Transposable element-mediated enhancement of gene expression in Saccharomyces cerevisiae involves sequence-specific binding of a trans-acting factor

In our studies on the regulation of adjacent-gene expression by Ty sequences, we demonstrated that a single-base-pair change (T-A----C-G) in the epsilon sequence of Ty917-derived elements is primarily responsible for enhancement of beta-galactosidase expression from lacZ fusion plasmids. Using an electrophoretic gel mobility assay, we showed that the same base pair transition is required for binding of a trans-acting factor, TyBF, from crude cell extracts in vitro. We identified the site of TyBF binding and determined the guanine nucleotide contact sites required for TyBF interaction. We propose that TyBF binding to cis-acting Ty2 sequences activates adjacent-gene transcription.

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A Ty1 cell-type-specific regulatory sequence is a recognition element for a constitutive binding factor.

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5299 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5299-5309 ◽

Cited By ~ 17

Author(s):

M Company ◽

B Errede

Keyword(s):

Gene Expression ◽

Binding Site ◽

Reporter Gene ◽

Base Pair ◽

Adjacent Gene ◽

Cell Type ◽

Cis Acting ◽

Cell Extracts ◽

Diploid Cells ◽

Constitutive Factor

Ty transposable-element insertion mutations of Saccharomyces cerevisiae can cause cell-type-dependent activation of adjacent-gene expression. Several cis-acting regulatory regions within Ty1 are responsible for the effect of Ty1 on adjacent-gene expression. One of these is the block II sequence that was defined by its homology to mammalian enhancers and to the yeast a1-alpha 2 control site. Tandem copies of a 57-base-pair region encompassing block II caused an additive increase in expression of the CYC7 reporter gene in the absence of other Ty1 sequences. The activation of gene expression by the multiple repeats was abolished in a/alpha diploid cells. A specific complex between a constitutive factor in whole-cell extracts and the DNA regulatory element was observed. The protein-binding site for the constitutive factor coincided with the block II element. Base-pair substitutions within the binding site abolished the ability of the block II element to function as a component of the Ty1 activator and to form the factor-DNA complex. The correlation between complex formation and reporter gene expression indicates that factor binding to the cis-acting element is essential for this element to function as a component of the Ty1 activator.

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A Ty1 cell-type-specific regulatory sequence is a recognition element for a constitutive binding factor

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5299-5309.1988 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5299-5309

Author(s):

M Company ◽

B Errede

Keyword(s):

Gene Expression ◽

Binding Site ◽

Reporter Gene ◽

Base Pair ◽

Adjacent Gene ◽

Cell Type ◽

Cis Acting ◽

Cell Extracts ◽

Diploid Cells ◽

Constitutive Factor

Ty transposable-element insertion mutations of Saccharomyces cerevisiae can cause cell-type-dependent activation of adjacent-gene expression. Several cis-acting regulatory regions within Ty1 are responsible for the effect of Ty1 on adjacent-gene expression. One of these is the block II sequence that was defined by its homology to mammalian enhancers and to the yeast a1-alpha 2 control site. Tandem copies of a 57-base-pair region encompassing block II caused an additive increase in expression of the CYC7 reporter gene in the absence of other Ty1 sequences. The activation of gene expression by the multiple repeats was abolished in a/alpha diploid cells. A specific complex between a constitutive factor in whole-cell extracts and the DNA regulatory element was observed. The protein-binding site for the constitutive factor coincided with the block II element. Base-pair substitutions within the binding site abolished the ability of the block II element to function as a component of the Ty1 activator and to form the factor-DNA complex. The correlation between complex formation and reporter gene expression indicates that factor binding to the cis-acting element is essential for this element to function as a component of the Ty1 activator.

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cis- and trans-acting regulatory elements of the yeast URA3 promoter

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5257-5270.1990 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5257-5270

Author(s):

A Roy ◽

F Exinger ◽

R Losson

Keyword(s):

Specific Binding ◽

Point Mutations ◽

Regulatory Elements ◽

Cis Acting ◽

Cell Extracts ◽

Dnase I Footprinting ◽

Pair Sequence ◽

Dyad Symmetry

Expression of the yeast pyrimidine biosynthetic gene, URA3, is induced three- to fivefold in response to uracil starvation, and this regulation is mediated by the transcriptional activator PPR1 (pyrimidine pathway regulator 1). In this study, we have analyzed the regulatory elements of the URA3 promoter by DNase I footprinting, using partially purified yeast cell extracts, by deletion mutagenesis, and by 5'-end mapping of RNA transcripts. Two DNA-binding activities have been detected, and at least four distinct cis-acting regions have been identified. A region rich in poly(dA-dT) serves as an upstream promoter element necessary for the basal level of URA3 expression. A 16-base-pair sequence with dyad symmetry acts acts as a uracil-controlled upstream activating site (UASURA) and shows a specific binding only with cell extracts from strains overproducing PPR1. This in vitro binding does not require dihydroorotic acid, the physiological inducer of URA3. The TATA region appears to be composed of two functionally distinct (constitutive and regulatory) elements. Two G + A-rich regions surrounding this TATA box bind an unidentified factor called GA-binding factor. The 5' copy, GA1, is involved in PPR1 induction and overlaps the constitutive TATA region. The 3' region, GA2, is necessary for maximal expression. Neither of these GA sequences acts as a UAS in a CYC1-lacZ context. The promoters of the unlinked but coordinately regulated URA1 and URA4 genes contain highly conserved copies of the UASURA sequence, which prompted us to investigate the effects of many point mutations within this UASURA sequence on PPR1-dependent binding. In this way, we have identified the most important residues of this binding site and found that a nonsymmetrical change of these bases is sufficient to prevent the specific binding and to suppress the UASURA activity in vivo. In addition, we showed that UASURA contains a constitutive activating element which can stimulate transcription from a heterologous promoter independently of dihydroorotic acid and PPR1.

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cis- and trans-acting regulatory elements of the yeast URA3 promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5257 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5257-5270 ◽

Cited By ~ 47

Author(s):

A Roy ◽

F Exinger ◽

R Losson

Keyword(s):

Specific Binding ◽

Point Mutations ◽

Regulatory Elements ◽

Cis Acting ◽

Cell Extracts ◽

Dnase I Footprinting ◽

Pair Sequence ◽

Dyad Symmetry

Expression of the yeast pyrimidine biosynthetic gene, URA3, is induced three- to fivefold in response to uracil starvation, and this regulation is mediated by the transcriptional activator PPR1 (pyrimidine pathway regulator 1). In this study, we have analyzed the regulatory elements of the URA3 promoter by DNase I footprinting, using partially purified yeast cell extracts, by deletion mutagenesis, and by 5'-end mapping of RNA transcripts. Two DNA-binding activities have been detected, and at least four distinct cis-acting regions have been identified. A region rich in poly(dA-dT) serves as an upstream promoter element necessary for the basal level of URA3 expression. A 16-base-pair sequence with dyad symmetry acts acts as a uracil-controlled upstream activating site (UASURA) and shows a specific binding only with cell extracts from strains overproducing PPR1. This in vitro binding does not require dihydroorotic acid, the physiological inducer of URA3. The TATA region appears to be composed of two functionally distinct (constitutive and regulatory) elements. Two G + A-rich regions surrounding this TATA box bind an unidentified factor called GA-binding factor. The 5' copy, GA1, is involved in PPR1 induction and overlaps the constitutive TATA region. The 3' region, GA2, is necessary for maximal expression. Neither of these GA sequences acts as a UAS in a CYC1-lacZ context. The promoters of the unlinked but coordinately regulated URA1 and URA4 genes contain highly conserved copies of the UASURA sequence, which prompted us to investigate the effects of many point mutations within this UASURA sequence on PPR1-dependent binding. In this way, we have identified the most important residues of this binding site and found that a nonsymmetrical change of these bases is sufficient to prevent the specific binding and to suppress the UASURA activity in vivo. In addition, we showed that UASURA contains a constitutive activating element which can stimulate transcription from a heterologous promoter independently of dihydroorotic acid and PPR1.

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Protein phosphatase 2A potentiates activity of promoters containing AP-1-binding elements

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2104-2112.1993 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2104-2112

Author(s):

A S Alberts ◽

T Deng ◽

A Lin ◽

J L Meinkoth ◽

A Schönthal ◽

...

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Protein Phosphatase ◽

Signal Transduction Pathways ◽

Marker Genes ◽

Direct Mechanism ◽

Phosphatase 2A ◽

Beta Galactosidase ◽

Regulatory Sites

The involvement of serine/threonine protein phosphatases in signaling pathways which modulate the activity of the transcription factor AP-1 was examined. Purified protein phosphatase types 1 (PP1) and 2A (PP2A) were microinjected into cell lines containing stably transfected lacZ marker genes under the control of an enhancer recognized by AP-1. Microinjection of PP2A potentiated serum-stimulated beta-galactosidase expression from the AP-1-regulated promoter. Similarly, transient expression of the PP2A catalytic subunit with c-Jun resulted in a synergistic transactivation of an AP-1-regulated reporter gene. PP2A, but not PP1, potentiated serum-induced c-Jun expression, which has been previously shown to be autoregulated by AP-1 itself. Consistent with these results, PP2A dephosphorylated c-Jun on negative regulatory sites in vitro, suggesting one possible direct mechanism for the effects of PP2A on AP-1 activity. Microinjection of PP2A had no effect on cyclic AMP (cAMP)-induced expression of a reporter gene containing a cAMP-regulated promoter, while PP1 injection abolished cAMP-induced gene expression. Taken together, these results suggest a specific role for PP2A in signal transduction pathways that regulate AP-1 activity and c-Jun expression.

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Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex

Biochemistry Research International ◽

10.1155/2018/6406372 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9

Author(s):

Gwang Sik Kim ◽

Young Chul Lee

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Transcriptional Activation ◽

Reporter Gene Expression ◽

Temperature Sensitive ◽

Internal Deletion ◽

Cell Extracts ◽

Evolutionarily Conserved

Med6 protein (Med6p) is a hallmark component of evolutionarily conserved Mediator complexes, and the genuine role of Med6p in Mediator functions remains elusive. For the functional analysis ofSaccharomyces cerevisiaeMed6p (scMed6p), we generated a series of scMed6p mutants harboring a small internal deletion. Genetic analysis of these mutants revealed that three regions (amino acids 33–42 (Δ2), 125–134 (Δ5), and 157–166 (Δ6)) of scMed6p are required for cell viability and are located at highly conserved regions of Med6 homologs. Notably, the Med6p-Δ2 mutant was barely detectable in whole-cell extracts and purified Mediator, suggesting a loss of Mediator association and concurrent rapid degradation. Consistent with this, the recombinant forms of Med6p having these mutations partially (Δ2) restore or fail (Δ5 and Δ6) to restore in vitro transcriptional defects caused by temperature-sensitivemed6mutation. In an artificial recruitment assay, Mediator containing a LexA-fused wild-type Med6p or Med6p-Δ5 was recruited to thelexAoperator region with TBP and activated reporter gene expression. However, the recruitment of Mediator containing LexA-Med6p-Δ6 tolexAoperator region resulted in neither TBP recruitment nor reporter gene expression. This result demonstrates a pivotal role of Med6p in the postrecruitment function of Mediator, which is essential for transcriptional activation by Mediator.

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Cis-acting enhancement of RNA polymerase III gene expression in vitro

MGG Molecular & General Genetics ◽

10.1007/bf00259409 ◽

1990 ◽

Vol 221 (3) ◽

pp. 435-442 ◽

Cited By ~ 1

Author(s):

Jo Ann M. Sekiguchi ◽

Eric B. Kmiec

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Iii ◽

Cis Acting ◽

Polymerase Iii

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Control of yeast gene expression by transposable elements: maximum expression requires a functional Ty activator sequence and a defective Ty promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4009 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4009-4017 ◽

Cited By ~ 9

Author(s):

L R Coney ◽

G S Roeder

Keyword(s):

Gene Expression ◽

Adjacent Gene ◽

Multiple Sequence ◽

Sequence Element ◽

Cis Acting ◽

Ty Elements ◽

The Relationship ◽

Maximum Expression ◽

Modest Effect

Integration of a transposable element adjacent to a gene frequently results in an alteration in expression of the nearby gene. The purpose of our experiments was to identify cis-acting sequences within a yeast transposon (Ty) that are important for expression of the adjacent gene. The role of these sequences in Ty transcription was also analyzed in order to examine the relationship between Ty and adjacent gene expression. Three naturally occurring Ty elements located at the HIS4 locus were examined. These Ty elements differed by multiple sequence changes and had different effects on HIS4 expression. To determine which sequences were important to Ty and HIS4 expression, Ty::lacZ and Ty::HIS4::lacZ fusion genes were constructed and analyzed. Results of these experiments indicated that a sequence element is present in the Ty epsilon region that is necessary for HIS4 expression but which has only a modest effect on Ty transcription. Additionally, a mutation in the Ty promoter region decreased Ty transcription and increased HIS4 expression. The opposite effects of this mutation on Ty and adjacent gene expression were probably caused by promoter competition.

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