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Nucleic Acids Res. Mar 15, 2002; 30(6): 1306–1315.
PMCID: PMC101354
The CUP1 upstream repeated element renders CUP1 promoter activation insensitive to mutations in the RNA polymerase II transcription complex
Laura Badi and Alcide Barberisa
Institute of Molecular Biology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
aTo whom correspondence should be addressed at present address: ESBATech AG, Wagistrasse 21, CH-8952 Zürich-Schlieren, Switzerland. Tel: +41 1 635 3135; Fax: +41 1 635 6811; Email: Present address:Laura Badi, F.Hoffmann-La Roche Ltd, Pharmaceuticals Division, CH-4002 Basel, Switzerland
Received December 22, 2001; Accepted January 21, 2002.
Activation of transcription in eukaryotes requires the concerted action of numerous components of the RNA polymerase II transcriptional apparatus. The degree of dependence on many of these components varies from gene to gene and it is still largely unknown how the requirement for any particular component is determined at any given gene. We show that removal of Gal11 from the yeast transcription complex can affect activation from the CUP1 UAS in a manner dependent on its genomic context. Our results indicate a novel function for the CUP1 upstream repeated element (CURE) located upstream of the CUP1 UAS at the naturally multimerized CUP1 locus. The presence of CURE endowed the CUP1 UAS with a reduced susceptibility to the effects of deleting Gal11. Similar results were obtained with the Srb/mediator subunit Srb5. Restoration of activation from the CUP1 promoter to wild-type levels by the CURE correlated with changes in the accessibility of local chromatin to nucleases. The CURE sequence may serve to protect the stress-inducible CUP1 UAS–promoter elements against reduced activation that may result from crippled transcription complexes under stress conditions.
Genes transcribed by RNA polymerase II (pol II) are generally silent unless activated by sequence-specific DNA-binding proteins called transcriptional activators. The function of activators at specific promoters can be counteracted by another class of DNA-binding proteins known as gene-specific repressors. Despite the pivotal role of these two types of regulatory proteins in controlling the process of differential gene expression (1,2), it is believed that they alone cannot account for the high degree of specificity and coordination of gene regulation in any given eukaryotic cell at any time during the cell cycle or in rapid response to changes in the environment (35).
The eukaryotic transcriptional machinery is composed of a large number of proteins that include, in addition to the pol II core enzyme, the general transcription factors, the Srb/mediator complex, the Srb10 CDK complex, the SAGA complex and the Swi/Snf complex, among others (3). It is conceivable that the raison d’être of such a large and complicated apparatus in eukaryotes (as compared to the relatively simple transcriptional apparatus in bacteria) is linked to the complexity of the combinatorial control of gene expression in eukaryotic cells (4). Recent results have shown that the RNA pol II transcription complex participates in transcriptional regulation by providing different activities that can be targets of certain signaling pathways and ultimately modulate the action of the pol II core enzyme (3). Genome-wide analysis of gene expression in yeast cells carrying mutations in a variety of components of the pol II transcription complex has shown that the requirement for many components varies from gene to gene (5). For example, while Srb4 protein is required for full expression of ~93% of yeast genes, Srb5, a partner of Srb4 in the Srb/mediator complex, is only required at ~16% of genes. In addition to simple requirement, the role of some of these components in the expression of a subset of genes may also vary. For example, while the chromatin remodeling Swi/Snf complex is required for activation of ~2% of yeast genes, other genes accounting for ~4% of yeast genes appear to be negatively regulated by this complex and thus show elevated expression in its absence (5). An additional example of a factor with a potential dual function is provided by Gal11, a component of the Srb/mediator complex. Gal11 is needed for full expression of ~40% of yeast genes, while its deletion causes an increase in the expression of ~3% of yeast genes (6).
It is still largely unknown how the requirement for any particular component and its role is determined at any given gene. In this work we have tested the possible role of chromosomal context in determining the dependence on the transcription factor Gal11 for activation of transcription from the CUP1 promoter. CUP1 was chosen because activation of transcription from this promoter seems to be exceptionally unaffected by the lack of transcription factors that are otherwise required for the activation of most yeast genes. For example, conditional ablation of the essential and widely required Srb4 protein and the TFIIH kinase subunit Kin28 or partial deletion of the C-terminal domain of the pol II enzyme did not affect transcription from the CUP1 promoter (79). The CUP1 gene encodes a metallothionein protein and is induced by stress conditions such as high levels of copper (Cu2+) and heat shock (10,11). Inducible and rapid production of the Cup1 protein in yeast is achieved by a combination of two systems: amplification of CUP1 gene copy number in the genome and its transcriptional regulation. The CUP1 locus contains a cluster of tandemly repeated units, which can be found in as many as 15 linearly arrayed copies in some yeast strains (12). Transcription from the CUP1 promoter is activated by sequence-specific transcription factors that bind the CUP1 upstream activating sequence (CUP1 UAS) (13). This sequence contains at least three binding sites for the Cu2+-dependent transcriptional activator Ace1/Cup2 (10) and one binding site for the heat shock-responsive factor Hsf1 (14). The CUP1 UAS is necessary and sufficient for rapid and robust activation of transcription (10,14).
The results presented here show that removal of Gal11 from the transcription complex significantly affected activation of transcription from the CUP1 UAS in a manner that was dependent on the genomic context surrounding the reporter gene. Further analysis of these genomic regions revealed a novel role for a DNA sequence in modulating CUP1 activation. This sequence, which we named the CUP1 upstream repeated element (CURE), is naturally present upstream of the CUP1 UAS and is co-amplified with the entire CUP1 transcription unit at the CUP1 locus (see Fig. Fig.3B).3B). We have found that the presence of CURE restored the otherwise impaired activation of transcription by the downstream CUP1 UAS in strains lacking Gal11. This effect was not specific for a Gal11 deletion, since the CURE also restored activation from the CUP1 promoter in the absence of Srb5. We suggest that this novel regulatory element may ensure efficient CUP1 transcription when cells experience stress conditions that may impair the function of some transcription factors.
Figure 3
Figure 3
Organization of the CUP1 locus in the reporter strains BLY91 and BLY97. (A) Southern blot analysis; empty arrows indicate the bands originating from KpnI/ScaI double digested CUP1 repeat units which contain the CUP1 UAS::lacZ reporter in strains (more ...)
Strains and genetic techniques
Yeast genetic techniques and media were as described (15). Yeast transformation was performed by the lithium acetate procedure (16). The Saccharomyces cerevisiae strains used in this study are shown in Table Table1.1. Mutant strains JPY8 (gal11Δ) (17) and BLY30 (srb5Δ) (this study) were obtained from the wild-type strain JPY5 (17) through replacement of the chromosomal GAL11 or SRB5 coding sequences, respectively, with a TRP1 targeting cassette. All reporter strains listed in Table Table11 were originated through transformation of the strain of interest with the corresponding integrative reporter plasmids and subsequent selection on leu selective plates. For each of the integrative plasmids described in Table Table22 XbaI linearization allowed insertion of the reporters at the CUP1 locus, while BstEII digestion made the reporters suitable for insertion at the LEU2 locus. After transformation with the targeting construct and growth on selective plates, the correct identity of each strain was confirmed by PCR and Southern blot analysis of purified genomic DNA (18).
Table 1.
Table 1.
Yeast strains used in this study
Table 2.
Table 2.
Yeast plasmids
Plasmids pBL11 and pBL12 are based on YCplac33 and contain the genome-derived expression cassettes for the GAL11 and SRB5 genes, respectively (19). A list of the episomal and integrative reporter constructs used in this study is shown in Table Table2.2. These plasmids are based on the LEU2-marked centromeric vector pSAL1::lacZ (a kind gift of A. Covarrubias, Institute of Biotechnology, UNAM, Cuernavaca), which contains the Cu2+-inducible promoter region (CUP1 UAS and core promoter) from –394 to +37 bp relative to the first (+1) of the two major transcription start sites of the CUP1 gene fused to the lacZ ORF (20). pSAL1::lacZint is an integrating derivative of pSAL1::lacZ obtained after removal of the ARS/CEN sequence. Plasmid pSAL1–1441::lacZint contains the CUP1 upstream repeated element (–1441 to –395) inserted at its natural location upstream of the CUP1 UAS–promoter region of the lacZ reporter gene. The sequences of all pSAL1::lacZ derivatives were checked for correct reconstitution of the CUP1 regulatory regions. Detailed plasmid maps are available upon request.
RNA analysis
Yeast total RNA was extracted from exponentially growing cells exposed for 1 h to 400 µm CuSO4 as inducing agent for the CUP1 promoter. For northern blot analysis (21), equal amounts (2.5 µg) of total RNA were assayed to estimate the quantity of specific transcripts derived from the lacZ reporter and from the SNR6 endogenous gene, which is constitutively transcribed by RNA polymerase III. The 32P-labeled probes used in these experiments were the full-length lacZ, CUP1 or SNR6 genes. S1 mapping was performed as described (22) using the lacZ-specific 32P-end-labeled oligonucleotide 5′-cgcacttttcggccaatggtcttggtaattcctttgcgctagaattgaactcaggtacaatcacttcttctgaatgagatttagtcatatatcaa. Quantitation of the radioactive signals was performed with the ImageQuant package (Molecular Dynamics). In all types of RNA mapping experiments the quantity of total input RNA, which was determined by measuring absorption at 260 nm and by ethidium bromide staining of a gel upon electrophoresis, was kept constant and used to normalize the specific signal.
β-Galactosidase liquid culture assay
The β-galactosidase liquid assay was performed as previously described (23). Yeast cells were exposed to 400 µm CuSO4 for 2 h to induce lacZ expression from the CUP1 promoter before protein extraction.
Micrococcal nuclease and restriction endonuclease analysis of intact chromatin
Nuclei from exponentially growing yeast cell cultures were extracted as described (24). For micrococcal nuclease (MNase) analysis, nuclei were digested with MNase (4 and 7 U/reaction) for 5 min. Deproteinized DNA samples (naked DNA) were processed in parallel as a control. Nuclei derived from 50 mg of cells (wet weight) were processed in a restriction endonuclease digestion by incubation for 2 h with 10 U of the chosen restriction enzyme. In both experiments, the DNA was purified and used in a secondary digestion reaction with ScaI/PvuII. The fragments of interest were identified through Southern blotting using a 32P-labeled probe obtained by PCR amplification of the NotI–PvuII fragment mapping within the lacZ gene of pSAL1::lacZ (indirect end-labeling technique).
The negative effect of a GAL11 deletion on CUP1 promoter activation depends on the chromosomal context of the UAS within the CUP1 repeat cluster
Biochemical analysis of the Srb/mediator complex has indicated that Gal11 is part of the Gal11/Rgr1/Sin4 subcomplex. Proteins of this subcomplex, unlike other Srb/mediator complex components, have been reported to be relevant cofactors for CUP1 activation (8). Our original interest was to investigate the mechanisms of transcriptional activation from the CUP1 promoter in gal11Δ yeast cells. Measurement and comparison of the CUP1 mRNA levels from Gal11 and Gal11+ cells revealed that the absence of this transcription factor did not significantly affect expression of the endogenous CUP1 genes (Fig. (Fig.1A).1A). To restrict the analysis to promoter function, we chose to quantitate the effects of the gal11Δ mutation on transcription levels from a series of lacZ reporter gene constructs based on the CUP1 UAS–promoter elements. The use of such a reporter system also allowed us to distinguish between transcripts produced upon activation of the analyzed CUP1 UAS from those synthesized from the highly duplicated endogenous CUP1 locus.
Figure 1
Figure 1
Lack of Gal11 significantly affects Cu2+-induced expression of an episomal CUP1 UAS::lacZ reporter gene but not that of the endogenous CUP1 genes. (A) Northern blot analysis of the relative levels of CUP1 and SNR6 transcripts isolated from JPY8 (more ...)
The yeast strains JPY5 (wt) and JPY8 (gal11Δ), which are isogenic except for the GAL11 locus, were transformed with the episomal reporter construct pSAL1::lacZ (see Materials and Methods). Expression of this lacZ reporter gene is controlled by a span of DNA that contains the Cu2+-inducible CUP1 UAS and promoter sequences from –394 to +37 bp relative to the first (+1) of the two major CUP1 transcription start sites (13,25). Measurement of β-galactosidase activity in protein extracts from these transformed strains revealed that the gal11Δ mutant had severely impaired Cu2+-induced lacZ expression levels in comparison to the wild-type strain. The extent of reduction in β-galactosidase activity caused by the lack of Gal11 was ~80%, as shown in Figure Figure1B.1B. This decrease in β-galactosidase activity was the direct consequence of a diminished level of Cu2+-induced transcription from the CUP1 UAS::lacZ reporter gene, as shown by measuring the relative lacZ mRNA levels by northern blot assay (Fig. (Fig.1C).1C). We therefore conclude that Gal11 is necessary for efficient transcription of a CUP1 UAS::lacZ reporter gene located on an episomal plasmid.
We next determined whether Gal11 was also required when the CUP1 UAS::lacZ reporter construct described above was integrated at the natural CUP1 locus within the chromosome. In order to target integration of this reporter gene to the CUP1 chromosomal region, we transformed JPY8 cells (gal11Δ) with an integrative plasmid (pSAL1::lacZint) that had been linearized by digestion at the single XbaI site in the CUP1 UAS sequence (see Materials and Methods). Homologous recombination was expected to occur between the XbaI-digested construct and any one, or more than one, of the repeated CUP1 genes in the yeast genome. Nine yeast clones that had undergone integration of pSAL1::lacZint were selected and analyzed for a requirement for Gal11 for induced activation of the various integrated CUP1 UAS::lacZ reporter genes. When we compared the Cu2+-induced β-galactosidase activities on a plate by colorimetric assay in strains containing either plasmid pBL11 (expressing Gal11) or YCplac33 (the empty parental plasmid) we observed that full activation of the lacZ reporter gene significantly required the presence of Gal11 in two of the nine yeast strains, while relative expression of the reporter gene in seven strains did not seem to be diminished in the absence of Gal11 (data not shown). Moreover, the Gal11-sensitive strains showed a stronger maximal level of lacZ expression than the Gal11-insensitive strains. Figure Figure2A2A presents the results of the β-galactosidase assays performed with strains BLY91 (one of the Gal11-sensitive reporter strains) and BLY97 (one of the Gal11-insensitive reporter strains). These results show that the absence of Gal11 caused a 3-fold reduction in Cu2+-induced CUP1 UAS::lacZ reporter gene expression in the BLY91 strain, while it did not significantly affect expression of the reporter gene in strain BLY97. We also compared the levels of Cu2+-induced transcription in these two strains by performing S1 mapping analysis of the lacZ transcript (Fig. (Fig.2B).2B). The results of these experiments show that the variations in lacZ mRNA levels upon copper induction correlated with the changes in β-galactosidase activity described above. S1 mapping analysis of the lacZ transcript under non-inducing conditions showed that the CUP1 UAS::lacZ basal expression level in the two strains followed a similar pattern of Gal11 dependence as the induced level of transcription (Fig. (Fig.2B).2B). The results of the S1 mapping assays were confirmed by primer extension analysis of the lacZ mRNA (data not shown). The data from the S1 mapping and primer extension analyses of Cu2+-induced UAS::lacZ gene transcription were combined and the average reduction in transcription in gal11Δ cells was calculated as a percentage of expressed lacZ in Gal11 relative to Gal11+ cells. These values were 35.1 ± 0.3% for BLY91 and 87.4 ± 6.3% for BLY97.
Figure 2
Figure 2
The effect of gal11Δ on activation of the CUP1 promoter is dependent on the chromosomal context of the integrated CUP1 UAS::lacZ reporter. (A) β-Galactosidase assay to measure lacZ expression in strains BLY91 and BLY97, either containing (more ...)
The CUP1 UAS::lacZ reporter genes in strains BLY91 and BLY97 showed remarkable differences both in their dependence on Gal11 for maximal expression and in the absolute levels of lacZ transcripts that were synthesized. One possible cause for such differences might be the precise location within the CUP1 repeat cluster of the integrated reporter plasmids and the potentially variable copy number. Indeed, the highly duplicated CUP1 locus provides several potential sites for integration of the reporter construct. We therefore performed Southern blot analysis of reporter constructs integrated at the CUP1 locus of strains BLY91 and BLY97. Genomic DNA was prepared from both strains and double digested with KpnI and ScaI restriction nucleases. Upon electrophoresis and blotting, the resultant filters were probed with a radiolabeled CUP1 UAS fragment (see Materials and Methods). Figure Figure3A3A shows that the two strains differed in the pattern of integration of the pSAL1::lacZint reporter construct within the CUP1 locus. The deduced pattern of integration is outlined in Figure Figure3B.3B. Both the BLY91 and BLY97 strains had integrated the CUP1 UAS::lacZ reporter construct at the CUP1 locus, although the two strains displayed differences in copy number and chromosomal context of the integrated plasmid. The BLY91 strain had integrated at least two copies of the lacZ reporter construct that generated a cluster of direct repeats, while the BLY97 strain had inserted a single copy of the lacZ reporter at the 3′-most position available for homologous recombination within the highly duplicated CUP1 locus. This integration site could be identified as the CUP1 UAS belonging to the most downstream CUP1 transcription unit of the repeat (Fig. (Fig.3B).3B). Southern blot analysis of the other CUP1 reporter strains revealed that the additional Gal11-sensitive strain, similarly to BLY91, had also integrated at least two copies of the lacZ reporter construct as direct repeats, while the remaining six Gal11-insensitive strains had integrated single copies of the reporter plasmid in one of the endogenous CUP1 genes (data not shown).
These results, taken together, suggest that the organization of the reporter plasmid within the CUP1 locus and its copy number might influence the dependence on Gal11 of expression of the CUP1 UAS::lacZ gene.
The CUP1 upstream repeated element drastically reduces the dependence on Gal11 for transcriptional activation from the CUP1 UAS
The comparative analysis of the CUP1 UAS::lacZ chromosomal locations described above suggested that the different DNA regions present upstream of the respective CUP1 UAS::lacZ genes might determine the difference between these Gal11-sensitive and Gal11-insensitive reporter strains. Since the Gal11-sensitive BLY91 strain contains at least two copies of the pSAL1::lacZint construct in consecutive positions (Fig. (Fig.3B),3B), one or more copies of the reporter CUP1 UAS elements are located immediately downstream of plasmid DNA sequences. These CUP1 UAS elements are therefore located in the same sequence context as the CUP1 UAS of the episomal reporter construct described above (see Fig. Fig.1B).1B). In contrast, the CUP1 UAS of the single pSAL1::lacZint copy in the Gal11-insensitive BLY97 strain is located immediately downstream of the chromosomal DNA sequence that is naturally present upstream of the endogenous CUP1 genes. As indicated in Figure Figure3B,3B, these sequences correspond to the ORF denoted YHR054C, which is part of the CUP1 amplification unit, and which we refer to as the CURE. Thus, a plausible hypothesis is that the CURE sequence is necessary and sufficient to counteract the sensitivity of the CUP1 UAS to deletions in GAL11 (gal11Δ). To test this hypothesis, we reconstituted the different reporter gene structures observed in BLY91 and BLY97 elsewhere in the yeast genome. We engineered a new integrative reporter plasmid, pSAL1–1441::lacZint, by inserting the CURE sequence upstream of the CUP1 UAS in the original pSAL1::lacZint plasmid. This cloning procedure generated the same sequence as found up to 1441 bp upstream of the transcription initiation site of the BLY97 reporter gene. Both plasmids were linearized by digestion at a unique BstEII site within the LEU2 marker gene of the plasmid to target homologous integration at the LEU2 locus of the JPY8 (gal11Δ) strain (see Materials and Methods). The resulting strains were named BLY18, which carries the original pSAL1::lacZint reporter gene integrated at the LEU2 locus, and BLY22, which carries the pSAL1–1441::lacZint reporter construct (including the CURE sequence) at the same chromosomal site. We then measured the transcriptional activity of Cu2+-activated lacZ reporter genes in BLY18 and BLY22 cells transformed with either a Gal11-expressing or an empty plasmid. Reporter gene transcription was monitored by measuring β-galactosidase activity (Fig. (Fig.4A)4A) and by northern blot analysis (Fig. (Fig.4B).4B). This analysis revealed that full activation of the CUP1 UAS::lacZ reporter gene that lacks the CURE sequence was strongly dependent on Gal11 in strain BLY18. In contrast, in the BLY22 strain the presence of the CURE sequence upstream of the CUP1 UAS element almost completely abolished the dependence on Gal11 for full transcriptional activation (Fig. (Fig.4A4A and B). The CURE drastically reduced the Gal11 requirement without significantly affecting the maximal level of reporter gene expression observed in the presence of Gal11 (Fig. (Fig.4A4A and B).
Figure 4
Figure 4
Insertion of the CURE 5′ of the CUP1 UAS::lacZ reporter confers insensitivity to gal11Δ. (A) β-Galactosidase assay to assess the level of Cu2+-induced lacZ expression in BLY18 and BLY22 either containing Gal11 (Gal11+ (more ...)
The results of these experiments show that the CURE sequence, which is naturally located upstream of the CUP1 UAS, renders activation of transcription from this promoter almost completely insensitive to the lack of Gal11 in a manner that is independent of the surrounding chromosomal context of the CUP1 locus.
The CURE sequence renders activation of transcription from the CUP1 UAS–promoter region independent of Srb5
To follow up the work on Gal11, we tested whether the presence of the CURE sequence upstream of the CUP1 UAS could rescue a transcriptional defect caused by a mutation in another component of the transcription complex. We thus extended our analysis of the effect of the CURE sequence on activation of transcription from the CUP1 UAS–promoter region to a yeast strain deleted for Srb5 (srb5Δ). As in the case of GAL11, SRB5 is not an essential gene and therefore it is possible to use similar experimental conditions to test its requirement in gene transcription. Indeed, since a srb5Δ mutant strain is viable, no temperature shift is needed to inactivate the function of Srb5. We therefore constructed the mutant strain BLY29 with a complete deletion of the SRB5 locus (srb5Δ). We first measured Cu2+-induced β-galactosidase activities from the BLY29 strain transformed with the episomal reporter construct pSAL1::lacZ along with either the plasmid pBL12, expressing Srb5, or the empty parental plasmid YCplac33. The results of these experiments were similar to those obtained by monitoring the effect of deleting Gal11 on the expression of this reporter plasmid and indicated that Srb5 was also required for full activation of this episomal CUP1 UAS::lacZ reporter gene (data not shown). We subsequently integrated the pSAL1::lacZint and pSAL1–1441::lacZint reporter constructs at the LEU2 locus of BLY29 and, after Cu2+ induction, monitored expression of these reporter genes in the presence or absence of Srb5. The northern blot in Figure Figure55 shows that the negative effect of srb5Δ on CUP1 UAS-mediated activation of transcription persisted even when the CUP1 UAS::lacZ reporter gene was integrated at the LEU2 locus. Similarly to our previous observations with the gal11Δ mutant, the negative effect of srb5Δ on transcription was almost completely rescued by addition of the CURE sequence upstream of the CUP1 UAS without significantly affecting the maximal level of reporter gene expression observed in the presence of Srb5 protein (Fig. (Fig.55).
Figure 5
Figure 5
Low levels of CUP1 UAS activation in srb5Δ are rescued by addition of the CURE 5′ of the CUP1 UAS::lacZ reporter. Northern blot analysis showing the radioactive signals corresponding to lacZ and SNR6 transcripts detected for the reporter (more ...)
We conclude that the ‘protective’ effect of the CURE sequence on the efficiency of transcriptional activation from the CUP1 UAS–promoter elements is not specific for the gal11Δ mutation, but can be extended to the deletion of at least one other component of the Srb/mediator complex, i.e. Srb5.
Presence of the CURE sequence influences the local chromatin configuration of the CUP1 promoter region
The results presented so far show that the CURE sequence, which is present upstream of the CUP1 UAS, is able to rescue impaired transcription in strains carrying mutations in the transcription complex components Gal11 and Srb5. To gain insights into how the CURE sequence might influence the activity of the downstream CUP1 UAS–promoter elements, we performed a series of chromatin assays in which we measured the degree of DNA accessibility to nucleases of the CUP1 UAS–promoter region in the presence or absence of the upstream CURE sequence. The BLY18 strain, which lacks the CURE sequence, and the BLY22 strain, which bears the CURE upstream of the integrated CUP1 UAS::lacZ construct, were analyzed for their chromatin organization at the sites of the LEU2-integrated CUP1 UAS reporter genes. This analysis was performed using two different nuclease hypersensitivity assays on the intact chromatin.
We first analyzed the overall chromatin structure of the upstream regions of the CUP1 reporter genes by MNase digestion followed by gel electrophoresis and blotting. Indirect end-labeling of the blotted DNA fragments was utilized to identify chromatin-specific, and possibly CURE-dependent, MNase-hypersensitive sites. This assay was performed on chromatin obtained after the exposure of yeast cultures to copper ions to induce activation of the CUP1 enhancer–promoter region. Four relatively strong MNase-hypersensitive sites were found upstream of the BLY18 CUP1 UAS within a region of the reporter construct that lacked the CURE sequence. These sites were not, or only weakly, detectable within the CURE sequence at a similar distance upstream of the BLY22 CUP1 UAS (Fig. (Fig.6A,6A, denoted by the open circles on the left). Three moderately hypersensitive sites appeared instead only within the CURE sequence (Fig. (Fig.6A,6A, closed circles). The overall chromatin structure of the CUP1 UAS–promoter region did not appear to be affected by the presence or absence of the upstream CURE sequence with the exception of a possible change revealed by the diminished hypersensitivity to MNase of one site in the 5′-region of the CUP1 UAS (denoted by an asterisk in Fig. Fig.6A)6A) in the presence of the CURE.
Figure 6
Figure 6
Figure 6
Figure 6
Alterations in local DNA accessibility within the CUP1 regulatory regions. (A) MNase analysis. A schematic representation of the ScaI (S)–PvuII (P) genomic regions considered for analysis is shown beside the autoradiogram. Lanes in which the (more ...)
To more precisely map and quantitate changes in DNA accessibility of the CUP1 UAS–promoter region, chromatin from untreated (Fig. (Fig.6B)6B) and Cu2+-treated BLY18 and BLY22 cells (Fig. (Fig.6C)6C) was analyzed by restriction nuclease digestion. These experiments show that presence of the CURE sequence upstream of the CUP1 UAS region causes alterations in the accessibility of a set of restriction nuclease sites within the CUP1 UAS–promoter region. The pattern of this altered accessibility does not significantly change for chromatin prepared from Cu2+-treated (induced) as compared to untreated cells. However, as expected (26), the overall degree of accessibility clearly increased upon Cu2+ induction, with the exception of the MspA1I site that overlaps the Ace1-binding site (compare Fig. Fig.6B6B with C). Access to this DNA site for MspA1I is most likely blocked by the presence of Ace1, which has been shown to bind its recognition sequences only upon Cu2+ induction. The most dramatic changes in DNA accessibility occurred at positions that lie within the 5′-portion of the cloned CUP1 UAS region, which became less accessible in the presence of the upstream CURE sequence (Fig. (Fig.6B6B and C, HaeII, NdeI and DraI). These results are in agreement with those from the MNase assays (asterisk in Fig. Fig.6A).6A). The differences in accessibility between the two promoter configurations (plus or minus the CURE) were smaller or absent for positions immediately upstream and downstream of the Ace1-binding sites. However, for those sites where differences were detectable, the accessibility trend changed from diminished accessibility to slightly higher accessibility in the presence of the CURE sequence (Fig. (Fig.6B6B and C, from SpeI to MunI). We conclude that the presence of the CURE sequence upstream of the CUP1 UAS did not generate the formation of an obvious, specific nucleosome pattern. However, this sequence had an influence on the chromatin structure rendering the DNA region surrounding the binding sites for the activators AceI and Hsf1 slightly more accessible, while the more distal 5′-sequences became less accessible to restriction nuclease activity.
Our principal results can be summarized as follows. Efficient activation of transcription of a reporter gene bearing the CUP1 UAS–promoter region isolated from its natural chromosomal context required the function of the transcription factors Gal11 and Srb5, two components of the Srb/mediator complex of the yeast RNA pol II transcriptional machinery. Insertion of the CURE upstream of the isolated CUP1 UAS–promoter region drastically reduced the dependence on these transcription factors for efficient activation of transcription without affecting the maximal levels of gene expression. Reciprocally, integration of this reporter gene at the CUP1 locus such as to reconstitute the CURE sequence upstream of the CUP1 UAS–promoter region also rendered activation of transcription much less sensitive to the absence of Gal11 and Srb5. Thus, the degree of dependence on these transcription factors (i.e. their utilization) for full activation from the CUP1 UAS is determined by the CURE sequence, which is naturally present upstream of each multimerized CUP1 gene.
It has recently become clear that numerous components (transcription factors) of the pol II transcription complex are differentially utilized at different genes or classes of genes (5). The functional role of such differential utilization of transcription complex components has remained elusive. Young and collaborators have pointed out that the distinct expression signatures observed for a number of these components reveal a level of genome regulation that can be superimposed on that due to sequence-specific transcriptional activators and repressors (3,5). They have suggested that the expression of specific sets of genes might also be controlled by affecting the availability or function of specific components of the initiation complex, which, for example, may be targets of certain signal transduction pathways (5). It is also conceivable that some genes need to be rapidly and efficiently activated even in the absence of several transcription complex components, for example in response to drastic environmental changes that may impair the function of some transcription factors. CUP1 might be an example of such a gene.
What determines the requirement and the role of any particular component of the transcription complex at any given gene? It has been suggested that different sequence-specific regulatory proteins (activators or repressors) may work by interacting with different transcription factors and thus determine, at least in part, which components of the transcription complex are required for the set of genes they specifically regulate (3). For example, recent studies by Lis and collaborators, which were aimed at analysis of the mechanism whereby activation of CUP1 transcription is insensitive to the depletion of Srb4 from the transcription complex, have shown that the CUP1 UAS-binding activators Ace1 and Hsf1 need to establish functional contacts with only a subset of transcription factors, which includes Rgr1 but not Srb4, to work optimally. Thus, mutated transcription complexes that lacked the components not required by Ace1, such as Srb4, could still be efficiently engaged in the CUP1 activation process, while a mutated complex that lacked Rgr1 only poorly responded to these specific activators (8). Core promoter sequences may also contribute to the differential requirements for components of the transcription complex. Indeed, Green and collaborators have shown that core promoter sequences, not the activator-binding elements, determine whether a defined gene requires the TFIID subunit TAF145 for activation of transcription in yeast (27). Similarly, genomic sequences that are not activator-binding sites appear to determine the dependence on the Swi/Snf complex for activation of the HO gene in yeast. Indeed, it has been shown that substitution of the binding sites for the HO-specific Swi5 activator by those for the Gal4 activator maintains the dependence on Swi/Snf, even though Gal4 does not usually require Swi/Snf for its function, as, for example, activation of the natural Gal4 target gene GAL1 is completely Swi/Snf independent (28).
The results of our analysis of CUP1 activation show that the requirement for the transcription factors Gal11 and Srb5 at the CUP1 promoter is strongly dependent on the presence of the CURE sequence upstream of the CUP1 UAS. Thus, in this case the control for the requirement of these transcription factors at the CUP1 promoter is not exercised by the CUP1 UAS, but rather by a functionally novel sequence that lies outside the CUP1 UAS–promoter region.
What is the mechanism by which the CURE sequence can drastically reduce the dependence on Gal11 and Srb5, and perhaps other transcription factors, for efficient activation from the CUP1 UAS–promoter region? Possible mechanisms implicate the presence of binding sites for transcription factors within the CURE sequence that may facilitate activation from the downstream CUP1 UAS by providing additional contacts to help recruit a crippled pol II machinery that lacks Gal11 or Srb5. In our experiments, such a putative helping effect of CURE-binding factors is not detectable in the presence of a wild-type pol II machinery, as the level of transcription in this case is not influenced by the CURE. It is also possible that the CURE sequence itself or specialized DNA-binding factors induce a transcriptionally more active chromatin structure in its vicinity. In agreement with this hypothesis, Grunstein and collaborators have shown that CUP1 and several stress-inducible genes, like HSP26, HSP70 (SSA3) and HSP70 (SSA4), can be activated upon nucleosome depletion in a UAS-independent fashion (29). Our analysis of the in vivo chromatin structure of the CUP1 UAS–promoter region showed that although there was no remarkable alteration in the overall nucleosomal pattern, the presence of the CURE caused changes in the DNA accessibility of this region (see Fig. Fig.6).6). We are considering the possibility that the DNA accessibility pattern we observe near the CUP1 UAS might be a consequence of the recognition of sequences in the CURE region by trans-acting factors. The HMG1/2-like DNA architectural factors NHP6A/B are among the plausible candidates for such factors. Indeed, deletion of the genes encoding NHP6A/B was shown to negatively affect expression of a subset of genes, including CUP1 (30). These two proteins bind DNA without apparent sequence specificity and induce an overall bending of the helix through their HMG box DNA-binding domain (30,31). Thus, it is conceivable that the CURE sequence might provide a docking point for such architectural factors that would ultimately facilitate the accessibility of the CUP1 promoter to the transcription complex.
In this work we consider the function of the CURE sequence with respect to its influence on CUP1 transcription. This region has so far been referred to as ORF YHR054C. The sequence of YHR054C shows strong similarity with the 3′-part of the neighboring YHR056C, from which it probably originated through duplication of this gene fragment. Despite the presence of an ORF, the ability of YHR054C to encode a functional protein product remains questionable (32). Some hypotheses regarding the necessity for co-amplification of YHR054C and CUP1 have been put forward. The amplification of YHR054C might lead to some selective advantage other than copper resistance, as copper-resistant strains seem to maintain multiple copies of the locus even in the absence of this particular selective pressure (33). Our results suggest that the sequence of YHR054C, namely the CURE, might provide a selective advantage through its role on CUP1 transcription rather than by giving rise to an independent gene product. Indeed, the CURE sequence positively acts on activation of CUP1 by turning a promoter that is dependent on transcription factors such as Gal11 and Srb5 into a promoter that is less sensitive to their deletion. We propose that the presence of the CURE within the CUP1 amplification unit sequence has been maintained through evolution to protect the stress-inducible CUP1 UAS–promoter elements against reduced activation efficiency that would otherwise result from crippled transcription complexes under stress conditions.
We thank Drs A. Auf der Maur, L. R. Martin, M. Petrascheck and J. F. Roth for their helpful comments on the manuscript and F. Ochsenbein for artwork. We are also grateful to Dr  A. Covarrubias for providing materials and to Dr M. Bodmer-Glavas for help with the chromatin analysis. This work was supported by research grants from the Helmut Horten Foundation and the Swiss National Research Fund.
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