Project description:Body cells in multi-cellular organisms are in the G0 state, in which cells are arrested and terminally differentiated. To understand how the G0 state is maintained, the genes that are specifically expressed or repressed in G0 must be identified, as they control G0. In the fission yeast Schizosaccharomyces pombe, haploid cells are completely arrested under nitrogen source starvation with high viability. We examined the global transcriptome of G0 cells and cells on the course to resume vegetative growth. Approximately 20% of the transcripts of ~5000 genes increased or decreased more than 4-fold in the two-step transitions that occur prior to replication. Of the top 30 abundant transcripts in G0, 23 were replaced by ribosome- and translation-related transcripts in the dividing vegetative state. Eight identified clusters with distinct alteration patterns of ~2700 transcripts were annotated by Gene Ontology. Disruption of 53 genes indicated that 9 of them were necessary to support the proper G0 state. These 9 genes included two C2H2 zinc finger transcription factors, a cyclin-like protein implicated in phosphorylation of RNA polymerase II, two putative autophagy regulators, a G-protein activating factor, and two CBS domain proteins, possibly involved in AMP-activated kinase. Keywords: Keywards: Time course, Nitrogen starvation, G0 state, Nitrogen replenishment, Nutrient signal, Cell cycle, Cell proliferation
Project description:Fission yeast Schizosaccharomyces pombe is a model for studying cellular quiescence. Shifting to medium without a nitrogen-source induces proliferative cells to enter long-term G0 quiescence. Klf1 is a Kruppel-like transcription factor with a 7-amino acid-spaced C2H2-type zinc finger motif. The deletion mutant ∆klf1 normally divides in vegetative medium, but proliferation is not restored after long-term G0 quiescence. Cell biologic, transcriptomic, and metabolomic analyses revealed a unique phenotype of the ∆klf1 mutant in quiescence. Mutant cells had diminished transcripts related to signaling molecules for switching to differentiation. In contrast, proliferative metabolites for cell-wall assembly and antioxidants significantly increased. Further, the size of the ∆klf1 cells is markedly increased during quiescence due to the aberrant accumulation of calcofluor-positive chitin-like materials beneath the cell wall. After 4 weeks quiescence, the ability for reversible proliferation is lost, but energy metabolism is maintained. Klf1 thus plays a role in G0 phase longevity through enhancing the differentiation signal and suppressing metabolism for growth. If Klf1 is lost, S. pombe fails to maintain a constant cell size during quiescence.
Project description:Fission yeast Schizosaccharomyces pombe is a model for studying cellular quiescence. Shifting to medium without a nitrogen-source induces proliferative cells to enter long-term G0 quiescence. Klf1 is a Kruppel-like transcription factor with a 7-amino acid-spaced C2H2-type zinc finger motif. The deletion mutant M-bM-^HM-^Fklf1 normally divides in vegetative medium, but proliferation is not restored after long-term G0 quiescence. Cell biologic, transcriptomic, and metabolomic analyses revealed a unique phenotype of the M-bM-^HM-^Fklf1 mutant in quiescence. Mutant cells had diminished transcripts related to signaling molecules for switching to differentiation. In contrast, proliferative metabolites for cell-wall assembly and antioxidants significantly increased. Further, the size of the M-bM-^HM-^Fklf1 cells is markedly increased during quiescence due to the aberrant accumulation of calcofluor-positive chitin-like materials beneath the cell wall. After 4 weeks quiescence, the ability for reversible proliferation is lost, but energy metabolism is maintained. Klf1 thus plays a role in G0 phase longevity through enhancing the differentiation signal and suppressing metabolism for growth. If Klf1 is lost, S. pombe fails to maintain a constant cell size during quiescence. Gene expression profile of fission yeast wild type cells and klf1-gene disruptant cells in proliferating state and in quiescent state. Type of experiment: Comparing between wild type cells and klf1-gene disruptant cells in proliferating condition and in quiescent condition. Experimental factor: Exponentially proliferating state in synthetic medium, EMM2, and quiescent G0 state in nitrogen-depleted synthetic medium, EMM2-N. Quality control steps taken: All experiments were repeated twice in each condition.
Project description:Tristetraprolin is a vertebrate CCCH tandem zinc finger protein that can bind to and destabilize certain mRNAs containing AU-rich element binding sites. zfs1 is the single gene in the fission yeast, Schizosaccharomyces pombe, that encodes a protein containing the critical features of the tristetraprolin zinc finger domain. zfs1 has been linked to pheromone signal transduction control and to the coordination of mitosis, but no biological function has been ascribed to the zfs1 protein. Through a functional genomics approach we compared transcript levels in wild-type and zfs1-deficient S. pombe strains; those elevated in the zfs1-deficient strain were examined for the presence of potential tristetraprolin-like binding sites. One such potential target transcript was encoded by arz1, a gene encoding a protein of unknown function that contains armadillo repeats. arz1 mRNA decay was inhibited in the zfs1-deficient strain when it was expressed under the control of a thiamine-repressible promoter. Mutations within one AU-rich element present in the arz1 3’-untranslated region protected this transcript from zfs1-promoted decay, whereas mutating another potential binding site had no effect. Binding assays confirmed a direct interaction between zfs1 and arz1 mRNA-based probes; this interaction was eliminated when key residues were mutated in either zfs1 zinc finger. zfs1 and its targets in S. pombe represent a useful model system for studies of zinc finger protein/AU-rich element interactions that result in mRNA decay. Keywords: knockout comparison, steady-state analysis
Project description:C2H2 zinc finger proteins represent the largest and most enigmatic class of human transcription factors. Their C2H2 arrays are highly variable, indicating that most will have unique DNA binding motifs. However, most of the binding motifs have not been directly determined. We have determined the binding sites and motifs of 119 C2H2 zinc finger proteins and the expression pattern of 80 cell lines overexpressing C2H2 zinc finger proteins in order to study the role of C2H2 zinc finger proteins in gene regulation.
Project description:C2H2 zinc finger proteins represent the largest and most enigmatic class of human transcription factors. Their C2H2 arrays are highly variable, indicating that most will have unique DNA binding motifs. However, most of the binding motifs have not been directly determined. We have determined the binding sites and motifs of 119 C2H2 zinc finger proteins and the expression pattern of 80 cell lines overexpressing C2H2 zinc finger proteins in order to study the role of C2H2 zinc finger proteins in gene regulation. We expressed GFP-tagged C2H2-ZF proteins in stable transgenic HEK293 cells. Total RNA was isolated using Trizol and sequencing libraries were constructed using TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold or TruSeq RNA Library Preparation Kit v2.
Project description:RNA sequencing was performed to investigate the the response mechanism of tomato response to drought stress. C2H2-type zinc finger proteins are classic and extensively studied members of the zinc finger family. C2H2-type zinc finger proteins participate in plant growth, development and stress responses. In this study, 99 C2H2-type zinc finger protein genes were identified and classified into four groups, and many functionally related cis-elements were identified. Differential C2H2-ZFP gene expression and specific responses were analyzed under drought, cold, salt and pathogen stresses based on RNA-Seq data. Thirty-two C2H2 genes were identified in response to multiple stresses. Seven, 3, 5, and 8 genes were specifically expressed under drought, cold, salt and pathogenic stresses, respectively. Five glycometabolism and sphingolipid-related, pathways and the endocytosis pathway were enriched by KEGG analysis. The results of this study represent a foundation for further study of the function of C2H2-type zinc finger proteins and will provide us with genetic resources for stress tolerance breeding.