Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

Download Full-text

ABF1 is a phosphoprotein and plays a role in carbon source control of COX6 transcription in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.4197-4208.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 4197-4208

Author(s):

S Silve ◽

P R Rhode ◽

B Coll ◽

J Campbell ◽

R O Poyton

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Glucose Repression ◽

Carbon Sources ◽

Growth Conditions ◽

Source Control ◽

Specific Target Gene ◽

Phosphorylation Pattern ◽

Nonfermentable Carbon Source ◽

In Cells

Previously, we have shown that the Saccharomyces cerevisiae DNA-binding protein ABF1 exists in at least two different electrophoretic forms (K. S. Sweder, P. R. Rhode, and J. L. Campbell, J. Biol. Chem. 263: 17270-17277, 1988). In this report, we show that these forms represent different states of phosphorylation of ABF1 and that at least four different phosphorylation states can be resolved electrophoretically. The ratios of these states to one another differ according to growth conditions and carbon source. Phosphorylation of ABF1 is therefore a regulated process. In nitrogen-starved cells or in cells grown on nonfermentable carbon sources (e.g., lactate), phosphorylated forms predominate, while in cells grown on fermentable carbon sources (e.g., glucose), dephosphorylated forms are enriched. The phosphorylation pattern is affected by mutations in the SNF1-SSN6 pathway, which is involved in glucose repression-depression. Whereas a functional SNF1 gene, which encodes a protein kinase, is not required for the phosphorylation of ABF1, a functional SSN6 gene is required for itsd ephosphorylation. The phosphorylation patterns that we have observed correlate with the regulation of a specific target gene, COX6, which encodes subunit VI of cytochrome c oxidase. Transcription of COX6 is repressed by growth in medium containing a fermentable carbon source and is derepressed by growth in medium containing a nonfermentable carbon source. COX6 repression-derepression is under the control of the SNF1-SSN6 pathway. This carbon source regulation is exerted through domain 1, a region of the upstream activation sequence UAS6 that binds ABF1 (J. D. Trawick, N. Kraut, F. Simon, and R. O. Poyton, Mol. Cell Biol. 12:2302-2314, 1992). We show that the greater the phosphorylation of ABF1, the greater the transcription of COX6. Furthermore, the ABF1-containing protein-DNA complexes formed at domain 1 differ according to the phosphorylation state of ABF1 and the carbon source on which the cells were grown. From these findings, we propose that the phosphorylation of ABF1 is involved in glucose repression-derepression of COX6 transcription.

Download Full-text

ABF1 is a phosphoprotein and plays a role in carbon source control of COX6 transcription in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.4197 ◽

1992 ◽

Vol 12 (9) ◽

pp. 4197-4208 ◽

Cited By ~ 35

Author(s):

S Silve ◽

P R Rhode ◽

B Coll ◽

J Campbell ◽

R O Poyton

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Glucose Repression ◽

Carbon Sources ◽

Growth Conditions ◽

Source Control ◽

Specific Target Gene ◽

Phosphorylation Pattern ◽

Nonfermentable Carbon Source ◽

In Cells

Previously, we have shown that the Saccharomyces cerevisiae DNA-binding protein ABF1 exists in at least two different electrophoretic forms (K. S. Sweder, P. R. Rhode, and J. L. Campbell, J. Biol. Chem. 263: 17270-17277, 1988). In this report, we show that these forms represent different states of phosphorylation of ABF1 and that at least four different phosphorylation states can be resolved electrophoretically. The ratios of these states to one another differ according to growth conditions and carbon source. Phosphorylation of ABF1 is therefore a regulated process. In nitrogen-starved cells or in cells grown on nonfermentable carbon sources (e.g., lactate), phosphorylated forms predominate, while in cells grown on fermentable carbon sources (e.g., glucose), dephosphorylated forms are enriched. The phosphorylation pattern is affected by mutations in the SNF1-SSN6 pathway, which is involved in glucose repression-depression. Whereas a functional SNF1 gene, which encodes a protein kinase, is not required for the phosphorylation of ABF1, a functional SSN6 gene is required for itsd ephosphorylation. The phosphorylation patterns that we have observed correlate with the regulation of a specific target gene, COX6, which encodes subunit VI of cytochrome c oxidase. Transcription of COX6 is repressed by growth in medium containing a fermentable carbon source and is derepressed by growth in medium containing a nonfermentable carbon source. COX6 repression-derepression is under the control of the SNF1-SSN6 pathway. This carbon source regulation is exerted through domain 1, a region of the upstream activation sequence UAS6 that binds ABF1 (J. D. Trawick, N. Kraut, F. Simon, and R. O. Poyton, Mol. Cell Biol. 12:2302-2314, 1992). We show that the greater the phosphorylation of ABF1, the greater the transcription of COX6. Furthermore, the ABF1-containing protein-DNA complexes formed at domain 1 differ according to the phosphorylation state of ABF1 and the carbon source on which the cells were grown. From these findings, we propose that the phosphorylation of ABF1 is involved in glucose repression-derepression of COX6 transcription.

Download Full-text

Multicopy FZF1 (SUL1) Suppresses the Sulfite Sensitivity but Not the Glucose Derepression or Aberrant Cell Morphology of a grr1 Mutant of Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/144.2.511 ◽

1996 ◽

Vol 144 (2) ◽

pp. 511-521 ◽

Cited By ~ 3

Author(s):

Dorina Avram ◽

Alan T Bakalinsky

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Glucose Metabolism ◽

Cell Morphology ◽

Glucose Repression ◽

Carbon Sources ◽

Single Copy ◽

Aberrant Cell ◽

Growth Defects ◽

Putative Transcription Factor

Abstract An ssu2 mutation in Sacccharomyces cermisiae, previously shown to cause sulfite sensitivity, was found to be allelic to GRR1, a gene previously implicated in glucose repression. The suppressor rgt1, which suppresses the growth defects of grr1 strains on glucose, did not fully suppress the sensitivity on glucose or nonglucose carbon sources, indicating that it is not strictly linked to a defect in glucose metabolism. Because the Cln1 protein was previously shown to be elevated in grr1 mutants, the effect of CLN1 overexpression on sulfite sensitivity was investigated. Overexpression in GRR1 cells resulted in sulfite sensitivity, suggesting a connection between CLN1 and sulfite metabolism. Multicopy FZF1, a putative transcription factor, was found to suppress the sulfite sensitive phenotype of grr1 strains, but not the glucose derepression or aberrant cell morphology. Multicopy FZF1 was also found to suppress the sensitivity of a number of other unrelated sulfite-sensitive mutants, but not that of ssu1 or met20, implying that FZF1 may act through Ssulp and Met20p. Disruption of FZF1 resulted in sulfite sensitivity when the construct was introduced in single copy at the FZF1 locus in a GRR1 strain, providing evidence that FZF1 is involved in sulfite metabolism.

Download Full-text

Identification of a ras-activated enhancer in the mouse osteopontin promoter and its interaction with a putative ETS-related transcription factor whose activity correlates with the metastatic potential of the cell.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.476 ◽

1995 ◽

Vol 15 (1) ◽

pp. 476-487 ◽

Cited By ~ 54

Author(s):

X Guo ◽

Y P Zhang ◽

D A Mitchell ◽

D T Denhardt ◽

A F Chambers

Keyword(s):

Transcription Factor ◽

Protein Binding ◽

Uv Light ◽

Metastatic Potential ◽

Binding Motif ◽

Binding Assays ◽

Mobility Shift ◽

Related Transcription Factor ◽

Altered Gene ◽

Alpha V Beta 3

The role of RAS in transducing signals from an activated receptor into altered gene expression is becoming clear, though some links in the chain are still missing. Cells possessing activated RAS express higher levels of osteopontin (OPN), an alpha v beta 3 integrin-binding secreted phosphoprotein implicated in a number of developmental, physiological, and pathological processes. We report that in T24 H-ras-transformed NIH 3T3 cells enhanced transcription contributes to the increased expression of OPN. Transient transfection studies, DNA-protein binding assays, and methylation protection experiments have identified a novel ras-activated enhancer, distinct from known ras response elements, that appears responsible for part of the increase in OPN transcription in cells with an activated RAS. In electrophoretic mobility shift assays, the protein-binding motif GGAGGCAGG was found to be essential for the formation of several complexes, one of which (complex A) was generated at elevated levels by cell lines that are metastatic. Southwestern blotting and UV light cross-linking studies indicated the presence of several proteins able to interact with this sequence. The proteins that form these complexes have molecular masses estimated at approximately 16, 28, 32, 45, 80, and 100 kDa. Because the approximately 16-kDa protein was responsible for complex A formation, we have designated it MATF for metastasis-associated transcription factor. The GGANNNAGG motif is also found in some other promoters, suggesting that they may be similarly controlled by MATF.

Download Full-text

Factors involved in specific transcription by mammalian RNA polymerase II: purification and analysis of transcription factor IIA and identification of transcription factor IIJ.

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.413 ◽

1992 ◽

Vol 12 (1) ◽

pp. 413-421 ◽

Cited By ~ 66

Author(s):

P Cortes ◽

O Flores ◽

D Reinberg

Keyword(s):

Transcription Factor ◽

Rna Polymerase Ii ◽

Stable Complex ◽

Mobility Shift ◽

Yeast Saccharomyces Cerevisiae ◽

Gel Mobility Shift Assay ◽

Two Factors ◽

Mobility Shift Assay ◽

Gel Mobility Shift ◽

Tfiid Complex

The previously described transcription factor IIA (TFIIA) protein fraction was separated into two factors that affect transcription, TFIIA and TFIIJ. TFIIA was found to have a stimulatory effect, and TFIIJ was found to be required for transcription. The requirement of TFIIJ was observed when bacterially produced purified human or yeast (Saccharomyces cerevisiae) TATA-binding protein (TBP) was used in lieu of the endogenous HeLa cell TFIID complex, suggesting that TFIIJ may be part of the TFIID complex. The stimulatory activity of TFIIA was found also to be dependent on the source of the TBP. Transcription reactions reconstituted with TFIID were stimulated by TFIIA; however, when human or yeast TBP was used instead of TFIID, TFIIA had no effect. TFIIA was found to interact with the TBP and was extensively purified by the use of affinity chromatography on columns containing immobilized recombinant yeast TBP. TFIIA is a heterotrimer composed of polypeptides of 34, 19, and 14 kDa. These three polypeptides were required to isolate, by using the gel mobility shift assay, a stable complex between TBP and the TATA box sequence.

Download Full-text

The CDK7-cycH-p36 complex of transcription factor IIH phosphorylates p53, enhancing its sequence-specific DNA binding activity in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.5923 ◽

1997 ◽

Vol 17 (10) ◽

pp. 5923-5934 ◽

Cited By ~ 94

Author(s):

H Lu ◽

R P Fisher ◽

P Bailey ◽

A J Levine

Keyword(s):

Transcription Factor ◽

Excision Repair ◽

Binding Activity ◽

Mobility Shift ◽

Trimeric Complex ◽

Dna Binding Activity ◽

Terminal Amino ◽

Gel Mobility Shift ◽

Kinase Phosphorylation

Phosphorylation is believed to be one of the mechanisms by which p53 becomes activated or stabilized in response to cellular stress. Previously, p53 was shown to interact with three components of transcription factor IIH (TFIIH): excision repair cross-complementing types 2 and 3 (ERCC2 and ERCC3) and p62. This communication demonstrates that p53 is phosphorylated by the TFIIH-associated kinase in vitro. The phosphorylation was found to be catalyzed by the highly purified kinase components of TFIIH, the CDK7-cycH-p36 trimeric complex. The phosphorylation sites were mapped to the C-terminal amino acids located between residues 311 and 393. Serines 371, 376, 378, and 392 may be the potential sites for this kinase. Phosphorylation of p53 by this kinase complex enhanced the ability of p53 to bind to the sequence-specific p53-responsive DNA element as shown by gel mobility shift assays. These results suggest that the CDK7-cycH-p36 trimeric complex of TFIIH may play a role in regulating p53 functions in cells.

Download Full-text

Gene expression noise can promote the fixation of beneficial mutations in fluctuating environments

PLoS Computational Biology ◽

10.1371/journal.pcbi.1007727 ◽

2020 ◽

Vol 16 (10) ◽

pp. e1007727 ◽

Cited By ~ 1

Author(s):

Michael Schmutzer ◽

Andreas Wagner

Keyword(s):

Transcription Factor ◽

Carbon Source ◽

Carbon Sources ◽

High Growth ◽

Population Bottlenecks ◽

Gene Expression Noise ◽

Fluctuating Environments ◽

Speed Up ◽

Available Carbon ◽

Beneficial Allele

Nongenetic phenotypic variation can either speed up or slow down adaptive evolution. We show that it can speed up evolution in environments where available carbon and energy sources change over time. To this end, we use an experimentally validated model of Escherichia coli growth on two alternative carbon sources, glucose and acetate. On the superior carbon source (glucose), all cells achieve high growth rates, while on the inferior carbon source (acetate) only a small fraction of the population manages to initiate growth. Consequently, populations experience a bottleneck when the environment changes from the superior to the inferior carbon source. Growth on the inferior carbon source depends on a circuit under the control of a transcription factor that is repressed in the presence of the superior carbon source. We show that noise in the expression of this transcription factor can increase the probability that cells start growing on the inferior carbon source. In doing so, it can decrease the severity of the bottleneck and increase mean population fitness whenever this fitness is low. A modest amount of noise can also enhance the fitness effects of a beneficial allele that increases the fraction of a population initiating growth on acetate. Additionally, noise can protect this allele from extinction, accelerate its spread, and increase its likelihood of going to fixation. Central to the adaptation-enhancing principle we identify is the ability of noise to mitigate population bottlenecks, particularly in environments that fluctuate periodically. Because such bottlenecks are frequent in fluctuating environments, and because periodically fluctuating environments themselves are common, this principle may apply to a broad range of environments and organisms.

Download Full-text

A common bacterial metabolite elicits prion-based bypass of glucose repression

eLife ◽

10.7554/elife.17978 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 26

Author(s):

David M Garcia ◽

David Dietrich ◽

Jon Clardy ◽

Daniel F Jarosz

Keyword(s):

Saccharomyces Cerevisiae ◽

Lactic Acid ◽

Glucose Metabolism ◽

Carbon Source ◽

Glucose Repression ◽

Carbon Sources ◽

Yeast Cells ◽

Genetic Element ◽

Yeast Saccharomyces Cerevisiae ◽

The Common

Robust preference for fermentative glucose metabolism has motivated domestication of the budding yeast Saccharomyces cerevisiae. This program can be circumvented by a protein-based genetic element, the [GAR+] prion, permitting simultaneous metabolism of glucose and other carbon sources. Diverse bacteria can elicit yeast cells to acquire [GAR+], although the molecular details of this interaction remain unknown. Here we identify the common bacterial metabolite lactic acid as a strong [GAR+] inducer. Transient exposure to lactic acid caused yeast cells to heritably circumvent glucose repression. This trait had the defining genetic properties of [GAR+], and did not require utilization of lactic acid as a carbon source. Lactic acid also induced [GAR+]-like epigenetic states in fungi that diverged from S. cerevisiae ~200 million years ago, and in which glucose repression evolved independently. To our knowledge, this is the first study to uncover a bacterial metabolite with the capacity to potently induce a prion.

Download Full-text

Activation of the NF-κB Pathway by Inflammatory Stimuli in Human Neutrophils

Blood ◽

10.1182/blood.v89.9.3421 ◽

1997 ◽

Vol 89 (9) ◽

pp. 3421-3433 ◽

Cited By ~ 221

Author(s):

Patrick P. McDonald ◽

Anette Bald ◽

Marco A. Cassatella

Keyword(s):

Cell Activation ◽

Protein Complexes ◽

Binding Activity ◽

Human Neutrophils ◽

Resting Cells ◽

Mobility Shift ◽

Dna Binding Activity ◽

Cytokines And Chemokines ◽

Inflammatory Stimuli ◽

Gel Mobility Shift

Abstract Activated neutrophils have the ability to upregulate the expression of many genes, in particular those encoding cytokines and chemokines, and to subsequently release the corresponding proteins. Although little is known to date concerning the regulation of gene transcription in neutrophils, it is noteworthy that many of these genes depend on the activation of transcription factors, such as NF-κB, for inducible expression. We therefore investigated whether NF-κB/Rel proteins are expressed in human neutrophils, as well as their fate on cell activation. We now report that dimers consisting of p50 NFκB1, p65 RelA, and/or c-Rel are present in neutrophils and that the greater part of these protein complexes is physically associated with cytoplasmic IκB-α in resting cells. Following neutrophil stimulation with proinflammatory agonists (such as lipopolysaccharide [LPS], tumor necrosis factor-α [TNF-α], and fMet-Leu-Phe) that induce the production of cytokines and chemokines in these cells, NF-κB/Rel proteins translocated to nuclear fractions, resulting in a transient induction of NF-κB DNA binding activity, as determined in gel mobility shift assays. The onset of both processes was found to be closely paralleled by, and dependent on, IκB-α degradation. Proinflammatory neutrophil stimuli also promoted the accumulation of IκB-α mRNA transcripts, resulting in the reexpression of the IκB-α protein. To our knowledge, this constitutes the first indication that NF-κB activation may underlie the action of proinflammatory stimuli towards human neutrophil gene expression and, as such, adds a new facet to our understanding of neutrophil biology.

Download Full-text