Phosphorylation and Maximal Activity of Saccharomyces cerevisiae Meiosis-Specific Transcription Factor Ndt80 Is Dependent on Ime2

ABSTRACT The Saccharomyces cerevisiae meiosis-specific transcription factor Ndt80 is responsible for the induction of a class of genes referred to as middle sporulation genes. Among the members of this family are the B-type cyclins and other genes whose products are required for meiotic chromosome division and spore morphogenesis. Inactivation of NDT80 leads to a failure to induce the middle sporulation genes and a subsequent arrest in pachytene. The expression of NDT80 is itself highly regulated. The initial transcription of NDT80 is dependent upon the protein kinase Ime2; once Ndt80 protein accumulates, it activates its own promoter, thus generating an autoactivation loop. In addition to being transcriptionally regulated, Ndt80 protein is posttranslationally regulated. Phosphorylation of Ndt80 occurs coincident with its activation as a transcription factor. If expressed prematurely in meiosis, Ndt80 accumulates initially in an unmodified form that is subsequently modified by phosphorylation. In contrast, Ndt80 expressed in ime2 mutant strains does not become modified and has a reduced ability to activate transcription of its target genes. Ime2 can also phosphorylate Ndt80 in vitro, further supporting a direct role for Ime2 in the phosphorylation of Ndt80. These data indicate that Ime2 plays a novel and previously unexpected role in promoting chromosome dissemination and progress through meiotic development by activating Ndt80.

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Differential activation mechanisms of two isoforms of Gcr1 transcription factor generated from spliced and un-spliced transcripts in Saccharomyces cerevisiae

Nucleic Acids Research ◽

10.1093/nar/gkaa1221 ◽

2020 ◽

Author(s):

Seungwoo Cha ◽

Chang Pyo Hong ◽

Hyun Ah Kang ◽

Ji-Sook Hahn

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Target Genes ◽

Gene Encoding ◽

Metabolic Switch ◽

Differential Activation ◽

N Terminus ◽

Activation Mechanisms ◽

In Trans ◽

Separate Activation

Abstract Gcr1, an important transcription factor for glycolytic genes in Saccharomyces cerevisiae, was recently revealed to have two isoforms, Gcr1U and Gcr1S, produced from un-spliced and spliced transcripts, respectively. In this study, by generating strains expressing only Gcr1U or Gcr1S using the CRISPR/Cas9 system, we elucidate differential activation mechanisms of these two isoforms. The Gcr1U monomer forms an active complex with its coactivator Gcr2 homodimer, whereas Gcr1S acts as a homodimer without Gcr2. The USS domain, 55 residues at the N-terminus existing only in Gcr1U, inhibits dimerization of Gcr1U and even acts in trans to inhibit Gcr1S dimerization. The Gcr1S monomer inhibits the metabolic switch from fermentation to respiration by directly binding to the ALD4 promoter, which can be restored by overexpression of the ALD4 gene, encoding a mitochondrial aldehyde dehydrogenase required for ethanol utilization. Gcr1U and Gcr1S regulate almost the same target genes, but show unique activities depending on growth phase, suggesting that these isoforms play differential roles through separate activation mechanisms depending on environmental conditions.

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Cns1 Is an Essential Protein Associated with the Hsp90 Chaperone Complex in Saccharomyces cerevisiae That Can Restore Cyclophilin 40-Dependent Functions in cpr7ΔCells

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.7353 ◽

1998 ◽

Vol 18 (12) ◽

pp. 7353-7359 ◽

Cited By ~ 63

Author(s):

James A. Marsh ◽

Helen M. Kalton ◽

Richard F. Gaber

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Chaperone ◽

Single Gene ◽

Normal Growth ◽

Maximal Activity ◽

Negative Regulation ◽

Growth Defect ◽

Chaperone Complex ◽

Cyclophilin 40

ABSTRACT Saccharomyces cerevisiae harbors two cyclophilin 40-type enzymes, Cpr6 and Cpr7, which are components of the Hsp90 molecular chaperone machinery. Cpr7 is required for normal growth and is required for maximal activity of heterologous Hsp90-dependent substrates, including glucocorticoid receptor (GR) and the oncogenic tyrosine kinase pp60v-src . In addition, it has recently been shown that Cpr7 plays a major role in negative regulation of the S. cerevisiae heat shock transcription factor (HSF). To better understand functions associated with Cpr7, a search was undertaken for multicopy suppressors of the cpr7Δ slow-growth phenotype. The screen identified a single gene, designatedCNS1 (for cyclophilin seven suppressor), capable of suppressing the cpr7Δ growth defect. Overexpression ofCNS1 in cpr7Δ cells also largely restored GR activity and negative regulation of HSF. In vitro protein retention experiments in which Hsp90 heterocomplexes were precipitated resulted in coprecipitation of Cns1. Interaction between Cns1 and the carboxy terminus of Hsp90 was also shown by two-hybrid analysis. The functional consequences of CNS1 overexpression and its physical association with the Hsp90 machinery indicate that Cns1 is a previously unidentified component of molecular chaperone complexes. Thus far, Cns1 is the only tetratricopeptide repeat-containing component of Hsp90 heterocomplexes found to be essential for cell viability under all conditions tested.

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The Caenorhabditis elegans Heterochronic Regulator LIN-14 Is a Novel Transcription Factor That Controls the Developmental Timing of Transcription from the Insulin/Insulin-Like Growth Factor Gene ins-33 by Direct DNA Binding

Molecular and Cellular Biology ◽

10.1128/mcb.25.24.11059-11072.2005 ◽

2005 ◽

Vol 25 (24) ◽

pp. 11059-11072 ◽

Cited By ~ 30

Author(s):

Marta Hristova ◽

Darcy Birse ◽

Yang Hong ◽

Victor Ambros

Keyword(s):

Transcription Factor ◽

Caenorhabditis Elegans ◽

Growth Factor ◽

Dna Binding ◽

Dna Sequences ◽

Target Genes ◽

Specific Gene ◽

Insulin Like Growth Factor ◽

Consensus Binding Site

ABSTRACT A temporal gradient of the novel nuclear protein LIN-14 specifies the timing and sequence of stage-specific developmental events in Caenorhabditis elegans. The profound effects of lin-14 mutations on worm development suggest that LIN-14 directly or indirectly regulates stage-specific gene expression. We show that LIN-14 can associate with chromatin in vivo and has in vitro DNA binding activity. A bacterially expressed C-terminal domain of LIN-14 was used to select DNA sequences that contain a putative consensus binding site from a pool of randomized double-stranded oligonucleotides. To identify candidates for genes directly regulated by lin-14, we employed DNA microarray hybridization to compare the mRNA abundance of C. elegans genes in wild-type animals to that in mutants with reduced or elevated lin-14 activity. Five of the candidate LIN-14 target genes identified by microarrays, including the insulin/insulin-like growth factor family gene ins-33, contain putative LIN-14 consensus sites in their upstream DNA sequences. Genetic analysis indicates that the developmental regulation of ins-33 mRNA involves the stage-specific repression of ins-33 transcription by LIN-14 via sequence-specific DNA binding. These results reinforce the conclusion that lin-14 encodes a novel class of transcription factor.

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GA Binding Protein (GABP) Is Required for Development and Maturation of Myeloid Cells.

Blood ◽

10.1182/blood.v104.11.776.776 ◽

2004 ◽

Vol 104 (11) ◽

pp. 776-776

Author(s):

Zhongfa Yang ◽

Alan G. Rosmarin

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Target Genes ◽

Embryonic Stem ◽

Myeloid Cells ◽

Transactivation Domain ◽

Wild Type ◽

Floxed Allele

Abstract GABP is an ets transcription factor that regulates transcription of key myeloid genes, including CD18 (beta2 leukocyte integrin), neutrophil elastase, lysozyme, and other key mediators of the inflammatory response; it is also known to regulate important cell cycle control genes. GABP consists of two distinct and unrelated proteins that, together, form a functional transcription factor complex. GABPalpha (GABPa) is an ets protein that binds to DNA; it forms a tetrameric complex by recruiting its partner, GABPbeta (GABPb), which contains the transactivation domain. GABPa is a single copy gene in both the human and murine genomes and it is the only protein that can recruit GABPb to DNA. We cloned GABPa from a murine genomic BAC library and prepared a targeting vector in which exon 9 (which encodes the GABPa ets domain) was flanked by loxP (floxed) recombination sites. The targeting construct was electroporated into embryonic stem cells, homologous recombinants were implanted into pseudopregnant mice, heterozygous floxed GABPa mice were identified, and intercrossing yielded expected Mendelian ratios of wild type, heterozygous, and homozygous floxed GABPa mice. Breeding of heterozygous floxed GABPa mice to CMV-Cre mice (which express Cre recombinase in all tissues) yielded expected numbers of hemizygous mice (only one intact GABPa allele), but no nullizygous (GABPa−/−) mice among 64 pups; we conclude that homozygous deletion of GABPa causes an embryonic lethal defect. To determine the effect of GABPa deletion on myeloid cell development, we bred heterozygous and homozygous floxed mice to LysMCre mice, which express Cre only in myeloid cells. These mice had a normal complement of myeloid cells but, unexpectedly, PCR indicated that their Gr1+ myeloid cells retained an intact (undeleted) floxed GABPa allele. We detected similar numbers of in vitro myeloid colonies from bone marrow of wild type, heterozygous floxed, and homozygous floxed progeny of LysMCre matings. However, PCR of twenty individual in vitro colonies from homozygous floxed mice indicated that they all retained an intact floxed allele. Breeding of floxed GABPa/LysMCre mice with hemizygous mice indicated that retention of a floxed allele was not due to incomplete deletion by LysMCre; rather, it appears that only myeloid cells that retain an intact GABPa allele can survive to mature in vitro or in vivo. We prepared murine embryonic fibroblasts from homozygous floxed mice and efficiently deleted GABPa in vitro. We found striking abnormalities in proliferation and G1/S phase arrest. We used quantitative RT-PCR to identify mechanisms that account for the altered growth of GABPa null cells. We found dramatically reduced expression of known GABP target genes that regulate DNA synthesis and cell cycle that appear to account for the proliferative defect. We conclude that GABPa is required for growth and maturation of myeloid cells and we identified downstream targets that may account for their failure to proliferate and mature in vitro and in vivo.

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Activity of phosphoforms and truncated versions of Ndt80, a checkpoint-regulated sporulation-specific transcription factor of Saccharomyces cerevisiae

Molecular Genetics and Genomics ◽

10.1007/s00438-003-0922-3 ◽

2003 ◽

Vol 270 (4) ◽

pp. 324-336 ◽

Cited By ~ 11

Author(s):

G. Shubassi ◽

N. Luca ◽

J. Pak ◽

J. Segall

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Specific Transcription Factor

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Regulation of Gluconeogenesis in Saccharomyces cerevisiae Is Mediated by Activator and Repressor Functions of Rds2

Molecular and Cellular Biology ◽

10.1128/mcb.01055-07 ◽

2007 ◽

Vol 27 (22) ◽

pp. 7895-7905 ◽

Cited By ~ 46

Author(s):

Nitnipa Soontorngun ◽

Marc Larochelle ◽

Simon Drouin ◽

François Robert ◽

Bernard Turcotte

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Target Genes ◽

Tricarboxylic Acid Cycle ◽

Glyoxylate Cycle ◽

Dual Function ◽

Snf1 Kinase ◽

Zinc Cluster ◽

Chip Technology

ABSTRACT In Saccharomyces cerevisiae, RDS2 encodes a zinc cluster transcription factor with unknown function. Here, we unravel a key function of Rds2 in gluconeogenesis using chromatin immunoprecipitation-chip technology. While we observed that Rds2 binds to only a few promoters in glucose-containing medium, it binds many additional genes when the medium is shifted to ethanol, a nonfermentable carbon source. Interestingly, many of these genes are involved in gluconeogenesis, the tricarboxylic acid cycle, and the glyoxylate cycle. Importantly, we show that Rds2 has a dual function: it directly activates the expression of gluconeogenic structural genes while it represses the expression of negative regulators of this pathway. We also show that the purified DNA binding domain of Rds2 binds in vitro to carbon source response elements found in the promoters of target genes. Finally, we show that upon a shift to ethanol, Rds2 activation is correlated with its hyperphosphorylation by the Snf1 kinase. In summary, we have characterized Rds2 as a novel major regulator of gluconeogenesis.

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Molecular Characterization of Saccharomyces cerevisiae TFIID

Molecular and Cellular Biology ◽

10.1128/mcb.22.16.6000-6013.2002 ◽

2002 ◽

Vol 22 (16) ◽

pp. 6000-6013 ◽

Cited By ~ 78

Author(s):

Steven L. Sanders ◽

Krassimira A. Garbett ◽

P. Anthony Weil

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Molecular Mass ◽

Molecular Characterization ◽

Biochemical Characterization ◽

Gel Filtration ◽

Calculated Molecular Mass ◽

General Transcription Factor

ABSTRACT We previously defined Saccharomyces cerevisiae TFIID as a 15-subunit complex comprised of the TATA binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). In this report we give a detailed biochemical characterization of this general transcription factor. We have shown that yeast TFIID efficiently mediates both basal and activator-dependent transcription in vitro and displays TATA box binding activity that is functionally distinct from that of TBP. Analyses of the stoichiometry of TFIID subunits indicated that several TAFs are present at more than 1 copy per TFIID complex. This conclusion was further supported by coimmunoprecipitation experiments with a systematic family of (pseudo)diploid yeast strains that expressed epitope-tagged and untagged alleles of the genes encoding TFIID subunits. Based on these data, we calculated a native molecular mass for monomeric TFIID. Purified TFIID behaved in a fashion consistent with this calculated molecular mass in both gel filtration and rate-zonal sedimentation experiments. Quite surprisingly, although the TAF subunits of TFIID cofractionated as a single complex, TBP did not comigrate with the TAFs during either gel filtration chromatography or rate-zonal sedimentation, suggesting that TBP has the ability to dynamically associate with the TFIID TAFs. The results of direct biochemical exchange experiments confirmed this hypothesis. Together, our results represent a concise molecular characterization of the general transcription factor TFIID from S. cerevisiae.

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Activity of the Yap1 Transcription Factor in Saccharomyces cerevisiae Is Modulated by Methylglyoxal, a Metabolite Derived from Glycolysis

Molecular and Cellular Biology ◽

10.1128/mcb.24.19.8753-8764.2004 ◽

2004 ◽

Vol 24 (19) ◽

pp. 8753-8764 ◽

Cited By ~ 64

Author(s):

Kazuhiro Maeta ◽

Shingo Izawa ◽

Shoko Okazaki ◽

Shusuke Kuge ◽

Yoshiharu Inoue

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Disulfide Bond ◽

Target Genes ◽

Cysteine Residue ◽

Cysteine Residues ◽

Glyoxalase I ◽

Toxic Metabolite ◽

Basic Leucine Zipper

ABSTRACT Methylglyoxal (MG) is synthesized during glycolysis, although it inhibits cell growth in all types of organisms. Hence, it has long been asked why such a toxic metabolite is synthesized in vivo. Glyoxalase I is a major enzyme detoxifying MG. Here we show that the Yap1 transcription factor, which is critical for the oxidative-stress response in Saccharomyces cerevisiae, is constitutively concentrated in the nucleus and activates the expression of its target genes in a glyoxalase I-deficient mutant. Yap1 contains six cysteine residues in two cysteine-rich domains (CRDs), i.e., three cysteine residues clustering near the N terminus (n-CRD) and the remaining three cysteine residues near the C terminus (c-CRD). We reveal that any of the three cysteine residues in the c-CRD is sufficient for MG to allow Yap1 to translocate into the nucleus and to activate the expression of its target gene. A Yap1 mutant possessing only one cysteine residue in the c-CRD but no cysteine in the n-CRD and deletion of the basic leucine zipper domain can concentrate in the nucleus with MG treatment. However, substitution of all the cysteine residues in Yap1 abolishes the ability of this transcription factor to concentrate in the nucleus following MG treatment. The redox status of Yap1 is substantially unchanged, and protein(s) interaction with Yap1 through disulfide bond is hardly detected in cells treated with MG. Collectively, neither intermolecular nor intramolecular disulfide bond formation seems to be involved in Yap1 activation by MG. Moreover, we show that nucleocytoplasmic localization of Yap1 closely correlates with growth phase and intracellular MG level. We propose a novel regulatory pathway underlying Yap1 activation by a natural metabolite in the cell.

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