Reb1p-binding Site Research Articles

Regulation of Antisense Transcription by NuA4 Histone Acetyltransferase and Other Chromatin Regulatory Factors.

NuA4 histone lysine (K) acetyltransferase (KAT) promotes transcriptional initiation of TATA-binding protein (TBP)-associated factor (TAF)-dependent ribosomal protein genes. TAFs have also been recently found to enhance antisense transcription from the 3' end of the GAL10 coding sequence. However, it remains unknown whether, like sense transcription of the ribosomal protein genes, TAF-dependent antisense transcription of GAL10 also requires NuA4 KAT. Here, we show that NuA4 KAT associates with the GAL10 antisense transcription initiation site at the 3' end of the coding sequence. Such association of NuA4 KAT depends on the Reb1p-binding site that recruits Reb1p activator to the GAL10 antisense transcription initiation site. Targeted recruitment of NuA4 KAT to the GAL10 antisense transcription initiation site promotes GAL10 antisense transcription. Like NuA4 KAT, histone H3 K4/36 methyltransferases and histone H2B ubiquitin conjugase facilitate GAL10 antisense transcription, while the Swi/Snf and SAGA chromatin remodeling/modification factors are dispensable for antisense, but not sense, transcription of GAL10. Taken together, our results demonstrate for the first time the roles of NuA4 KAT and other chromatin regulatory factors in controlling antisense transcription, thus illuminating chromatin regulation of antisense transcription.

Transcription Factor Reb1p Regulates DGK1-encoded Diacylglycerol Kinase and Lipid Metabolism in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the DGK1-encoded diacylglycerol kinase catalyzes the CTP-dependent phosphorylation of diacylglycerol to form phosphatidate. This enzyme, in conjunction with PAH1-encoded phosphatidate phosphatase, controls the levels of phosphatidate and diacylglycerol for phospholipid synthesis, membrane growth, and lipid droplet formation. In this work, we showed that a functional level of diacylglycerol kinase is regulated by the Reb1p transcription factor. In the electrophoretic mobility shift assay, purified recombinant Reb1p was shown to specifically bind its consensus recognition sequence (CGGGTAA, -166 to -160) in the DGK1 promoter. Analysis of cells expressing the PDGK1-lacZ reporter gene showed that mutations (GT→TG) in the Reb1p-binding sequence caused an 8.6-fold reduction in β-galactosidase activity. The expression of DGK1(reb1), a DGK1 allele containing the Reb1p-binding site mutation, was greatly lower than that of the wild type allele, as indicated by analyses of DGK1 mRNA, Dgk1p, and diacylglycerol kinase activity. In the presence of cerulenin, an inhibitor of de novo fatty acid synthesis, the dgk1Δ mutant expressing DGK1(reb1) exhibited a significant defect in growth as well as in the synthesis of phospholipids from triacylglycerol mobilization. Unlike DGK1, the DGK1(reb1) expressed in the dgk1Δ pah1Δ mutant did not result in the nuclear/endoplasmic reticulum membrane expansion, which occurs in cells lacking phosphatidate phosphatase activity. Taken together, these results indicate that the Reb1p-mediated regulation of diacylglycerol kinase plays a major role in its in vivo functions in lipid metabolism.

Yeast Dgk1p diacylglycerol kinase is regulated by the transcription factor Reb1p

In the yeast Saccharomyces cerevisiae, Dgk1p diacylglycerol (DAG) kinase catalyzes the CTP‐dependent phosphorylation of DAG to form phosphatidate. The enzyme activity is required for phospholipid synthesis during growth resumption from stationary phase when de novo fatty acid synthesis is inhibited. Growth resumption is accompanied by the induction of DAG kinase activity, which implicates a potential transcriptional regulation of DGK1. The promoter of DGK1 contains a consensus sequence TTACCCG (−167 to −173) for the binding of the transcription factor Reb1p. Using purified recombinant His6‐Reb1p, we performed electrophoretic mobility shift assays to examine the interaction of Reb1p with the potential Reb1p binding site. The interaction of His6‐Reb1p with a radiolabeled oligonucleotide probe was dependent on the concentration of His6‐Reb1p, and non‐labeled probe competed with this interaction. Mutations in the Reb1p binding site reduced this interaction. The DGK1 gene with a mutation in the Reb1p binding site (DGK1reb1) was expressed in dgk1Δ mutant cells and caused a low constitutive level of DGK1 mRNA, Dgk1p, and DAG kinase activity. In addition, the dgk1Δ cells expressing DGK1reb1 showed a defect in growth resumption from stationary phase and the mobilization of triacylglycerol for membrane phospholipid synthesis was compromised. Supported by NIH grant GM‐28140.

Reb1p-dependent DNA Bending Effects Nucleosome Positioning and Constitutive Transcription at the Yeast Profilin Promoter

The molecular basis of constitutive gene activation is largely unknown. The yeast profilin gene (PFY1), encoding a housekeeping component of the actin cytoskeleton, is constitutively transcribed at a moderate level. The PFY1 promoter dispenses with classical transactivators and a consensus TATA box; however, it contains a canonic site for the abundant multifunctional nuclear factor rDNA enhancer-binding protein (Reb1p) combined with a dA.dT element. Reb1p binds specifically in vitro. Mutation of this site reduces PFY1 expression to about 35%. A nucleosome-free gap of about 190 bp is centered at the genomic Reb1p binding site in vivo and spans the presumptive core promoter and transcriptional initiation sites. Nucleosomes at the border of the gap are positioned. Mutation of the Reb1p motif in the genomic PFY1 promoter abolishes nucleosome positioning, fills the gap with a non-positioned nucleosome, and reduces transcription by a factor of 3. From permutation studies we conclude that Reb1p induces a strong bend into the DNA. Phasing analyses indicate that it is directed toward the major groove. The data suggest that Reb1p plays an architectural role on DNA and that Reb1p-dependent DNA bending leads to a DNA conformation that is incompatible with packaging into nucleosomes and concomitantly facilitates constitutive transcription. In the absence of other transcription activators, Reb1p excludes nucleosomes and moderately stimulates transcription by distorting DNA.

Permanent Nucleosome Exclusion from the Gal4p-inducible YeastGCY1 Promoter

The promoter of the galactose-inducible yeast GCY1 gene allows high rates of basal transcription and is kept free of nucleosomes regardless of growth conditions. The general regulatory factor, Reb1p, as well as the nucleotide sequence of a single Gal4p-binding site, structurally cooperate to exclude nucleosomes from about 480 bp of DNA that spans the UAS(GAL), the Reb1p-binding site, the TATA-box, and the transcriptional initiation sites. Gal4p, which induces transcription of GCY1 about 25-fold in the presence of galactose, is not required for the alteration in chromatin configuration in the promoter upstream region since the hypersensitive site is unchanged when Gal4p is inactive or absent. As soon as either the Reb1p-binding site or the UAS(GAL) or both are mutated, nucleosomes slip into the promoter of GCY1 paralleled by a reduction of basal transcription activity to about 30% in either single mutant and to <10% in the double mutant. In the mutant of the Reb1p-binding site, induction by galactose/Gal4p restores a nucleosome-free state to an extent resembling the GCY1 wild-type promoter, showing that, in principle, activated Gal4p can exclude nucleosomes on its own. Northern blots of GCY1 transcripts confirm that Reb1p modulates basal transcription and has little influence on the galactose-induced state.

Neither Reb1p nor Poly(dA·dT) Elements Are Responsible for the Highly Specific Chromatin Organization at the ILV1Promoter

Analysis of the chromatin structure at the yeast ILV1 locus revealed highly positioned nucleosomes covering the entire locus except for a hypersensitive site in the promoter region. All previously identified cis-acting elements required for GCN4-independent ILV1 basal level transcription, including a binding site for the REB1 protein (Reb1p), and a poly(dA*dT) element (26 As out of 32 nucleotides) situated 15 base pairs downstream of the Reb1p-binding site, reside within this hypersensitive site. The existence of a second A*T-rich element (25 As out of 33 nucleotides) present six base pairs upstream of the Reb1p-binding site, suggested that nucleosome exclusion from the hypersensitive site in the ILV1 promoter region might be dictated by synergistic action of the two poly(dA*dT) elements. Replacing one or both of them had, however, no effect on the chromatin structure of the ILV1 promoter, although drastically reduced basal transcription. Similarly, deletion of the Reb1p-binding site, albeit affecting ILV1 expression, had no detectable effect on chromatin at the ILV1 promoter. The absence of a good correlation between effects of these elements on gene activity and on chromatin structure at the ILV1 promoter indicates that the chromatin organization present at the ILV1 promoter is independent of the known regulatory elements and most likely dictated directly by the DNA sequence.

Cohabitation of insulators and silencing elements in yeast subtelomeric regions.

In budding yeast, the telomeric DNA is flanked by a combination of two subtelomeric repetitive sequences, the X and Y' elements. We have investigated the influence of these sequences on telomeric silencing. The telomere-proximal portion of either X or Y' dampened silencing when located between the telomere and the reporter gene. These elements were named STARs, for subtelomeric anti-silencing regions. STARs can also counteract silencer-driven repression at the mating-type HML locus. When two STARs bracket a reporter gene, its expression is no longer influenced by surrounding silencing elements, although these are still active on a second reporter gene. In addition, an intervening STAR uncouples the silencing of neighboring genes. STARs thus display the hallmarks of insulators. Protection from silencing is recapitulated by multimerized oligonucleotides representing Tbf1p- and Reb1p-binding sites, as found in STARs. In contrast, sequences located more centromere proximal in X and Y' elements reinforce silencing. They can promote silencing downstream of an insulated expressed domain. Overall, our results suggest that the silencing emanating from telomeres can be propagated in a discontinuous manner via a series of subtelomeric relay elements.

The chromatin structure of the GAL1 promoter forms independently of Reb1p in Saccharomyces cerevisiae.

Positive and negative regulation of the GAL1 promoter of the yeast Saccharomyces cerevisiae results from a network of interactions between transcription factors and chromatin. In this study we used footprinting procedures to characterize these interactions in vivo. DNase I analysis of the GAL1 upstream activating sequence (UAS(GAL1/10)) showed expected Gal4 activator protein binding during growth in galactose, and also revealed binding of the Reb1 protein (Reb1p) during growth in glucose. In addition, we mapped to nucleotide resolution a positioned nucleosome that, in the inactive promoter, packages DNA between the UAS(GAL1/10) and the GAL1 TATA sequence, leaving both of these elements nucleosome free. The nucleosome footprint was lost when the promoter was activated. Surprisingly, mutation of the Reb1p binding site had no effect on nucleosome positioning or on the kinetics or extent of activation or repression of either the GAL1 or GAL10 promoters under any of the conditions assayed.

Positive and negative autoregulation of REB1 transcription in Saccharomyces cerevisiae.

Reb1p is a DNA binding protein of Saccharomyces cerevisiae that has been implicated in the activation of transcription by polymerase (Pol) II, in the termination of transcription by Pol I, and in the organization of nucleosomes. Studies of the transcriptional control of the REB1 gene have led us to identify three Reb1p binding sites in the 5' region of the its gene, termed A, B, and C, at positions -110, -80, and +30 with respect to transcription initiation. In vitro, Reb1p binds to the three sites with the relative affinity of A >/= C > B. Kinetic parameters suggest that when both A and C sites are present on the same DNA molecule, the C site may recruit Reb1p for the A site. In vivo the A and B sites each contribute to the transcription activity of REB1 in roughly additive fashion. Mutation of both A and B sites abolishes transcription. On the other hand, the C site is a negative element, reducing transcription by 40%. In cells overexpressing Reb1p, the C site reduces transcription by more than 80%. This effect can be transposed to another transcription unit, demonstrating that the effect of Reb1p binding at the C site does not depend on interaction with upstream Reb1p molecules. Relocation of the C site to a position 105 bp downstream of the transcription initiation site abolishes its effect, suggesting that it does not act as a conventional attenuator of transcription. We conclude that binding of Reb1p at the C site hinders formation of the initiation complex. This arrangement of Reb1p binding sites provides a positive and negative mechanism to autoregulate the expression of REB1. Such an arrangement could serve to dampen the inevitable fluctuation in Rep1p levels caused by the intermittent presence of its mRNA within an individual cell.

Datin, a yeast poly(dA:dT)-binding protein, behaves as an activator of the wild-type ILV1 promoter and interacts synergistically with Reb1p.

A cis-acting element required for GCN4-independent basal-level transcription of ILV1 was previously identified in our laboratories as a binding site for the REB1 protein (Reb1p). Further deletion analysis of the ILV1 promoter region identified a second element also required for GCN4-independent basal-level ILV1 expression. This second element is an A.T-rich tract (26 As out of 32 nucleotides) situated 15 bp downstream of the Reb1p-binding site. Deletion of both the Reblp site and the poly(dA:dT) element totally eliminates basal activity of the ILV1 promoter. We show that the two elements act synergistically to control ILV1 expression and that the synergistic effect is distance dependent. We demonstrate that (i) datin (Dat1p), the only known poly (dA:dT)-binding protein in yeast, specifically binds to the ILV1 poly(dA:dT) element in vitro; (ii) Dat1p functions as a trans-activating factor in the ILV1 context; and (iii) the synergistic activation observed in vivo between the Reb1p site and the poly(dA:dT) element depends on the presence of the structural gene for Dat1p, DAT1.

Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Since DNA replication and transcription often temporally and spatially overlap each other, the impact of one process on the other is of considerable interest. We have reported previously that transcription is impeded at the replication termini of Escherichia coli and Bacillus subtilis in a polar mode and that, when transcription is allowed to invade a replication terminus from the permissive direction, arrest of replication fork at the terminus is abrogated. In the present report, we have addressed four significant questions pertaining to the mechanism of transcription impedance by the replication terminator proteins. Is transcription arrested at the replication terminus or does RNA polymerase dissociate from the DNA causing authentic transcription termination? How does transcription cause abrogation of replication fork arrest at the terminus? Are the points of arrest of the replication fork and transcription the same or are these different? Are eukaryotic RNA polymerases also arrested at prokaryotic replication termini? Our results show that replication terminator proteins of E. coli and B. subtilis arrest but do not terminate transcription. Passage of an RNA transcript through the replication terminus causes the dissociation of the terminator protein from the terminus DNA, thus causing abrogation of replication fork arrest. DNA and RNA chain elongation are arrested at different locations on the terminator sites. Finally, although bacterial replication terminator proteins blocked yeast RNA polymerases in a polar fashion, a yeast transcription terminator protein (Reb1p) was unable to block T7 RNA polymerase and E. coli DnaB helicase.

The general regulatory factor Reb1p controls basal, but not Gal4p-mediated, transcription of the GCY1 gene in yeast.

Expression of the gene GCY1 in Saccharomyces cerevisiae is induced by about 25-fold in the presence of galactose as a result of activation by Gal4p. In contrast to other Gal4p-regulated genes, such as GAL1 or GAL10, GCY1 is transcribed at a relatively high basal level. We have analysed the basis of this behaviour and have found that, in addition to a UASGAL, a binding site for the general regulatory factor Reb1p is localized 100 bp upstream of the TATA sequence and about 140 bp 3' to the UASGAL. Reb1p binds to this site with low affinity. Reb1p, an abundant, multifunctional DNA-binding protein in yeast, acts as a weak transcriptional activator in the control regions of several genes encoding unrelated functions. The action of Reb1p is assumed to be strongly position dependent. In the control region of GCY1. Reb1p acts independently of position and stimulates basal expression of GCY1 about threefold, whereas Gal4p-mediated activation is not influenced significantly. Promoter-proximal insertion of an additional Reb1p recognition site enhances basal transcription only marginally, but can largely compensate for deletion of the natural Reb1p-binding site. Either an Abf1p- or a Rap1p-binding site can substitute for the Reb1p recognition sequence, indicating that these general regulatory factors fulfill related functions in basal transcription, without affecting Gal4p-mediated activation. In addition to Reb1p, the sequence of the Gal4p-binding site influences basal transcription. This effect is independent of the Gal4 protein, as it operates in a gal4 mutant background as well. This finding suggests that the nucleotide sequence of the UASGAL in the GCY1 promoter has intrinsic properties, presumably a particular DNA structure, that influence basal transcription and act synergistically with Reb1p.

Similar upstream regulatory elements of genes that encode the two largest subunits of RNA polymerase II in Saccharomyces cerevisiae.

We have determined the location of cis-acting elements that are important for the expression of RPO21 and RPO22, genes that encode the two largest subunits of RNA polymerase II (RNAPII) in Saccharomyces cerevisiae. A series of 5'-end deletions and nucleotide substitutions in the upstream regions of RPO21 and RPO22 were tested for their effect on the expression of lacZ fusions of these genes. Deletion of sequences from -723 to -693 in RPO21, which disrupted two Reb1p-binding sites and an Abf1p-binding site, resulted in a 10-fold decrease in expression. A T-rich region downstream of these sites was also important for expression. Deletion of sequences from -437 to -392 in the RPO22-upstream, which resulted in a 30-fold decrease in expression, indicated that the Reb1p- and Abf1p-binding sites in this region were important for RPO22 expression, as was a T-rich sequence immediately downstream of these sites. The RPO21 and RPO22 upstream regions were capable of interacting in vitro (gel-mobility-shift assays) with Reb1p and Abf1p. The similarities in the type and organization of elements in the upstream regions of RPO21 and RPO22 suggest that expression of these genes may be regulated coordinately.

The multifunctional transcription factors Abf1p, Rap1p and Reb1p are required for full transcriptional activation of the chromosomal PGK gene in Saccharomyces cerevisiae.

We have identified two new transcription factor binding sites upstream of the previously defined UAS within the phosphoglycerate kinase (PGK) gene promoter in Saccharomyces cerevisiae. These sites are bound in vitro by the multifunctional factors Cpf1p and Reb1p. We have generated targeted deletions of Rap1p, Abf1p and Reb1p binding sites in the promoter of the chromosomal copy of the PGK gene. Northern blot analysis confirmed that most PGK promoter activity is mediated through the Rap1p binding site. However, significant effects are also mediated through both the Reb1p and Abf1p sites. In contrast, when the promoter is present on a high-copy-number plasmid, both the Abf1p and Reb1p sites play no role in transcriptional activation. The role of Cpf1p was examined using a cpf1 null strain. Cpf1p was found to have little if any, effect on activation of either the chromosomal or plasmid-borne PGK gene.

The Release Element of the Yeast Polymerase I Transcription Terminator Can Function Independently of Reb1p

The Saccharomyces cerevisiae polymerase I (polI) transcription terminator utilizes a DNA-binding protein (Reb1p) as part of a signal that causes the polymerase to pause prior to release from the template. To study the release element of the terminator, independent of the Reb1p pause signal, we have replaced the Reb1p binding site with the binding site for the lac repressor, which acts as a self-contained heterologous pause signal for polI. Release efficiency is maximal when the lac repressor causes polI to pause in exactly the same position that Reb1p would have caused it to pause, suggesting that polI must be precisely positioned for transcript release to occur. Mutational analysis shows that the release element is a region rich in T residues which codes for the extreme 3' end of the transcript and which has no apparent ability to form hairpins when transcribed into RNA. We discuss possible mechanisms whereby this polI release element might function.

Importance of General Regulatory Factors Rap1p, Abf1p and Reb1p for the Activation of Yeast Fatty Acid Synthase Genes FAS1 and FAS2

The fatty acid synthase genes FAS1 and FAS2 of the yeast Saccharomyces cerevisiae are under transcriptional control of pathway-specific regulators of phospholipid biosynthesis. However, site-directed mutagenesis of the respective cis-acting elements upstream of FAS1 and FAS2 revealed that additional sequences activating both genes must exist. A deletion analysis of the FAS1 promoter lacking the previously characterized inositol/choline-responsive-element motif defined a region (nucleotides -760 to -850) responsible for most of the remaining activation potency. Gel-retardation experiments and in-vitro DNase footprint studies proved the binding of the general regulatory factors Rap1p, Abf1p and Reb1p to this FAS1 upstream region. Mutation of the respective binding sites led to a drop of gene activation to 8% of the wild-type level. Similarly, we also demonstrated the presence of a Reb1p-binding site upstream of FAS2 and its importance for gene activation. Thus, in addition to the previously characterized FAS-binding factor 1 interacting with the inositol/choline-responsive-element motif, a second motif common to the promoter regions of both FAS genes could be identified. Transcription of yeast fatty acid synthase genes is therefore subjected to both the pathway-specific control affecting genes of phospholipid biosynthesis and to the activation by general transcription factors allowing a sufficiently high level of constitutive gene expression.

Defining the sequence specificity of the Saccharomyces cerevisiae DNA binding protein REB1p by selecting binding sites from random-sequence oligonucleotides.

We have used a random selection protocol to define the consensus and range of binding sites for the Saccharomyces cerevisiae REB1 protein. Thirty-five elements were sequenced which bound specifically to a GST-REB1p fusion protein coupled to glutathione-Sepharose under conditions in which more than 99.9% of the random sequences were not retained. Twenty-two of the elements contained the core sequence CGGGTRR, with all but one of the remaining elements containing only one deviation from the core. Of the core sequence, the only residues that were absolutely conserved were the three consecutive G residues. Statistical analysis of a nucleotide-use matrix suggested that the REB1p binding site also extends into flanking sequences with the optimal sequence for REB1p binding being GNGCCGGGGTAACNC. There was a positive correlation between the ability of the sites to bind in vitro and activate transcription in vivo; however, the presence of non-conformants suggests that the binding site may contribute more to transcriptional activation than simply allowing protein binding. Interestingly, one of the REB1p binding elements had a DNAse 1 footprint appreciably longer than other elements with similar affinity. Analysis of its sequence indicated the potential for a second REB1p binding site on the opposite strand. This suggests that two closely positioned low-affinity sites can function together as a highly active site. In addition, database searches with some of the randomly defined REB1p binding sites suggest that related elements are commonly found within 'TATA-less' promoters.