The EMBO Journal vol.9 no.8 pp.2543-2553, 1990
Regulation of a yeast HSP70 gene by a cAMP responsive transcriptional control element
William R.Boorstein1,3 Elizabeth A.Craig1,2 'Molecular and Cellular Biology Program, and 2Department of Physiological Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA
3Present address: HHMI, Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, USA Communicated by M.Ashburner
HSP7O genes exhibit complex regulation in response to stress and a variety of cellular and developmental events. The SSA3 HSP70 gene of Saccharomyces cerevisiae is activated at the transcriptional level under conditions of nutrient limitation. Analysis of deletions revealed that cisacting DNA sequences present immediately upstream and downstream of the previously identified heat shock elements (UASHS) mediate this regulation. A 35 bp region of SSA3, distinct from UASHS, contains sequences capable of activating a heterologous promoter following the diauxic shift and in the stationary phase of the yeast life cycle; this region has been designated an upstream activating sequence, UASPDS. Expression driven by UASpDS is regulated by the RAS/cAMIP pathway. Reduced cAMP dependent protein kinase activity results in UASpDS dependent activation of the SSA3 promoter while constitutive cAMP dependent protein kinase activity prevents UASPDS mediated transcription, even under growth conditions that would normally result in full activation. Although the heat shock element alone exhibits no UAS activity under conditions in which UASpDS promotes transcription, UASHS interacts positively with UASpDS to mediate high levels of SSA3 transcription in response to nutrient limitation and lowered intracellular cAMP concentration. This interaction is independent of the precise spacing and relative orientation of the two elements. Key words: cAMP dependent protein kinase/heat shock/ HSP701Saccharomyces cerevisiae/transcriptional regulation Introduction Saccharomyces cerevisiae is able to grow under diverse conditions by the coordinated regulation of growth rate, metabolic activity, and the cell cycle. cAMP plays a central role in both the control and integration of these processes (reviewed by Pringle and Hartwell, 1981; and by Hanes et al., 1986). The role of cAMP in allowing yeast to adapt and grow on fruit and vegetable matter in nature can be analyzed in the laboratory under conditions of limiting quantities of glucose. S. cerevisiae cultured on glucose and other fermentable carbon sources exhibits two distinct growth phases (reviewed by Kappeli, 1986 and by Lagunas, 1986). The initial phase of growth is exponential and rapid; cells meet their energy requirements primarily by fermentation. ©C
Oxford University Press
When the fermentable carbon source is depleted, growth stops for a period of several hours and then resumes at a much slower rate, this time fueled by aerobic metabolism of the products of fermentation, principally ethanol. Eventually the medium can no longer support growth and the cells become arrested in a resistant 'stationary' state. The transition to respiratory metabolism following the logarithmic phase is called the diauxic shift. Many changes occur at the diauxic shift (reviewed by Pringle and Hartwell, 1981). The generation time increases. Glycolytic enzymes are inactivated, catabolite repression is relieved (reviewed by Entian, 1986), and cells begin to accumulate storage carbohydrates. The cell wall becomes more resistant to degradative enzymes. When the cells can no longer continue to divide, they become arrested in an unbudded state at START A, analogous to the Go state of the mammalian cell cycle. Cells arrested in Go are metabolically active and express a small, characteristic set of proteins including a subset of heat shock proteins (Iida and Yahara, 1984; Boucherie, 1985). Go arrested cells are constitutively thermotolerant (SchenbergFrascino and Moustacchi, 1972; Plesset et al., 1987), a phenotype believed to be related to the expression of these heat shock proteins. They are also able to withstand extended periods of nutrient deprivation. The dramatic changes in growth rate and metabolic activities at the diauxic shift and Go cell cycle arrest are believed to be regulated primarily by decreases in cAMP dependent protein kinase (cAPK) activity via reduction of intracellular cAMP levels (Figure 7). cAMP levels are controlled by modulation of adenylate cyclase and cAMP-phosphodiesterase activities. The yeast RAS genes encode GTP binding proteins that activate adenylate cyclase (encoded by CRY] = CDC35). When RAS is activated, intracellular cAMP levels increase, causing the negative regulatory subunit of cAPK (encoded by BCYI) to dissociate from the catalytic kinase subunits (encoded by TPKJ, 2, and 3 ), thus activating them (reviewed by Gibbs and Marshall, 1989). The changes in growth and metabolism that occur in wild type cells at the diauxic shift do not occur- in mutant cells in which cAPK activity has become constitutive because of disruption of the BCYJ gene or because of mutations in which RAS is constitutively activated (Toda et al., 1985, 1987; Cannon and Tatchell, 1987). Cells with constitutive cAPK activity stop growing when glucose is exhausted because they are unable to utilize non-fermentable carbon sources. beyl cells are also unable to respond to this nutrient limitation by normal cell cycle arrest at Go; instead, starved bcyl cells are often multibudded and uninucleate. Consistent with the improper response to nutrient limitation, bcyl strains are highly sensitive to starvation, i.e. they become inviable rapidly upon cessation of growth (Matsumoto et al., 1983). In addition, beyl cells are heat shock sensitive, in contrast to the thermotqlerant phenotype of Go arrested cells. Conversely, cells with artificially depleted cAMP exhibit a 2543
W.R.Boorstein and E.A.Craig
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Fig. 1. Cell density (open symbols) and specific activity of (3-galactosidase (filled symbols) from SSA3 promoter constructs during growth to stationary phase in rich glucose medium at 23°C. Diamonds represent transformants of the SSA3-lacZ fusion gene plasmid pWB204A-583 (see Figure 2, top line) and circles represent transformants of the SSA3 UASHS/CYCJ hybrid promoter fused to lacZ (pWB226; see Figure 4) under identical conditions. The time at which the diauxic shift occurred is indicated by the plateau in the growth curve (see arrow); glucose was depleted at this time (data not shown). The cell density plot is from pWB204A-583 transformant, but is virtually indistinguishable from the corresponding pWB226 plot. (-galactosidase activities depicted here are from a typical experiment. Both hybrid genes are inducible by heat shock treatment (Boorstein and Craig, 1990).
nutrient deprivation phenotype, even in rich media. Strains carrying a mutant adenylate cyclase gene, cyr], require exogenous cAMP for growth (Matsumoto et al., 1982a). Removal of cAMP causes cyri cells to arrest in Go regardless of the nutrient conditions. Cells arrested in this manner express Go specific proteins and are thermotolerant (lida and Yahara, 1984; Plesset et al., 1987). The completion of START A, which requires a functional adenylate cyclase gene and appears to be correlated with a transient increase in cAMP, signals the first step of commitment to a new round of cell division (reviewed by Pringle and Hartwell, 1981 and by Boynton and Whitfield, 1983). START A events do not occur until critical steps of the previous cell cycle are completed and unless sufficient nutrients are available to complete a subsequent cycle. cAMP is a good candidate for coordinating metabolic and cell cycle controls as cAMP levels are believed to be depressed at the nutrient sensitive control point of the cell cycle and lowered cAMP is a major component of the intracellular signal of suboptimal nutrient conditions. In these studies we examined the regulation of SSA3, the only one of the eight members of the yeast multigene HSP70 family whose mRNA levels increase at the diauxic shift. The increase in SSA3 mRNA abundance, like other changes that occur at the diauxic shift and in response to nutrient limitation, appears to be dependent upon a decrease in cAPK activity; SSA3 mRNA does not accumulate in bcyl cells under these conditions and can be induced experimentally by decreasing intracellular cAMP levels in cyr] strains growing on exogenously supplied cAMP (WernerWashburne et al., 1989). SSA3 was also previously shown to exhibit a classic transcriptional heat shock response. Basal SSA3 expression is very low in rapidly growing cells in glucose media. SSA3 mRNA becomes abundant within 15 min of shifting such cultures from 230 to 39°C (Werner-Washburne et al., 1989). A 32 bp fragment of the SSA3 promoter, UASHS, mediates this response by positive control of transcription (Boorstein and Craig, 1990). The same upstream activating sequence also drives a low level of basal expression. In the absence of UASHS, other portions of the SSA3 promoter are virtually unable to drive either basal or heat inducible expression. UASHS exhibits extensive homology to the eukaryotic HSE consensus sequence and is presumed to 2544
function by interaction with the heat shock transcriptional factor, HSF. HSF from non-stressed yeast cells has been shown to bind HSE sequences both in vivo and in vitro (Sorger and Pelham, 1987; Jakobsen and Pelham, 1988). Stress activation via HSEs occurs post-translationally and is believed to involve phosphorylation of HSF (Zimarino and Wu, 1987; Sorger and Pelham, 1988). In this paper we demonstrate that SSA3 is activated at the transcriptional level at the diauxic shift, in stationary phase arrested cells, and in respect to decreased levels of cAPK activity. We identify a cis-acting regulatory element of the SSA3 promoter which governs this pattern of regulation and investigate its interaction with UASHS in controlling transcription of SSA3. Results Localization of sequences controlling post diauxic shift expression To study SSA3 regulation following the diauxic shift, we utilized an SSA3 -lacZ fusion gene that was previously shown to be activated like the native SSA3 gene in response to heat shock. Transcription from the hybrid gene initiates at the proper sites within the SSA3 sequences and ,B-galactosidase activity accurately reflects hybrid transcript levels (Boorstein and Craig, 1990). Strains containing the hybrid gene were grown in rich medium at a constant temperature of 23°C. The pronounced slowing of growth (Figure 1) and the exhaustion of glucose from the medium (data not shown) were used as indicators of the diauxic shift. The onset of 3-galactosidase expression was coincident with the diauxic shift. 3-galactosidase activity continued to increase to high levels as cultures progressed to stationary phase (Figure 1). To determine whether common promoter elements regulate SSA3 expression in response to growth state and to heat shock, we measured the activity after the diauxic shift of the previously defined heat inducible UAS (Boorstein and Craig, 1990). The SSA3-lacZ fusion gene was expressed after heat shock (Figure 2) and in stationary phase (Figure 1) similar to native SSA3 expression. However, UASHS alone was unable to mediate expression from the heterologous yeast CYCI promoter after the diauxic shift at 23°C (Figure 1). UASHS was only able to promote transcription in response to heat shock (Boorstein and Craig, 1990 and Figure 4).
HSP70 cAMP responsive transcriptional element B-galactosidase activity induction logarithmic PDS (PDS - log) -583
= =
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Fig. 2. 5' deletion analysis of the SSA3 promoter indicating basal, PDS and heat shock expression. The black lines represent SSA3 sequences that remain following the deletion. Sequences are numbered with respect to the most abundant 5' end of the hybrid mRNA (+1). Amino acid 3 (+ 34) of SSA3 is fused to lacZ. White diamonds represent matches to the dyad consensus heat shock element sequence, CnnGAAnnTTCnnG (Pelham, 1985). Only the HSEs centered at - 156 were previously shown to be functional. Black triangles represent repeated DNA sequences (Table III). Features of the SSA3 promoter region between -236 and -124 are detailed in Figure 3. The putative TATA element is indicated by the boxed 'T'. Vector sequences upstream of the deletion endpoints are identical in all constructs. (-galactosidase activity is presented in Miller units. (*) Induction is presented as the magnitude of increase in 3-galactosidase activity, calculated by subtracting the activity in logarithmic phase cultures from that in stationary (PDS) phase cultures at 23°C. Presentation of inductions as magnitudes facilitates identification of sequences regulating PDS expression by minimizing the impact of small variations in low basal activity. Such variations can result in large changes of induction ratios. -236
_,D_C3 ~_-124
GGCGCi~TATCATCAAACGTATTTGACTTGATGCCTATGGhGTTTGGTCCTTAATTAGGGATCGCTGTGGAAAGTTATAGAATATTACACGAAGCAGCCACAAGGGTGACCG A CGT T A C CG LTC AC, ,GTCGA.C Ml 43 M2 heme||IsosaesaseusuIsuIHIssausem *- PDS OlIgo -* uemeuIeml§||@||IstuIumnumeuou*em* AAAAAA
AA
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*-
AAA
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A
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AA
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_175 -166
Fig. 3. The DNA sequence of SSA3 from -236 through -124 (the 113mer) indicating sequence features and details of constructions used in these studies. Triangles represent matches to -GAA- modules that are positioned and oriented appropriately to comprise HSF binding sites (Amin et al., 1988; Perisic et al., 1989); black triangles mark exact matches, grey triangles mark sequences with a single base mismatch, and the white triangle is included to indicate appropriate spacing between flanking GAA elements. Arrows indicate positions of inverted repeats; adjacent repeats differ from each other by a single base pair. The boxed sequence resembles the consensus GRFI binding site: 5'-RRTGNNTGGGTKY-3' (R = A or G, Y = T or C, K = T or G) (Buchman et al., 1988a); this region also matches, in 11 of 13 positions, the exact GRFI binding site in the RNR2 (ribonucleotide reductase small subunit) promoter (Hurd and Roberts, 1989). The nucleotide substitutions in the Ml, M2, M3 and hse- mutations are shown immediately below the native sequence. The position and extent of the 35 bp PDS and 32 bp HSE oligonucleotides, used to test UAS function in the CYCI -lacZ vector (pWB220), are indicated with respect to the DNA sequence by the striped bars. Solid black bars at the bottom of the figure indicate the positions of 5' deletion endpoints and SSA3 sequences remaining in these constructions.
These results demonstrate that sequences capable of mediating post diauxic shift (PDS) activation are located within 617 nucleotides upstream of the third codon of the SSA3 gene, and that UASHS is not sufficient to drive this pattern of gene expression. 5' deletions of the SSA3 -lacZ hybrid gene were assayed to identify sequences that control the expression of SSA3 following the diauxic shift (Figure 2). Removal of increasing amounts of SSA3 sequence between -200 and -111 resulted in progressive loss of PDS f-galactosidase activity, suggesting that a single small discrete DNA element is not solely responsible for PDS expression of SSA3. Rather, the results indicate that multiple or extended sequences that exhibit some degree of functional redundancy mediate PDS expression. Deletions of two short adjacent regions immediately upstream of UASHs each decrease PDS activity of the fusion gene by two thirds, defining a domain of primary importance to this mode of regulation between -200 and -166. Loss of the upstream part of this domain (-200 to - 175) results in the largest decrease in PDS expression, > 200 3-galactosidase units, with no effect on
heat shock expression. This region includes a sequence that resembles GRFI (also referred to as RAP], and TUF) binding sites, which are known to function in both activation and repression of transcription of yeast genes (Brand et al., 1987; Shore and Nasmyth, 1987; Buchman et al., 1988a; Kimmerly et al., 1988). It also includes three repeated sequences that are present in alternating orientations (see Figure 3); inverted repeats often characterize binding sites for transcriptional regulatory factors (e.g. Vinson et al., 1989). This deletion removes all but a portion of the downstream repeat. Deletion of the downstream portion of this domain (- 175 to -166) decreased heat shock as well as PDS expression, although the effect on PDS activity was twice as large. The phenotype of this mutation may result from removal of part of the HSE, from removal of a flanking regulatory sequence, or both. It is not known whether the partial HSE match that extends into this region comprises part of the functional HSE (see Figure 3 and Boorstein and
Craig, 1990). Additional sequences, both upstream and downstream of the region discussed above, were also identified as import2545
W.R Boorstein and E.A.Craig
ant for regulation of SSA3 expression (Figure 2). A-124, which lacks UASHs, is induced 2-fold in stationary phase. Further 5' deletion to -111 abolishes PDS induction indicating that sequences involved in PDS expression are also found downstream of UASHS. A 7 bp exact match to one of the three sequences repeated between -195 and -174 is found in this 13 bp downstream region (see Table HI). The presence of sequences that may function to modulate SSA3 expression negatively in stationary phase is suggested by the increase of 180 units of expression upon deletion of upstream sequences between -583 and -200. Sequences in addition to UASHS that affect basal and heat inducible SSA3 expression in exponentially growing cells are also revealed in the 5' deletion analysis shown in Figure 2; these have been previously discussed (Boorstein and Craig, 1990). Identification of UASpDs To confirm that PDS regulation of SSA3 occurs at the level of transcription and to identify upstream activating sequences that mediate PDS activation, we tested short regions of the SSA3 promoter (Figure 3) for their ability to activate a CYCI -lacZ translational fusion gene (Figure 4). The hybrid gene includes CYCI 'downstream' promoter elements, TATA and initiation sequences; expression is entirely dependent upon activation by sequences with UAS function cloned into a restriction site immediately upstream of the CYCI sequences. A 113 bp fragment from -236 to -124, containing most of the region shown by 5' deletion analysis to contribute to PDS expression and including UASHS, was
able to activate transcription from the CYC] promoter almost 30-fold, both upon heat shock and after the diauxic shift. To attempt to separate heat shock and post diauxic shift regulatory elements, a synthetic 35 bp oligonucleotide that was identical to sequences immediately upstream of the UASHS (and within the 1 13mer sequence) was tested. This short sequence mediated a 60-fold induction of the reporter gene in stationary phase. Basal levels of transcription driven by the 35mer were 10-fold lower than those of the 113mer; this difference in basal activity contributed to the higher induction ratio of the shorter fragment. In contrast, the short oligonucleotide caused only a marginal (0.3 unit) increase in expression in response to heat shock. The 35mer construct mediated PDS expression in both orientations. The ability of the 35 bp SSA3 fragment to drive transcription of the CYC] -lacZ fusion gene, from a site slightly further (28 bp) than its native location with respect to the transcriptional initiation region, demonstrates at least a limited degree of position independence of this transcriptional regulatory element. These results demonstrate that the region between -205 and -171 contains an upstream activating sequence. We designate this element 'UASPDS'. The results of analysis of 5' deletion mutations and the fact that the SSA3 113mer drives 5-fold higher levels of transcription than the component 35 bp oligonucleotide in stationary phase cells suggest that the HSEs may contribute to SSA3 expression following the diauxic shift even though UASHS is not sufficient to drive expression in stationary phase. We performed two sets of experiments to test this
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Fig. 4. UAS activity of SSA3 promoter region fragments under PDS and heat shock conditions. The CYCI -lacZ vector lacking a UAS is depicted XhoI cloning site (-178) immediately upstream of the CYCI sequence; arrowheads at the end of the lines indicate orientation of the inserts with respect to their native orientation in the SSA3 promoter. '113mer' extends from the A-236 endpoint to the BstEll site at -124. White triangles represent -GAA- modules that comprise functional heat shock elements. The three small black arrows in the PDS region represent inverted repeat sequences. The striped rectangular box denotes similarity to GRFI binding sites. The SSA3 113mer DNA sequence and features depicted here are presented in detail in Figure 3 along with the 32 bp HSE and 35 bp PDS oligonucleotides. 'HSE + PDS' and 'HSE + PDS(rev)' include two SSA3 oligonucleotides immediately adjacent to each other, upstream of the CYC] proximal promoter elements; the relative positions of the two sequences is reversed with respect to their order in the SSA3 promoter. The plasmids utilized here differ from the SSA3-lacZ fusion constructs (Figure 2) only in the regions depicted. Units of activity and induction are presented as described in Figure 2. on the top line. Wide black lines represent sequences from the promoter region of SSA3 that are inserted into the
2546
HSP70 cAMP responsive transcriptional element
hypothesis. First, we cloned both UASPDS- and UASHScontaining oligonucleotides into the UAS test vector. Mutual enhancement of the activity of each element by the presence of the other resulted; 3-galactosidase activity in stationary phase was increased 2- to 5-fold and heat induction was increased 2.5-fold compared with that of constructs containing UASpDS and UASHS alone, respectively (Figure 4). The apparent positive interaction between the UAS activities of these sequences was observed despite the fact that UASPDS was downstream of UASHS, unlike the native orientation; furthermore, the enhancement was observed when UASpDs was present in either orientation downstream of UASHS. In complementary experiments, disruption of UASHS in the 113mer fragment resulted in a promoter with 86% less PDS activity than that of the non-mutant 113mer construct (Figure 5A). PDS expression from the hse1 13mer mutant was comparable with that from the UASPDS oligonucleotide (Figure 4). Similar results (71 % reduction of PDS levels) were obtained when the same hse- mutation was analyzed in the context of the 'complete' SSA3 promoter (in pWB204A-236) (Figure SB). However, expression from both UASHS disrupted 113mer and A-236 constructs was induced at least 85-fold after the diauxic shift over their respective basal levels, further demonstrating that UASHS is not essential for PDS activation. Due to the role of UASHS in basal expression (Boorstein and Craig, 1990), the
decreased basal activity from constructs lacking UASHS resulted in an increased induction ratio, although the magnitude of the induction was substantially reduced. Since evidence for interaction between UASHS and UASPDS was obtained in three different relative orientations and spacings, the initial observations suggesting HSE involvement in PDS expression could not have resulted from partially overlapping, but not functionally interacting, regulatory sequences. We previously demonstrated that UASHS drives a low basal level of transcription (Boorstein and Craig, 1990). Here we observed that both the 113mer, and the UASHS plus UASPDS synthetic constructs result in lower basal f-galactosidase activity than do constructs driven by UASHS alone. The UASPDS region appears to repress transcription driven by the HSE during logarithmic phase growth under nonstressed conditions. Mutational analysis of the UASpDs region To define the PDS control element more precisely and to assess potential function of the GRFI-like recognition site and the inverted repeat sequences we constructed clustered multiple substitution mutations within the 35 bp that were demonstrated to exhibit UASPDS activity. The DNA sequences of the 113 bp fragment and mutant derivatives
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Regulation of a yeast HSP70 gene by a cAMP responsive transcriptional control element
William R.Boorstein1,3 Elizabeth A.Craig1,2 'Molecular and Cellular Biology Program, and 2Department of Physiological Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA
3Present address: HHMI, Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, USA Communicated by M.Ashburner
HSP7O genes exhibit complex regulation in response to stress and a variety of cellular and developmental events. The SSA3 HSP70 gene of Saccharomyces cerevisiae is activated at the transcriptional level under conditions of nutrient limitation. Analysis of deletions revealed that cisacting DNA sequences present immediately upstream and downstream of the previously identified heat shock elements (UASHS) mediate this regulation. A 35 bp region of SSA3, distinct from UASHS, contains sequences capable of activating a heterologous promoter following the diauxic shift and in the stationary phase of the yeast life cycle; this region has been designated an upstream activating sequence, UASPDS. Expression driven by UASpDS is regulated by the RAS/cAMIP pathway. Reduced cAMP dependent protein kinase activity results in UASpDS dependent activation of the SSA3 promoter while constitutive cAMP dependent protein kinase activity prevents UASPDS mediated transcription, even under growth conditions that would normally result in full activation. Although the heat shock element alone exhibits no UAS activity under conditions in which UASpDS promotes transcription, UASHS interacts positively with UASpDS to mediate high levels of SSA3 transcription in response to nutrient limitation and lowered intracellular cAMP concentration. This interaction is independent of the precise spacing and relative orientation of the two elements. Key words: cAMP dependent protein kinase/heat shock/ HSP701Saccharomyces cerevisiae/transcriptional regulation Introduction Saccharomyces cerevisiae is able to grow under diverse conditions by the coordinated regulation of growth rate, metabolic activity, and the cell cycle. cAMP plays a central role in both the control and integration of these processes (reviewed by Pringle and Hartwell, 1981; and by Hanes et al., 1986). The role of cAMP in allowing yeast to adapt and grow on fruit and vegetable matter in nature can be analyzed in the laboratory under conditions of limiting quantities of glucose. S. cerevisiae cultured on glucose and other fermentable carbon sources exhibits two distinct growth phases (reviewed by Kappeli, 1986 and by Lagunas, 1986). The initial phase of growth is exponential and rapid; cells meet their energy requirements primarily by fermentation. ©C
Oxford University Press
When the fermentable carbon source is depleted, growth stops for a period of several hours and then resumes at a much slower rate, this time fueled by aerobic metabolism of the products of fermentation, principally ethanol. Eventually the medium can no longer support growth and the cells become arrested in a resistant 'stationary' state. The transition to respiratory metabolism following the logarithmic phase is called the diauxic shift. Many changes occur at the diauxic shift (reviewed by Pringle and Hartwell, 1981). The generation time increases. Glycolytic enzymes are inactivated, catabolite repression is relieved (reviewed by Entian, 1986), and cells begin to accumulate storage carbohydrates. The cell wall becomes more resistant to degradative enzymes. When the cells can no longer continue to divide, they become arrested in an unbudded state at START A, analogous to the Go state of the mammalian cell cycle. Cells arrested in Go are metabolically active and express a small, characteristic set of proteins including a subset of heat shock proteins (Iida and Yahara, 1984; Boucherie, 1985). Go arrested cells are constitutively thermotolerant (SchenbergFrascino and Moustacchi, 1972; Plesset et al., 1987), a phenotype believed to be related to the expression of these heat shock proteins. They are also able to withstand extended periods of nutrient deprivation. The dramatic changes in growth rate and metabolic activities at the diauxic shift and Go cell cycle arrest are believed to be regulated primarily by decreases in cAMP dependent protein kinase (cAPK) activity via reduction of intracellular cAMP levels (Figure 7). cAMP levels are controlled by modulation of adenylate cyclase and cAMP-phosphodiesterase activities. The yeast RAS genes encode GTP binding proteins that activate adenylate cyclase (encoded by CRY] = CDC35). When RAS is activated, intracellular cAMP levels increase, causing the negative regulatory subunit of cAPK (encoded by BCYI) to dissociate from the catalytic kinase subunits (encoded by TPKJ, 2, and 3 ), thus activating them (reviewed by Gibbs and Marshall, 1989). The changes in growth and metabolism that occur in wild type cells at the diauxic shift do not occur- in mutant cells in which cAPK activity has become constitutive because of disruption of the BCYJ gene or because of mutations in which RAS is constitutively activated (Toda et al., 1985, 1987; Cannon and Tatchell, 1987). Cells with constitutive cAPK activity stop growing when glucose is exhausted because they are unable to utilize non-fermentable carbon sources. beyl cells are also unable to respond to this nutrient limitation by normal cell cycle arrest at Go; instead, starved bcyl cells are often multibudded and uninucleate. Consistent with the improper response to nutrient limitation, bcyl strains are highly sensitive to starvation, i.e. they become inviable rapidly upon cessation of growth (Matsumoto et al., 1983). In addition, beyl cells are heat shock sensitive, in contrast to the thermotqlerant phenotype of Go arrested cells. Conversely, cells with artificially depleted cAMP exhibit a 2543
W.R.Boorstein and E.A.Craig
100
150 0
0.10 r-
CM.
co O °..
50
1
0.1
=
=
0
0
20
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60 Time (hr)
80
100
120
Fig. 1. Cell density (open symbols) and specific activity of (3-galactosidase (filled symbols) from SSA3 promoter constructs during growth to stationary phase in rich glucose medium at 23°C. Diamonds represent transformants of the SSA3-lacZ fusion gene plasmid pWB204A-583 (see Figure 2, top line) and circles represent transformants of the SSA3 UASHS/CYCJ hybrid promoter fused to lacZ (pWB226; see Figure 4) under identical conditions. The time at which the diauxic shift occurred is indicated by the plateau in the growth curve (see arrow); glucose was depleted at this time (data not shown). The cell density plot is from pWB204A-583 transformant, but is virtually indistinguishable from the corresponding pWB226 plot. (-galactosidase activities depicted here are from a typical experiment. Both hybrid genes are inducible by heat shock treatment (Boorstein and Craig, 1990).
nutrient deprivation phenotype, even in rich media. Strains carrying a mutant adenylate cyclase gene, cyr], require exogenous cAMP for growth (Matsumoto et al., 1982a). Removal of cAMP causes cyri cells to arrest in Go regardless of the nutrient conditions. Cells arrested in this manner express Go specific proteins and are thermotolerant (lida and Yahara, 1984; Plesset et al., 1987). The completion of START A, which requires a functional adenylate cyclase gene and appears to be correlated with a transient increase in cAMP, signals the first step of commitment to a new round of cell division (reviewed by Pringle and Hartwell, 1981 and by Boynton and Whitfield, 1983). START A events do not occur until critical steps of the previous cell cycle are completed and unless sufficient nutrients are available to complete a subsequent cycle. cAMP is a good candidate for coordinating metabolic and cell cycle controls as cAMP levels are believed to be depressed at the nutrient sensitive control point of the cell cycle and lowered cAMP is a major component of the intracellular signal of suboptimal nutrient conditions. In these studies we examined the regulation of SSA3, the only one of the eight members of the yeast multigene HSP70 family whose mRNA levels increase at the diauxic shift. The increase in SSA3 mRNA abundance, like other changes that occur at the diauxic shift and in response to nutrient limitation, appears to be dependent upon a decrease in cAPK activity; SSA3 mRNA does not accumulate in bcyl cells under these conditions and can be induced experimentally by decreasing intracellular cAMP levels in cyr] strains growing on exogenously supplied cAMP (WernerWashburne et al., 1989). SSA3 was also previously shown to exhibit a classic transcriptional heat shock response. Basal SSA3 expression is very low in rapidly growing cells in glucose media. SSA3 mRNA becomes abundant within 15 min of shifting such cultures from 230 to 39°C (Werner-Washburne et al., 1989). A 32 bp fragment of the SSA3 promoter, UASHS, mediates this response by positive control of transcription (Boorstein and Craig, 1990). The same upstream activating sequence also drives a low level of basal expression. In the absence of UASHS, other portions of the SSA3 promoter are virtually unable to drive either basal or heat inducible expression. UASHS exhibits extensive homology to the eukaryotic HSE consensus sequence and is presumed to 2544
function by interaction with the heat shock transcriptional factor, HSF. HSF from non-stressed yeast cells has been shown to bind HSE sequences both in vivo and in vitro (Sorger and Pelham, 1987; Jakobsen and Pelham, 1988). Stress activation via HSEs occurs post-translationally and is believed to involve phosphorylation of HSF (Zimarino and Wu, 1987; Sorger and Pelham, 1988). In this paper we demonstrate that SSA3 is activated at the transcriptional level at the diauxic shift, in stationary phase arrested cells, and in respect to decreased levels of cAPK activity. We identify a cis-acting regulatory element of the SSA3 promoter which governs this pattern of regulation and investigate its interaction with UASHS in controlling transcription of SSA3. Results Localization of sequences controlling post diauxic shift expression To study SSA3 regulation following the diauxic shift, we utilized an SSA3 -lacZ fusion gene that was previously shown to be activated like the native SSA3 gene in response to heat shock. Transcription from the hybrid gene initiates at the proper sites within the SSA3 sequences and ,B-galactosidase activity accurately reflects hybrid transcript levels (Boorstein and Craig, 1990). Strains containing the hybrid gene were grown in rich medium at a constant temperature of 23°C. The pronounced slowing of growth (Figure 1) and the exhaustion of glucose from the medium (data not shown) were used as indicators of the diauxic shift. The onset of 3-galactosidase expression was coincident with the diauxic shift. 3-galactosidase activity continued to increase to high levels as cultures progressed to stationary phase (Figure 1). To determine whether common promoter elements regulate SSA3 expression in response to growth state and to heat shock, we measured the activity after the diauxic shift of the previously defined heat inducible UAS (Boorstein and Craig, 1990). The SSA3-lacZ fusion gene was expressed after heat shock (Figure 2) and in stationary phase (Figure 1) similar to native SSA3 expression. However, UASHS alone was unable to mediate expression from the heterologous yeast CYCI promoter after the diauxic shift at 23°C (Figure 1). UASHS was only able to promote transcription in response to heat shock (Boorstein and Craig, 1990 and Figure 4).
HSP70 cAMP responsive transcriptional element B-galactosidase activity induction logarithmic PDS (PDS - log) -583
= =
~
=
+1 ATG
-156
-468 -236
=
J
_
.200 -175
=~~
-166 -124
Vector
-_ _
-111
= =
-93
SSA3
230
T390
230
3.7 3.5 6.7 17 16 11 14 5.8 6.9
60 61 100
150 250 290 330 100 37
84 80 54 13 6.3
7.0
23°
146 247 283 313
84 26
28
14
8.6 9.8
2.8 2.9
lacZ
Fig. 2. 5' deletion analysis of the SSA3 promoter indicating basal, PDS and heat shock expression. The black lines represent SSA3 sequences that remain following the deletion. Sequences are numbered with respect to the most abundant 5' end of the hybrid mRNA (+1). Amino acid 3 (+ 34) of SSA3 is fused to lacZ. White diamonds represent matches to the dyad consensus heat shock element sequence, CnnGAAnnTTCnnG (Pelham, 1985). Only the HSEs centered at - 156 were previously shown to be functional. Black triangles represent repeated DNA sequences (Table III). Features of the SSA3 promoter region between -236 and -124 are detailed in Figure 3. The putative TATA element is indicated by the boxed 'T'. Vector sequences upstream of the deletion endpoints are identical in all constructs. (-galactosidase activity is presented in Miller units. (*) Induction is presented as the magnitude of increase in 3-galactosidase activity, calculated by subtracting the activity in logarithmic phase cultures from that in stationary (PDS) phase cultures at 23°C. Presentation of inductions as magnitudes facilitates identification of sequences regulating PDS expression by minimizing the impact of small variations in low basal activity. Such variations can result in large changes of induction ratios. -236
_,D_C3 ~_-124
GGCGCi~TATCATCAAACGTATTTGACTTGATGCCTATGGhGTTTGGTCCTTAATTAGGGATCGCTGTGGAAAGTTATAGAATATTACACGAAGCAGCCACAAGGGTGACCG A CGT T A C CG LTC AC, ,GTCGA.C Ml 43 M2 heme||IsosaesaseusuIsuIHIssausem *- PDS OlIgo -* uemeuIeml§||@||IstuIumnumeuou*em* AAAAAA
AA
AAA
AAA
AA
A
*enasmiemsuseuseumuuaeoonssu
*-
AAA
A
A
A
HSE 0Ngo .:- I......n..ae.
AA
a.o u
I-2 36 -200
_175 -166
Fig. 3. The DNA sequence of SSA3 from -236 through -124 (the 113mer) indicating sequence features and details of constructions used in these studies. Triangles represent matches to -GAA- modules that are positioned and oriented appropriately to comprise HSF binding sites (Amin et al., 1988; Perisic et al., 1989); black triangles mark exact matches, grey triangles mark sequences with a single base mismatch, and the white triangle is included to indicate appropriate spacing between flanking GAA elements. Arrows indicate positions of inverted repeats; adjacent repeats differ from each other by a single base pair. The boxed sequence resembles the consensus GRFI binding site: 5'-RRTGNNTGGGTKY-3' (R = A or G, Y = T or C, K = T or G) (Buchman et al., 1988a); this region also matches, in 11 of 13 positions, the exact GRFI binding site in the RNR2 (ribonucleotide reductase small subunit) promoter (Hurd and Roberts, 1989). The nucleotide substitutions in the Ml, M2, M3 and hse- mutations are shown immediately below the native sequence. The position and extent of the 35 bp PDS and 32 bp HSE oligonucleotides, used to test UAS function in the CYCI -lacZ vector (pWB220), are indicated with respect to the DNA sequence by the striped bars. Solid black bars at the bottom of the figure indicate the positions of 5' deletion endpoints and SSA3 sequences remaining in these constructions.
These results demonstrate that sequences capable of mediating post diauxic shift (PDS) activation are located within 617 nucleotides upstream of the third codon of the SSA3 gene, and that UASHS is not sufficient to drive this pattern of gene expression. 5' deletions of the SSA3 -lacZ hybrid gene were assayed to identify sequences that control the expression of SSA3 following the diauxic shift (Figure 2). Removal of increasing amounts of SSA3 sequence between -200 and -111 resulted in progressive loss of PDS f-galactosidase activity, suggesting that a single small discrete DNA element is not solely responsible for PDS expression of SSA3. Rather, the results indicate that multiple or extended sequences that exhibit some degree of functional redundancy mediate PDS expression. Deletions of two short adjacent regions immediately upstream of UASHs each decrease PDS activity of the fusion gene by two thirds, defining a domain of primary importance to this mode of regulation between -200 and -166. Loss of the upstream part of this domain (-200 to - 175) results in the largest decrease in PDS expression, > 200 3-galactosidase units, with no effect on
heat shock expression. This region includes a sequence that resembles GRFI (also referred to as RAP], and TUF) binding sites, which are known to function in both activation and repression of transcription of yeast genes (Brand et al., 1987; Shore and Nasmyth, 1987; Buchman et al., 1988a; Kimmerly et al., 1988). It also includes three repeated sequences that are present in alternating orientations (see Figure 3); inverted repeats often characterize binding sites for transcriptional regulatory factors (e.g. Vinson et al., 1989). This deletion removes all but a portion of the downstream repeat. Deletion of the downstream portion of this domain (- 175 to -166) decreased heat shock as well as PDS expression, although the effect on PDS activity was twice as large. The phenotype of this mutation may result from removal of part of the HSE, from removal of a flanking regulatory sequence, or both. It is not known whether the partial HSE match that extends into this region comprises part of the functional HSE (see Figure 3 and Boorstein and
Craig, 1990). Additional sequences, both upstream and downstream of the region discussed above, were also identified as import2545
W.R Boorstein and E.A.Craig
ant for regulation of SSA3 expression (Figure 2). A-124, which lacks UASHs, is induced 2-fold in stationary phase. Further 5' deletion to -111 abolishes PDS induction indicating that sequences involved in PDS expression are also found downstream of UASHS. A 7 bp exact match to one of the three sequences repeated between -195 and -174 is found in this 13 bp downstream region (see Table HI). The presence of sequences that may function to modulate SSA3 expression negatively in stationary phase is suggested by the increase of 180 units of expression upon deletion of upstream sequences between -583 and -200. Sequences in addition to UASHS that affect basal and heat inducible SSA3 expression in exponentially growing cells are also revealed in the 5' deletion analysis shown in Figure 2; these have been previously discussed (Boorstein and Craig, 1990). Identification of UASpDs To confirm that PDS regulation of SSA3 occurs at the level of transcription and to identify upstream activating sequences that mediate PDS activation, we tested short regions of the SSA3 promoter (Figure 3) for their ability to activate a CYCI -lacZ translational fusion gene (Figure 4). The hybrid gene includes CYCI 'downstream' promoter elements, TATA and initiation sequences; expression is entirely dependent upon activation by sequences with UAS function cloned into a restriction site immediately upstream of the CYCI sequences. A 113 bp fragment from -236 to -124, containing most of the region shown by 5' deletion analysis to contribute to PDS expression and including UASHS, was
able to activate transcription from the CYC] promoter almost 30-fold, both upon heat shock and after the diauxic shift. To attempt to separate heat shock and post diauxic shift regulatory elements, a synthetic 35 bp oligonucleotide that was identical to sequences immediately upstream of the UASHS (and within the 1 13mer sequence) was tested. This short sequence mediated a 60-fold induction of the reporter gene in stationary phase. Basal levels of transcription driven by the 35mer were 10-fold lower than those of the 113mer; this difference in basal activity contributed to the higher induction ratio of the shorter fragment. In contrast, the short oligonucleotide caused only a marginal (0.3 unit) increase in expression in response to heat shock. The 35mer construct mediated PDS expression in both orientations. The ability of the 35 bp SSA3 fragment to drive transcription of the CYC] -lacZ fusion gene, from a site slightly further (28 bp) than its native location with respect to the transcriptional initiation region, demonstrates at least a limited degree of position independence of this transcriptional regulatory element. These results demonstrate that the region between -205 and -171 contains an upstream activating sequence. We designate this element 'UASPDS'. The results of analysis of 5' deletion mutations and the fact that the SSA3 113mer drives 5-fold higher levels of transcription than the component 35 bp oligonucleotide in stationary phase cells suggest that the HSEs may contribute to SSA3 expression following the diauxic shift even though UASHS is not sufficient to drive expression in stationary phase. We performed two sets of experiments to test this
B-galactosidase activity induction locgarithmic Insert
(plasmid)
E SCYC1
Vector
none.
(pWB2)
CZ
,ffi-"s
PDS (PDS - log) 230 230 0.8 1.0
230
t390
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59
62
60
0.2
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12
12
0.2
0.4
5.4
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31
28
26
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26
24
,
---
113-mer
HSE
(pWB230)
PDS
(pWB240) PDS (rev)
(pWB240-R) HSE
(pWB2) HSE (rev)
(pWB226-R)
HSE + PDS
(PWB241)
4-
-,-aw
HSE + PDS (rev)
(pWB241-R)
Fig. 4. UAS activity of SSA3 promoter region fragments under PDS and heat shock conditions. The CYCI -lacZ vector lacking a UAS is depicted XhoI cloning site (-178) immediately upstream of the CYCI sequence; arrowheads at the end of the lines indicate orientation of the inserts with respect to their native orientation in the SSA3 promoter. '113mer' extends from the A-236 endpoint to the BstEll site at -124. White triangles represent -GAA- modules that comprise functional heat shock elements. The three small black arrows in the PDS region represent inverted repeat sequences. The striped rectangular box denotes similarity to GRFI binding sites. The SSA3 113mer DNA sequence and features depicted here are presented in detail in Figure 3 along with the 32 bp HSE and 35 bp PDS oligonucleotides. 'HSE + PDS' and 'HSE + PDS(rev)' include two SSA3 oligonucleotides immediately adjacent to each other, upstream of the CYC] proximal promoter elements; the relative positions of the two sequences is reversed with respect to their order in the SSA3 promoter. The plasmids utilized here differ from the SSA3-lacZ fusion constructs (Figure 2) only in the regions depicted. Units of activity and induction are presented as described in Figure 2. on the top line. Wide black lines represent sequences from the promoter region of SSA3 that are inserted into the
2546
HSP70 cAMP responsive transcriptional element
hypothesis. First, we cloned both UASPDS- and UASHScontaining oligonucleotides into the UAS test vector. Mutual enhancement of the activity of each element by the presence of the other resulted; 3-galactosidase activity in stationary phase was increased 2- to 5-fold and heat induction was increased 2.5-fold compared with that of constructs containing UASpDS and UASHS alone, respectively (Figure 4). The apparent positive interaction between the UAS activities of these sequences was observed despite the fact that UASPDS was downstream of UASHS, unlike the native orientation; furthermore, the enhancement was observed when UASpDs was present in either orientation downstream of UASHS. In complementary experiments, disruption of UASHS in the 113mer fragment resulted in a promoter with 86% less PDS activity than that of the non-mutant 113mer construct (Figure 5A). PDS expression from the hse1 13mer mutant was comparable with that from the UASPDS oligonucleotide (Figure 4). Similar results (71 % reduction of PDS levels) were obtained when the same hse- mutation was analyzed in the context of the 'complete' SSA3 promoter (in pWB204A-236) (Figure SB). However, expression from both UASHS disrupted 113mer and A-236 constructs was induced at least 85-fold after the diauxic shift over their respective basal levels, further demonstrating that UASHS is not essential for PDS activation. Due to the role of UASHS in basal expression (Boorstein and Craig, 1990), the
decreased basal activity from constructs lacking UASHS resulted in an increased induction ratio, although the magnitude of the induction was substantially reduced. Since evidence for interaction between UASHS and UASPDS was obtained in three different relative orientations and spacings, the initial observations suggesting HSE involvement in PDS expression could not have resulted from partially overlapping, but not functionally interacting, regulatory sequences. We previously demonstrated that UASHS drives a low basal level of transcription (Boorstein and Craig, 1990). Here we observed that both the 113mer, and the UASHS plus UASPDS synthetic constructs result in lower basal f-galactosidase activity than do constructs driven by UASHS alone. The UASPDS region appears to repress transcription driven by the HSE during logarithmic phase growth under nonstressed conditions. Mutational analysis of the UASpDs region To define the PDS control element more precisely and to assess potential function of the GRFI-like recognition site and the inverted repeat sequences we constructed clustered multiple substitution mutations within the 35 bp that were demonstrated to exhibit UASPDS activity. The DNA sequences of the 113 bp fragment and mutant derivatives
A. -178
Vector
I
Insert (plasmid)
-*
11 3-mor
lcZ
cyc1
-
B-galactosidase activity Induction
lotparithmic
PDS (PDS - log)
230
1390
230
230
2.2
59
62
60
m
