Yamei Wang

Overlapping and distinct functions for a Caenorhabditis elegans SIR2 and DAF-16/FOXO

The conserved SIR2 protein regulates life span in both yeast and worms: in both organisms overexpression of SIR2 can extend life span and in Caenorhabditis elegans this life span extension is dependent on the forkhead transcription factor, DAF-16. Here, we have done extensive genetic analysis with sir-2.1(ok434), a null mutant of C. elegans sir-2.1, the closest homolog to yeast Sir2p and human SIRT1 to further elucidate its function in life span regulation. sir-2.1(ok434) mutants show a slight decrease in life span as well as sensitivity to various stresses. Our genetic analysis suggests that sir-2.1 is required for life span extension by caloric restriction, independent of the insulin/IGF-1 signaling pathway. Importantly, analysis with unc-13 mutants indicates that sir-2.1 and daf-16 have overlapping and distinct roles in life span regulation. Our expression analysis shows that sir-2.1 has overlapping and distinct expression pattern compared with daf-16, consistent with the results from our genetic data. Our data defines a central role for C. elegans SIR2 in regulation of life span by caloric restriction and demonstrates that sir-2.1 and daf-16 have both overlapping and distinct functions in regulation of C. elegans life span.

C. elegans 14-3-3 proteins regulate life span and interact with SIR-2.1 and DAF-16/FOXO

14-3-3 proteins are evolutionarily conserved and ubiquitous proteins that function in a wide variety of biological processes. Here we define a new role for C. elegans 14-3-3 proteins in life span regulation. We identify two C. elegans 14-3-3 proteins as interacting proteins of a major life span regulator, the C. elegans SIR2 ortholog, SIR-2.1. Similar to sir-2.1, we find that overexpression of either 14-3-3 protein (PAR-5 or FTT-2) extends life span and that this is dependent on DAF-16, a forkhead transcription factor (FOXO), another important life span regulator in the insulin/IGF-1 signaling pathway. Furthermore, we show that both 14-3-3 proteins are co-expressed with DAF-16 and SIR-2.1 in the tissues critical for life span regulation. Finally, we show that DAF-16/FOXO also physically interacts with the 14-3-3 proteins. These results suggest that C. elegans 14-3-3 proteins can regulate longevity by cooperating with both SIR-2.1 and DAF-16/FOXO.

Cross-Talk between the KNOX and Ethylene Pathways Is Mediated by Intron-Binding Transcription Factors in Barley

In the barley (Hordeum vulgare) Hooded (Kap) mutant, the duplication of a 305-bp intron sequence leads to the overexpression of the Barley knox3 (Bkn3) gene, resulting in the development of an extra flower in the spikelet. We used a one-hybrid screen to identify four proteins that bind the intron-located regulatory element (Kap intron-binding proteins). Three of these, Barley Ethylene Response Factor1 (BERF1), Barley Ethylene Insensitive Like1 (BEIL1), and Barley Growth Regulating Factor1 (BGRF1), were characterized and their in vitro DNA-binding capacities verified. Given the homology of BERF1 and BEIL1 to ethylene signaling proteins, we investigated if these factors might play a dual role in intron-mediated regulation and ethylene response. In transgenic rice (Oryza sativa), constitutive expression of the corresponding genes produced phenotypic alterations consistent with perturbations in ethylene levels and variations in the expression of a key gene of ethylene biosynthesis. In barley, ethylene treatment results in partial suppression of the Kap phenotype, accompanied by up-regulation of BERF1 and BEIL1 expression, followed by down-regulation of Bkn3 mRNA levels. In rice protoplasts, BEIL1 activates the expression of a reporter gene driven by the 305-bp intron element, while BERF1 can counteract this activation. Thus, BEIL1 and BERF1, likely in association with other Kap intron-binding proteins, should mediate the fine-tuning of Bkn3 expression by ethylene. We propose a hypothesis for the cross talk between the KNOX and ethylene pathways.

Genetics of barley Hooded suppression

The molecular basis of the barley dominant Hooded (K) mutant is a duplication of 305 bp in intron IV of the homeobox gene Bkn3. A chemical mutagenesis screen was carried out to identify genetical factors that participate in Bkn3 intron-mediated gene regulation. Plants from recurrently mutagenized KK seeds were examined for the suppression of the hooded awn phenotype induced by the K allele and, in total, 41 suK (suppressor of K) recessive mutants were identified. Complementation tests established the existence of five suK loci, and alleles suKB-4, suKC-33, suKD-25, suKE-74, and suKF-76 were studied in detail. All K-suppressed mutants showed a short-awn phenotype. The suK loci have been mapped by bulked segregant analysis nested in a standard mapping procedure based on AFLP markers. K suppressor loci suKB, B, E, and F all map in a short interval of chromosome 7H, while the locus suKD is assigned to chromosome 5H. A complementation test between the four suK mutants mapping on chromosome 7H and the short-awn mutant lks2, located nearby, excluded the allelism between suK loci and lks2. The last experiment made clear that the short-awn phenotype of suK mutants is due to a specific dominant function of the K allele, a function that is independent from the control on hood formation. The suK loci are discussed as candidate participants in the regulation of Bkn3 expression.

A Novel Role of Dma1 in Regulating Forespore Membrane Assembly and Sporulation in Fission Yeast

By characterizing the fission yeast Dma1s function during meiosis, we revealed that Dma1 is required for spore formation, while it is dispensable for fidelity of nuclear divisions. We also found that Dma1 is functionally related to SIN pathway and meiosis-specific kinase Slk1 during sporulation.

Facile manipulation of protein localization in fission yeast through binding of GFP-binding protein to GFP

ABSTRACT GFP-binding protein (or GBP) has been recently developed in various systems and organisms as an efficient tool to purify GFP-fusion proteins. Due to the high affinity between GBP and GFP or GFP variants, this GBP-based approach is also ideally suited to alter the localization of functional proteins in live cells. In order to facilitate the wide use of the GBP-targeting approach in the fission yeast Schizosaccharomyces pombe, we developed a set of pFA6a-, pJK148- and pUC119-based vectors containing GBP- or GBP–mCherry-coding sequences and variants of inducible nmt1 or constitutive adh1 promoters that result in different levels of expression. The GBP or GBP–mCherry fragments can serve as cassettes for N- or C-terminal genomic tagging of genes of interest. We illustrated the application of these vectors in the construction of yeast strains with Dma1 or Cdc7 tagged with GBP–mCherry and efficient targeting of Dma1– or Cdc7–GBP–mCherry to the spindle pole body by Sid4–GFP. This series of vectors should help to facilitate the application of the GBP-targeting approach in manipulating protein localization and the analysis of gene function in fission yeast, at the level of single genes, as well as at a systematic scale. Summary: A set of vectors containing GBP- or GBP–mCherry-coding sequences and variants of inducible nmt1 or constitutive adh1 promoters were constructed. They should allow easier artificial manipulation of protein localization in fission yeast.

Fission yeast nucleolar protein Dnt1 regulates G2/M transition and cytokinesis by downregulating Wee1 kinase

Summary Cytokinesis involves temporally and spatially coordinated action of the cell cycle, cytoskeletal and membrane systems to achieve separation of daughter cells. The septation initiation network (SIN) and mitotic exit network (MEN) signaling pathways regulate cytokinesis and mitotic exit in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Previously, we have shown that in fission yeast, the nucleolar protein Dnt1 negatively regulates the SIN pathway in a manner that is independent of the Cdc14-family phosphatase Clp1/Flp1, but how Dnt1 modulates this pathway has remained elusive. By contrast, it is clear that its budding yeast relative, Net1/Cfi1, regulates the homologous MEN signaling pathway by sequestering Cdc14 phosphatase in the nucleolus before mitotic exit. In this study, we show that dnt1+ positively regulates G2/M transition during the cell cycle. By conducting epistasis analyses to measure cell length at septation in double mutant (for dnt1 and genes involved in G2/M control) cells, we found a link between dnt1+ and wee1+. Furthermore, we showed that elevated protein levels of the mitotic inhibitor Wee1 kinase and the corresponding attenuation in Cdk1 activity is responsible for the rescuing effect of dnt1&Dgr; on SIN mutants. Finally, our data also suggest that Dnt1 modulates Wee1 activity in parallel with SCF-mediated Wee1 degradation. Therefore, this study reveals an unexpected missing link between the nucleolar protein Dnt1 and the SIN signaling pathway, which is mediated by the Cdk1 regulator Wee1 kinase. Our findings also define a novel mode of regulation of Wee1 and Cdk1, which is important for integration of the signals controlling the SIN pathway in fission yeast.

Dnt1 acts as a mitotic inhibitor of the spindle checkpoint protein dma1 in fission yeast

The interaction between Dma1 and Dnt1 in fission yeast is characterized. The results show that, similar to its homologue Chfr in higher eukaryotes, Dma1 in fission yeast can also affect factors required for microtubule nucleation and spindle formation at early mitosis.

A vector system for efficient and economical switching of a ura4+ module to three commonly used antibiotic marker cassettes in Schizosaccharomyces pombe

We describe here the development of a set of plasmid vectors that allow simple, efficient and economical switching of a ura4+ module in existing Schizosaccharomyces pombe strains to any of the three routinely used antibiotic marker cassettes, kanMX6, hphMX6 and natMX6. In principle, the applications of this system can also be extended to switching ura4+ for additional MX6 module‐based cassettes, such as bleMX6, as long as the antibiotic marker has been cloned into an ura4+ module‐switching vector. We illustrate the application of this set of vectors in exchange of the ura4+ marker in existing strains with three antibiotic marker cassettes with high efficiency. Copyright

F-box proteins Pof3 and Pof1 regulate Wee1 degradation and mitotic entry in fission yeast

ABSTRACT The key cyclin-dependent kinase Cdk1 (Cdc2) promotes irreversible mitotic entry, mainly by activating the phosphatase Cdc25 while suppressing the tyrosine kinase Wee1. Wee1 needs to be downregulated at the onset of mitosis to ensure rapid activation of Cdk1. In human somatic cells, one mechanism of suppressing Wee1 activity is mediated by ubiquitylation-dependent proteolysis through the Skp1/Cul1/F-box protein (SCF) ubiquitin E3 ligase complex. This mechanism is believed to be conserved from yeasts to humans. So far, the best-characterized human F-box proteins involved in recognition of Wee1 are β-TrCP (BTRCP) and Tome-1 (CDCA3). Although fission yeast Wee1 was the first identified member of its conserved kinase family, the F-box proteins involved in recognition and ubiquitylation of Wee1 have not been identified in this organism. In this study, our screen using Wee1–Renilla luciferase as the reporter revealed that two F-box proteins, Pof1 and Pof3, are required for downregulating Wee1 and are possibly responsible for recruiting Wee1 to SCF. Our genetic analyses supported a functional relevance between Pof1 and Pof3 and the rate of mitotic entry, and Pof3 might play a major role in this process. Summary: Wee1 stability in fission yeast is controlled by F-box proteins Pof3 and Pof1.