scholarly journals Functional Domains of Rep2, a Transcriptional Activator Subunit for Res2–Cdc10, Controlling the Cell Cycle “Start”

1998 ◽  
Vol 9 (6) ◽  
pp. 1577-1588 ◽  
Author(s):  
Sayaka Tahara ◽  
Koichi Tanaka ◽  
Yasuhito Yuasa ◽  
Hiroto Okayama
Keyword(s):  
Cell Cycle ◽  
Amino Acid ◽  
Fission Yeast ◽  
Transcriptional Activation ◽  
Transcriptional Activator ◽  
S Phase ◽  
Activation Domains ◽  
Multicopy Suppressor ◽  
C Terminus

In the fission yeast Schizosaccharomyces pombe, passage from G1 to S-phase requires the execution of the transcriptional factor complex that consists of the Cdc10 and Res1/2 molecules. This complex activates the MluI cell cycle box cis-element contained in genes essential for S-phase onset and progression. The rep2 + gene, isolated as a multicopy suppressor of a temperature-sensitivecdc10 mutant, has been postulated to encode a putative transcriptional activator subunit for the Res2–Cdc10 complex. To identify the rep2 + function and molecularly define its domain organization, we reconstituted the Res2–Cdc10 complex-dependent transcriptional activation in Saccharomyces cerevisiae. Reconstitution experiments, deletion analyses using one and two hybrid systems, and in vivo Res2 coimmunoprecipitation assays show that the Res2–Cdc10 complex itself can recognize but cannot activate MluI cell cycle box without Rep2, and that consistent with its postulated function, Rep2 contains 45-amino acid Res2 binding and 22-amino acid transcriptional activation domains in the middle and C terminus of the molecule, respectively. The functional essentiality of these domains is also demonstrated by their requirement for rescue of the cold-sensitive rep2deletion mutant of fission yeast.

scholarly journals Negative Regulation of Cdc18 DNA Replication Protein by Cdc2

1998 ◽  
Vol 9 (1) ◽  
pp. 63-73 ◽  
Author(s):  
Antonia Lopez-Girona ◽  
Odile Mondesert ◽  
Janet Leatherwood ◽  
Paul Russell
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Fission Yeast ◽  
Phosphorylation Site ◽  
Constitutive Expression ◽  
S Phase ◽  
Negative Regulation ◽  
Chromosomal Dna

Fission yeast Cdc18, a homologue of Cdc6 in budding yeast and metazoans, is periodically expressed during the S phase and required for activation of replication origins. Cdc18 overexpression induces DNA rereplication without mitosis, as does elimination of Cdc2-Cdc13 kinase during G2 phase. These findings suggest that illegitimate activation of origins may be prevented through inhibition of Cdc18 by Cdc2. Consistent with this hypothesis, we report that Cdc18 interacts with Cdc2 in association with Cdc13 and Cig2 B-type cyclins in vivo. Cdc18 is phosphorylated by the associated Cdc2 in vitro. Mutation of a single phosphorylation site, T104A, activates Cdc18 in the rereplication assay. The cdc18-K9 mutation is suppressed by a cig2 mutation, providing genetic evidence that Cdc2-Cig2 kinase inhibits Cdc18. Moreover, constitutive expression of Cig2 prevents rereplication in cells lacking Cdc13. These findings identify Cdc18 as a key target of Cdc2-Cdc13 and Cdc2-Cig2 kinases in the mechanism that limits chromosomal DNA replication to once per cell cycle.

scholarly journals Interaction of cdc2 and rum1 regulates Start and S-phase in fission yeast

1995 ◽  
Vol 108 (10) ◽  
pp. 3285-3294 ◽  
Author(s):  
K. Labib ◽  
S. Moreno ◽  
P. Nurse
Keyword(s):  
Cell Cycle ◽  
Yeast Cell ◽  
Fission Yeast ◽  
Critical Mass ◽  
Genetic Data ◽  
S Phase ◽  
G1 Phase ◽  
Mitotic Entry ◽  
Important Regulator

The p34cdc2 kinase is essential for progression past Start in the G1 phase of the fission yeast cell cycle, and also acts in G2 to promote mitotic entry. Whilst very little is known about the G1 function of cdc2, the rum1 gene has recently been shown to encode an important regulator of Start in fission yeast, and a model for rum1 function suggests that it inhibits p34cdc2 activity. Here we present genetic data suggesting that rum1 maintains p34cdc2 in a pre-Start G1 form, inhibiting its activity until the cell achieves the critical mass required for Start, and find that in the absence of rum1 p34cdc2 has increased Start activity in vivo. It is also known that mutation of cdc2, or overexpression of rum1, can disrupt the dependency of S-phase upon mitosis, resulting in an extra round of S-phase in the absence of mitosis. We show that cdc2 and rum1 interact in this process, and describe dominant cdc2 mutants causing multiple rounds of S-phase in the absence of mitosis. We suggest that interaction of rum1 and cdc2 regulates Start, and this interaction is important for the regulation of S-phase within the cell cycle.

scholarly journals Two Distinct Domains in Staf To Selectively Activate Small Nuclear RNA-Type and mRNA Promoters

1998 ◽  
Vol 18 (5) ◽  
pp. 2650-2658 ◽  
Author(s):  
Catherine Schuster ◽  
Alain Krol ◽  
Philippe Carbon
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Repeat Unit ◽  
Transcriptional Activator ◽  
Small Nuclear Rna ◽  
Pol Ii ◽  
Activation Domains ◽  
Transcription Complexes ◽  
Pol Iii

ABSTRACT Staf is a transcriptional activator of prime importance for enhanced transcription of small nuclear (snRNA) and snRNA-type genes transcribed by RNA polymerases II and III (Pol II and III). In addition to this activity, it also possesses the capacity to stimulate expression from an RNA polymerase II mRNA promoter. This promiscuous activator thus provides a useful model system for studying the mechanism by which one single transcription factor can activate a large variety of promoters. Here, we report the use of in vivo assays to identify the Staf activation domains involved in promoter selectivity. Analysis of Staf mutants reveals the existence of two physically and functionally distinct regions, outside of the DNA binding domain, responsible for mediating selective transcriptional activation. While a 93-amino-acid domain, with the striking presence of four repeated units, is specialized for transcriptional activation of an mRNA promoter, a segment of only 18 amino acids, with a critical Leu-213 residue, acts specifically on Pol II and Pol III snRNA and snRNA-type promoters. In addition, this study disclosed the fundamental importance of invariant leucine and aspartic acid residues located in each repeat unit of the mRNA activation domain. Staf is therefore the first transcriptional activator described so far to harbor two physically and functionally distinct activator domains. This finding suggests that the same activator can contact different, specialized transcription complexes formed on different types of basal promoters through promoter-specific transactivation pathways.

scholarly journals The transcriptional repressor gene Mad3 is a novel target for regulation by E2F1

2003 ◽  
Vol 370 (1) ◽  
pp. 307-313 ◽  
Author(s):  
Elizabeth J. FOX ◽  
Stephanie C. WRIGHT
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
S Phase ◽  
Mobility Shift ◽  
Binding Factor ◽  
Flanking Region ◽  
Novel Target ◽  
Electrophoretic Mobility Shift Assays

Mad family proteins are transcriptional repressors that antagonize the activity of the c-Myc proto-oncogene product. Mad3 is expressed specifically during the S-phase of the cell cycle in both proliferating and differentiating cells, suggesting that its biological function is probably linked to processes that occur during this period. To determine the mechanisms that regulate the cell-cycle-specific transcription of Mad3, we used reporter gene assays in stably transfected fibroblasts. We show that the activation of Mad3 at the G1—S boundary is mediated by a single E2F (E2 promoter binding factor)-binding site within the 5′-flanking region of the gene. Mutation of this element eliminated transcriptional activation at S-phase, suggesting that the positively acting E2F proteins play a role in Mad3 regulation. Using electrophoretic mobility-shift assays and chromatin immunoprecipitation, we show that E2F1 binds to the Mad3 5′-flanking region both in vitro and in vivo. We thus identify Mad3 as a novel transcriptional target of E2F1.

scholarly journals Histone gene expression is regulated by histone H2B ubiquitylation in fission yeast

2016 ◽  
Author(s):  
Viviane Pagé ◽  
David Grabowski ◽  
C. David Allis ◽  
Jason C. Tanny
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
S Phase ◽  
Histone Gene ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Histone Genes ◽  
Histone H2b

AbstractCell cycle-regulated expression of histone genes is vital for coordination of DNA replication with chromatin assembly. In yeast, histone genes are regulated by transcriptional activation in S phase and transcriptional repression in other phases of the cell cycle, although the mechanisms are poorly understood. Here we describe a role for histone H2B monoubiquitylation (H2Bub1), a histone mark that is linked to RNA polymerase II transcription elongation, in the activation of replication-dependent histone genes in the fission yeast Schizosaccharomyces pombe. Loss of H2Bub1 is also associated with a delay in S phase progression and reduced expression of the replication initiation factor Cdc18 (ortholog of Cdc6). We provide evidence that H2Bub1 impacts histone gene transcription and acts through a mechanism that involves the 3’UTR. Consistent with our previous finding that H2Bub1 is functionally opposed to the elongation factor Cdk9, we also find that the effects of H2Bub1 on histone genes are suppressed by reduction in the activity of Cdk9. Our data suggest that H2Bub1 promotes cell growth by activating cell cycle-regulated genes.

scholarly journals A Pcl-like Cyclin Activates the Res2p-Cdc10p Cell Cycle “Start” Transcriptional Factor Complex in Fission Yeast

2000 ◽  
Vol 11 (9) ◽  
pp. 2845-2862 ◽  
Author(s):  
Koichi Tanaka ◽  
Hiroto Okayama
Keyword(s):  
Cell Cycle ◽  
Genetic Analysis ◽  
Fission Yeast ◽  
Budding Yeast ◽  
Mitotic Cell ◽  
Mating Pheromone ◽  
Yeast Saccharomyces Cerevisiae ◽  
Multicopy Suppressor ◽  
Pheromone Signaling

In the fission yeast Schizosaccharomyces pombe, the “start” of the cell cycle is controlled by the two functionally redundant transcriptional regulator complexes, Res1p-Cdc10p and Res2p-Cdc10p, that activate genes essential for the onset and progression of S phase. The activity of the Res2p-Cdc10p complex is regulated at least by the availability of the Rep2trans-activator subunit in the mitotic cell cycle. We have recently isolated the pas1+gene as a multicopy suppressor of the res1 null mutant. This gene encodes a novel cyclin that shares homology with the Pho85 kinase–associated cyclins of the budding yeast Saccharomyces cerevisiae. Genetic analysis reveals that Pas1 cyclin is unrelated to phosphate metabolism and stimulates the G1-S transition by specifically activating the Res2p-Cdc10p complex independently of Rep2p. Pas1 cyclin also controls mating pheromone signaling. Cells lacking pas1+are highly sensitive to mating pheromone, responding with facilitated G1arrest and premature commitment to conjugation. Pas1 cyclin associates in vivo with both Cdc2 and Pef1 kinases, the latter of which is a fission yeast counterpart of the budding yeast Pho85 kinase, but genetic analysis indicates that the Pef1p-associated Pas1p is responsible for the activation of Res2p-Cdc10p during the G1-S transition.

scholarly journals Transcriptional Activation in Yeast Cells Lacking Transcription Factor IIA

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1573-1581 ◽  
Author(s):  
Susanna Chou ◽  
Sukalyan Chatterjee ◽  
Mark Lee ◽  
Kevin Struhl
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Large Subunit ◽  
Yeast Cells ◽  
Specific Cell ◽  
Wild Type ◽  
Activation Domains ◽  
Cycle Arrest ◽  
General Transcription Factor

Abstract The general transcription factor IIA (TFIIA) forms a complex with TFIID at the TATA promoter element, and it inhibits the function of several negative regulators of the TATA-binding protein (TBP) subunit of TFIID. Biochemical experiments suggest that TFIIA is important in the response to transcriptional activators because activation domains can interact with TFIIA, increase recruitment of TFIID and TFIIA to the promoter, and promote isomerization of the TFIID-TFIIA-TATA complex. Here, we describe a double-shut-off approach to deplete yeast cells of Toa1, the large subunit of TFIIA, to <1% of the wild-type level. Interestingly, such TFIIA-depleted cells are essentially unaffected for activation by heat shock factor, Ace1, and Gal4-VP16. However, depletion of TFIIA causes a general two- to threefold decrease of transcription from most yeast promoters and a specific cell-cycle arrest at the G2-M boundary. These results indicate that transcriptional activation in vivo can occur in the absence of TFIIA.

scholarly journals Spanning the gap: unraveling RSC dynamics in vivo

2021 ◽  
Author(s):  
Heinz Neumann ◽  
Bryan J. Wilkins
Keyword(s):  
Cell Cycle ◽  
Posttranslational Modifications ◽  
Transcriptional Activation ◽  
Binding Mode ◽  
Binding Modes ◽  
Binding Domains ◽  
Binding Interface ◽  
The Many ◽  
And Function

AbstractMultiple reports over the past 2 years have provided the first complete structural analyses for the essential yeast chromatin remodeler, RSC, providing elaborate molecular details for its engagement with the nucleosome. However, there still remain gaps in resolution, particularly within the many RSC subunits that harbor histone binding domains.Solving contacts at these interfaces is crucial because they are regulated by posttranslational modifications that control remodeler binding modes and function. Modifications are dynamic in nature often corresponding to transcriptional activation states and cell cycle stage, highlighting not only a need for enriched spatial resolution but also temporal understanding of remodeler engagement with the nucleosome. Our recent work sheds light on some of those gaps by exploring the binding interface between the RSC catalytic motor protein, Sth1, and the nucleosome, in the living nucleus. Using genetically encoded photo-activatable amino acids incorporated into histones of living yeast we are able to monitor the nucleosomal binding of RSC, emphasizing the regulatory roles of histone modifications in a spatiotemporal manner. We observe that RSC prefers to bind H2B SUMOylated nucleosomes in vivo and interacts with neighboring nucleosomes via H3K14ac. Additionally, we establish that RSC is constitutively bound to the nucleosome and is not ejected during mitotic chromatin compaction but alters its binding mode as it progresses through the cell cycle. Our data offer a renewed perspective on RSC mechanics under true physiological conditions.

scholarly journals RAE-1 ligands for the NKG2D receptor are regulated by E2F transcription factors, which control cell cycle entry

2012 ◽  
Vol 209 (13) ◽  
pp. 2409-2422 ◽  
Author(s):  
Heiyoun Jung ◽  
Benjamin Hsiung ◽  
Kathleen Pestal ◽  
Emily Procyk ◽  
David H. Raulet
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Stress Responses ◽  
Transcriptional Activation ◽  
Posttranscriptional Regulation ◽  
Cellular Proliferation ◽  
Control Cell ◽  
T Cell Subsets ◽  
Cell Cycle Entry

The NKG2D stimulatory receptor expressed by natural killer cells and T cell subsets recognizes cell surface ligands that are induced on transformed and infected cells and facilitate immune rejection of tumor cells. We demonstrate that expression of retinoic acid early inducible gene 1 (RAE-1) family NKG2D ligands in cancer cell lines and proliferating normal cells is coupled directly to cell cycle regulation. Raet1 genes are directly transcriptionally activated by E2F family transcription factors, which play a central role in regulating cell cycle entry. Induction of RAE-1 occurred in primary cell cultures, embryonic brain cells in vivo, and cells in healing skin wounds and, accordingly, wound healing was delayed in mice lacking NKG2D. Transcriptional activation by E2Fs is likely coordinated with posttranscriptional regulation by other stress responses. These findings suggest that cellular proliferation, as occurs in cancer cells but also other pathological conditions, is a key signal tied to immune reactions mediated by NKG2D-bearing lymphocytes.

scholarly journals p53 upregulated by HIF-1α promotes hypoxia-induced G2/M arrest and renal fibrosis in vitro and in vivo

2018 ◽  
Vol 11 (5) ◽  
pp. 371-382 ◽  
Author(s):  
Limin Liu ◽  
Peng Zhang ◽  
Ming Bai ◽  
Lijie He ◽  
Lei Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Renal Fibrosis ◽  
Intraperitoneal Administration ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
P53 Expression ◽  
Tubular Cells

Abstract Hypoxia plays an important role in the genesis and progression of renal fibrosis. The underlying mechanisms, however, have not been sufficiently elucidated. We examined the role of p53 in hypoxia-induced renal fibrosis in cell culture (human and rat renal tubular epithelial cells) and a mouse unilateral ureteral obstruction (UUO) model. Cell cycle of tubular cells was determined by flow cytometry, and the expression of profibrogenic factors was determined by RT-PCR, immunohistochemistry, and western blotting. Chromatin immunoprecipitation and luciferase reporter experiments were performed to explore the effect of HIF-1α on p53 expression. We showed that, in hypoxic tubular cells, p53 upregulation suppressed the expression of CDK1 and cyclins B1 and D1, leading to cell cycle (G2/M) arrest (or delay) and higher expression of TGF-β, CTGF, collagens, and fibronectin. p53 suppression by siRNA or by a specific p53 inhibitor (PIF-α) triggered opposite effects preventing the G2/M arrest and profibrotic changes. In vivo experiments in the UUO model revealed similar antifibrotic results following intraperitoneal administration of PIF-α (2.2 mg/kg). Using gain-of-function, loss-of-function, and luciferase assays, we further identified an HRE3 region on the p53 promoter as the HIF-1α-binding site. The HIF-1α–HRE3 binding resulted in a sharp transcriptional activation of p53. Collectively, we show the presence of a hypoxia-activated, p53-responsive profibrogenic pathway in the kidney. During hypoxia, p53 upregulation induced by HIF-1α suppresses cell cycle progression, leading to the accumulation of G2/M cells, and activates profibrotic TGF-β and CTGF-mediated signaling pathways, causing extracellular matrix production and renal fibrosis.

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