Interestingly, Atf1 physically binds the APC/C in vivo. Furthermore, we show that addition of purified Atf1 proteins into a cell-free system stimulates ubiquitylation of cyclin B and securin by the APC/C. These results reveal a novel role for Atf1 in cell cycle control through protein-protein interaction.
  • Loss of regulators of vacuolar ATPase function and ceramide synthesis results in multidrug sensitivity in schizosaccharomyces pombe.

    Dawson, Keren; Toone, W Mark; Jones, Nic; Wilkinson, Caroline R M; Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, United Kingdom. (2008-06)
    We undertook a screen to isolate determinants of drug resistance in fission yeast and identified two genes that, when mutated, result in sensitivity to a range of structurally unrelated compounds, some of them commonly used in the clinic. One gene, rav1, encodes the homologue of a budding yeast protein which regulates the assembly of the vacuolar ATPase. The second gene, lac1, encodes a homologue of genes that are required for ceramide synthesis. Both mutants are sensitive to the chemotherapeutic agent doxorubicin, and using the naturally fluorescent properties of this compound, we found that both rav1 and lac1 mutations result in an increased accumulation of the drug in cells. The multidrug-sensitive phenotype of rav1 mutants can be rescued by up-regulation of the lag1 gene which encodes a homologue of lac1, whereas overexpression of either lac1 or lag1 confers multidrug resistance on wild-type cells. These data suggest that changing the amount of ceramide synthase activity in cells can influence innate drug resistance. The function of Rav1 appears to be conserved, as we show that SpRav1 is part of a RAVE-like complex in fission yeast and that loss of rav1 results in defects in vacuolar (H(+))-ATPase activity. Thus, we conclude that loss of normal V-ATPase function results in an increased sensitivity of Schizosaccharomyces pombe cells to drugs. The rav1 and lac1 genes are conserved in both higher eukaryotes and various pathogenic fungi. Thus, our data could provide the basis for strategies to sensitize tumor cells or drug-resistant pathogenic fungi to drugs.
  • Suppressor role of activating transcription factor 2 (ATF2) in skin cancer.

    Bhoumik, Anindita; Fichtman, Boris; Derossi, Charles; Breitwieser, Wolfgang; Kluger, Harriet M; Davis, Sean; Subtil, Antonio; Meltzer, Paul; Krajewski, Stan; Jones, Nic; Ronai, Ze'ev; Burnham Institute for Medical Research, La Jolla, CA 92037, USA. (2008-02-05)
    Activating transcription factor 2 (ATF2) regulates transcription in response to stress and growth factor stimuli. Here, we use a mouse model in which ATF2 was selectively deleted in keratinocytes. Crossing the conditionally expressed ATF2 mutant with K14-Cre mice (K14.ATF2(f/f)) resulted in selective expression of mutant ATF2 within the basal layer of the epidermis. When subjected to a two-stage skin carcinogenesis protocol [7,12-dimethylbenz[a]anthracene/phorbol 12-tetradecanoate 13-acetate (DMBA/TPA)], K14.ATF2(f/f) mice showed significant increases in both the incidence and prevalence of papilloma development compared with the WT ATF2 mice. Consistent with these findings, keratinocytes of K14.ATF2(f/f) mice exhibit greater anchorage-independent growth compared with ATF2 WT keratinocytes. Papillomas of K14.ATF2(f/f) mice exhibit reduced expression of presenilin1, which is associated with enhanced beta-catenin and cyclin D1, and reduced Notch1 expression. Significantly, a reduction of nuclear ATF2 and increased beta-catenin expression were seen in samples of squamous and basal cell carcinoma, as opposed to normal skin. Our data reveal that loss of ATF2 transcriptional activity serves to promote skin tumor formation, thereby indicating a suppressor activity of ATF2 in skin tumor formation.
  • Fission yeast MAP kinase Sty1 is recruited to stress-induced genes.

    Reiter, Wolfgang; Watt, Stephen; Dawson, Keren; Lawrence, Clare L; Bähler, Jürg; Jones, Nic; Wilkinson, Caroline R M; Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, UK. (2008-04-11)
    The stress-induced expression of many fission yeast genes is dependent upon the Sty1 mitogen-activated protein kinase (MAPK) and Atf1 transcription factor. Atf1 is phosphorylated by Sty1 yet this phosphorylation is not required for stress-induced gene expression, suggesting another mechanism exists whereby Sty1 activates transcription. Here we show that Sty1 associates with Atf1-dependent genes and is recruited to both their promoters and coding regions. This occurs in response to various stress conditions coincident with the kinetics of the activation of Sty1. Association with promoters is not a consequence of increased nuclear accumulation of Sty1 nor does it require the phosphorylation of Atf1. However, recruitment is completely abolished in a mutant lacking Sty1 kinase activity. Both Atf1 and its binding partner Pcr1 are required for association of Sty1 with Atf1-dependent promoters, suggesting that this heterodimer must be intact for optimal recruitment of the MAPK. However, many Atf1-dependent genes are still expressed in a pcr1Delta mutant but with significantly delayed kinetics, thus providing an explanation for the relatively mild stress sensitivity displayed by pcr1Delta. Consistent with this delay, Sty1 and Atf1 cannot be detected at these promoters in this condition, suggesting that their association with chromatin is weak or transient in the absence of Pcr1.
  • Multiple pathways differentially regulate global oxidative stress responses in fission yeast.

    Chen, Dongrong; Wilkinson, Caroline R M; Watt, Stephen; Penkett, Christopher J; Toone, W Mark; Jones, Nic; Bähler, Jürg; Cancer Research UK Fission Yeast Functional Genomics Group, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, United Kingdom. (2008-01)
    Cellular protection against oxidative damage is relevant to ageing and numerous diseases. We analyzed the diversity of genome-wide gene expression programs and their regulation in response to various types and doses of oxidants in Schizosaccharomyces pombe. A small core gene set, regulated by the AP-1-like factor Pap1p and the two-component regulator Prr1p, was universally induced irrespective of oxidant and dose. Strong oxidative stresses led to a much larger transcriptional response. The mitogen-activated protein kinase (MAPK) Sty1p and the bZIP factor Atf1p were critical for the response to hydrogen peroxide. A newly identified zinc-finger protein, Hsr1p, is uniquely regulated by all three major regulatory systems (Sty1p-Atf1p, Pap1p, and Prr1p) and in turn globally supports gene expression in response to hydrogen peroxide. Although the overall transcriptional responses to hydrogen peroxide and t-butylhydroperoxide were similar, to our surprise, Sty1p and Atf1p were less critical for the response to the latter. Instead, another MAPK, Pmk1p, was involved in surviving this stress, although Pmk1p played only a minor role in regulating the transcriptional response. These data reveal a considerable plasticity and differential control of regulatory pathways in distinct oxidative stress conditions, providing both specificity and backup for protection from oxidative damage.
  • Int6/eIF3e promotes general translation and Atf1 abundance to modulate Sty1 MAPK-dependent stress response in fission yeast.

    Udagawa, T; Nemoto, N; Wilkinson, Caroline R M; Narashimhan, J; Jiang, L; Watt, S; Zook, A; Jones, Nic; Wek, R; Bähler, J; Asano, K; Molecular Cellular and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, Kansas 66506, USA. (2008-08-08)
    int-6 is one of the frequent integration sites for mouse mammary tumor viruses. Although its product is the e-subunit of translation initiation factor eIF3, other evidence indicates that it interacts with proteasomes or other proteins to regulate protein stability. Here we report that the fission yeast int6(+) is required for overcoming stress imposed by histidine starvation, using the drug 3-aminotriazole (3AT). Microarray and complementary Northern studies using wild-type, int6Delta or gcn2Delta mutants indicate that 3AT-treated wild-type yeast induces core environmental stress response (CESR) genes in addition to typical general amino acid control (GAAC) genes whose transcription depends on the eIF2 kinase, Gcn2. In agreement with this, Sty1 MAPK and its target transcription factor Atf1, which signal the CESR, are required for overcoming 3AT-induced starvation. We find that Int6 is required for maintaining the basal level of Atf1 and for rapid transcriptional activation of the CESR on 3AT-insult. Pulse labeling experiments indicate that int6Delta significantly slows down de novo protein synthesis. Moreover, Atf1 protein half-life was reduced in int6Delta cells. These effects would account for the compromised Atf1 activity on 3AT-induced stress. Thus, the robust protein synthesis promoted by intact eIF3 appears to be a part of the requisites for sound Sty1 MAPK-dependent signaling governed by the activity of the Atf1 transcription factor.