Identification of a DNA binding factor involved in cell-cycle control of the yeast HO gene

Cell, Vol. 57, 21-29.

April 7, 1989, Copyright

0 1989 by Cell Press

Identification of a DNA Binding Factor Involved in Cell-Cycle Control of the Yeast HO Gene Brenda J. Andrews and Ira Herskowitz Department of Biochemistry and Biophysics University of California, San Francisco San Francisco, California 94143-0448

Summary Transcription of the HO gene is controlled by at least eleven frans-acting regulators, which are responsible for limiting expression to mother cells and to one period in the cell cycle. A subset of these regulators and a repeated element (the cell-cycle box, CCB) are required for expression of HO during late Gl. We describe gel retardation and footprinting experiments that identify a factor, CCBF (cell-cycle box factor), that binds to multiple cell-cycle box elements. We show that the SW/4 and SW16 genes are required for formation of the CCBF-promoter complex in vitro, either as components of CCBF or as modulators of CCBF activity. The other SW/ genes (SWM, 2,3, and 5) are not required. We also show that SW14 and SW16 are the only SW/ genes required for expression in vivo from the CCB sequences. These observations indicate that the CCBF is responsible for cell-cycle regulation of HO and lead to the view that two of the SW/ genes, SW/4 and SW16, are specifically involved in this regulatory event. Introduction Genes that are regulated in the cell cycle provide an opportunity to study how the eukaryotic cell cycle is controlled. The regulation of these genes must be sensitive, directly or indirectly, to the molecules that control the cell cycle. The induction or repression of genes in response to physiological cues is commonly mediated by specific transcription factors that recognize defined sequence elements within upstream regulatory regions (Maniatis et al., 1987; Guarente, 1988). The upstream regulatory region of the yeast HO gene contains a repeated sequence element that is responsible for the cell-specific activation of HO (Nasmyth, 1985a; Breeden and Nasmyth, 1987), as well as other sequence elements that mediate additional controls. The HO gene encodes a site-specific endonuclease that initiates mating-type interconversion, the process by which haploid cells switch between a and a cell types (reviewed in Herskowitz and Oshima, 1981, Nasmyth and Shore, 1987; Herskowitz, 1988). The pattern of interconversion is determined by the regulation of HO transcription (Jensen et al., 1983; Nasmyth, 1983). HO expression and mating-type interconversion are restricted to a short period in late Gl, after the cell has committed itself to another mitotic cell cycle (Nasmyth, 1983). The commitment step (START) is dependent on a number of genes, including a protein kinase encoded by the C/X28 gene (Reed

et al., 1985). In addition to cell-cycle control, HO is repressed in diploid (a/a) cells (Jensen et al., 1983), and is transcribed only in mother cells (Nasmyth, 1983). Thus, HO transcription is sensitive to whether a cell is haploid or diploid, whether it is a mother or daughter, and its stage in the cell cycle. A region extending at least 1400 base pairs (bp) upstream of the start of transcription is required for the correct expression and regulation of HO (Figure 1; Nasmyth, 1985a). Functional dissection of the promoter has revealed that the far upstream sequences, URSl (upstream regulatory sequence l), are sufficient to activate transcription in the absence of the downstream sequences (URS2), and can act as an upstream activating sequence (UAS) to promote transcription of heterologous genes (Breeden and Nasmyth, 1985). The URSl region contains a site of action for the principal determinants of mother cell specificity (Sternberg et al., 1987; Stillman et al., 1988). In contrast, the URSP region, which lies between URSl and the TATA element, is not essential for transcription and does not activate a test promoter. Instead, the URSP region is involved in restricting HO expression to late Gl of the cell cycle. This complex control region contains ten copies of a sequence (PuNNPyCACGAAAA) which, when removed from the context of the remainder of the URS2 region and tandemly repeated, promotes transcription of a reporter gene that is periodic in the cell cycle and is dependent on START (Nasmyth, 1985b; Breeden and Nasmyth, 1987). Thus, these cell-cycle box (CCB) elements can act as positive regulatory elements and are sufficient to confer cellcycle regulation. A number of regulators that have sites of action within the HO upstream region have been identified genetically. Activation of HO requires the products of the SW/ genes, SW/l through SW16 (Stern et al., 1984; Breeden and Nasmyth, 1987). In addition, a number of SW/-independent (SIN) genes encoding possible repressors of HO transcription have been identified (Sternberg et al., 1987, Nasmyth et al., 1987). Several observations suggest that two of the SW/ genes, SW/4 and SW/f& are required for STARTdependent, periodic transcription via the CCB sequences within URS2. When the URS2 region, containing the CCB sequences, is removed from the HO promoter, cell-cycle regulation (CDC28-dependence) and the need for SW/4 and SW/6 are coordinately lost (Nasmyth, 1985b). Furthermore, the CCB sequences are unable to function as activating sequences in swi4- and swi6- mutant strains (Breeden and Nasmyth, 1987). Thus, SW14 and SW16 are important components of an activation pathway acting at the CCB sequences to promote cell-cycle regulation. The molecular nature of the transient activation by SW14 and SW/6 and the mechanism by which the activation of transcription is dependent on passage through the START step of Gl are unknown. In this paper, we show that the cell-cycle box sequences within the HO promoter represent recognition sites for a specific DNA binding activity. The promoter binding activ-

Cell 22

mother/daughter

ceil-cycle

HO TATA -1400

-940

H

I=CACGAAAA, Figure

1. A Schematic

of the Upstream

Regulatory

-

-mo CCS element Region

1234567

of HO

URSI and URSP are “upstream regulatory sequences” as defined by Nasmyth (1965a). URSl can function as an upstream activating sequence (UAS) and contains a site of aotion for the determinants of mother-cell specificity (mother/daughter control). URSP contains the repeated sequence motif (consensus PuNNPyCACGAAAA) that is responsible for cell cycle-regulated transcription of HO. We refer to these elements as cell cycle boxes (CCB). The black bar beneath the URSP region illustrates the fragment used as a probe for specific DNA-protein complexes in the experiments described in this paper. Numbers are given with respect to the start of translation (scheme of Russell et al., 1966).

-CCBF

-free ity is dependent on the SW!4 and SW/6 genes but not on SW/l, 2, 3, and 5, indicating that the unique requirement for SW/4 and SW/6 in cell-cycle regulation of HO is due to the involvement of SW/4 and SW/6 in the formation of a DNA-protein complex at the CCB elements. This profile of SW/ gene dependence represents a biochemical correlate of in vivo studies in which we demonstrate an absolute requirement for only SW14 and SW/6 in the activation of transcription via the CC6 sequences. Results Yeast Extracts Contain an Activity that Binds to the CCB Sequences CCB sequences are responsible for the cell-cycle control of HO transcription. To identify proteins that may play a role in this process, we have used the gel retardation assay (Fried and Crothers, 1981; Garner and Revzin, 1981) to search for protein-DNA complexes specific to DNA segments containing CCB sequences. The HO fragment used in the assay extends from -507 to -394 with respect to the presumed translation initiation codon, and contains three CCS repeats as well as adjacent URS2 sequences (see Figure 1). We showed in other experiments that this segment brings a test promoter under the control of several SW/ and SIN genes (B. Andrews, unpublished data). When a crude yeast extract from wild-type cells was incubated with the URSP probe, a number of complexes were formed, one of which was specifically sensitive to the presence of excess URS2 competitor DNA (Figure 2, lanes l-3). Formation of this complex was also prevented by the addition of heterologous DNA fragments containing a single copy (lanes 4 and 5, Figure 2) or multiple copies (lanes 6 and 7, Figure 2) of a synthetic CCB sequence. A non-CCB-containing fragment of similar size did not compete for complex formation (see Figure 4, lane 9). The fragment containing a single CCB element competed less effectively for protein binding than the competitor probe

Figure 2. A DNA quences

Binding

Activity

That

Recognizes

the

CCB

Se-

A 120 bp 32P-labeled fragment containing 3 CCB elements and adjacent URSP sequences was incubated with a crude yeast extract in the presence or absence of excess, unlabeled competitor DNA fragments. Bound and free DNA were separated by electrophoresis and visualized by autoradiography of the gel. The reactions contained the following competitors: lane 1, no competitor; lanes 2 and 3, unlabeled URSP probe; lanes 4 and 5, a fragment containing a single synthetic CCB; lanes 6 and 7, a fragment containing three tandem CCB elements. The unlabeled competitor was in V&fold molar excess over labeled DNA in the reactions of lanes 2,4, and 6 and 20-fold molar excess in the reactions of lanes 3,5, and 7 (as indicated above the lanes). A reaction that contained competitor DNA lacking CCB elements is shown in lane 9 of Figure 4. All of the reactions contained approximately 10 ug of crude yeast extract.

with three CCB sequences. These results indicate that the CCB sequences represent binding sites for the factor detected in this assay. We refer to this protein as CCBF (cellcycle box factor). The CCBF binding site was probed in greater detail by copper-phenanthroline (OP/Cu+) chemical footprinting. This technique exploits the nucleic acid cleavage activity of orthophenanthroline and copper ions, which can operate upon DNA-protein complexes in polyacrylamide gels (Kuwabara and Sigman, 1987). As in conventional footprinting techniques, OP/Cu+ protection identifies sequences rendered inaccessible to cleavage by the nuclease because of protein binding. We used the chemical footprinting method since the DNA-protein complex is treated with nuclease after resolution on nondenaturing gels. This method can detect protein-DNA complexes with relatively large dissociation rate constants (Peterson and Calame, 1987) apparently due to the stabilization of the complexes within the acrylamide gel matrix. We found

Cell-Cycle 23

Regulation

of HO Expression

G t;

C + T

(-)

U

B

-410-4201

-4609

-480-

Figure 3. CCBF Digestion

Protects

the CCB Sequences

in URSP from Nuclease

Protection of the URS2 probe described in Figure 1 from attack by OP/Cu+ chemical nuclease is shown. The numbers at the left of the figure indicate the distance from the ATG in the HO upstream region (numbering scheme of Russell et al., 1999). The arrows indicate the positions of the CCB sequences within the URSP probe. A nondenaturing gel on which the CCBF complex had been resolved was incubated with OP/Cu+ reagents. The bound and unbound material was excised, eluted, and electrophoresed on the gel shown. The lane marked (-) illustrates the ladder of nuclease cleavage obtained when the URS2 probe was incubated in the absence of protein. The lane labeled “U” contains DNA isolated from the free region (unbound) of the preparative assay gel, while the ‘B” lane contains DNA isolated from the CCBF complex. The G+A and C+T lanes illustrate purine and pyrimidine sequencing ladders of the URSP probe (Experimental Procedures).

this approach highly suitable for use with crude yeast extracts. A preparative nondenaturing gel on which the CCBFURSP complex had been resolved was incubated with OP/ Cu+ reagents (see Experimental Procedures for details). The treated complexed and unbound material was identi-

fied by autoradiography, excised from the gel, and extracted. The pattern of nuclease protection due to the binding of CCBF was visualized by running the reactions on a denaturing gel adjacent to sequencing standards followed by autoradiography. The CCBF complex yielded two distinct regions of protection, one of 10 bp (-466 to -457) and the other of approximately 19 bp (-441 and -422; Figure 3, lane “B”). The protected sequences corresponded closely to the positions of the three CCB elements with the URS2 fragment. The smaller protected region matched the position of the single CCB element within the URSP fragment precisely. The larger protected region was slightly offset with respect to two tandem CCB elements. Thus, the CCBF complex detected in this assay appears to reflect specific recognition of dispersed cell-cycle box elements. Genetic Requirements for the In Vivo Activity of the CCB Sequences Six regulators (SW/l-s) that have sites of action in the HO upstream region have been genetically defined. To test which of these regulators might act at the CCB sequences and hence be involved in CCB complex formation, we made use of the ability of synthetic CCB elements to activate transcription of a test gene (Breeden and Nasmyth, 1987). We have constructed plasmids similar to those used by Breeden and Nasmyth in which two, three, or four copies of the CCB element were tandemly arranged in front of a CYC-/acZ fusion gene lacking its own UAS sequence (PASS, Sternberg et al., 1987; Guarente and Hoar, 1984). These constructs were transformed into each of the six swi mutant strains (swil-, swi2-, swi3-, swi4-, SW&, SW@), which are unable to transcribe the HO gene. CCB-mediated transcription was assayed by measuring f3-galactosidase levels in the yeast transformants. The data in Table 1 show that the CCB sequence was unable to function as a UAS in swi4- and swi6 strains, whereas UAS activity was found in all other swi mutants examined. For example, pBA161, which contains four CCB sequences in the upstream region of the test gene, exhibited less than 1% activity in swi4- and swig mutants, compared with SW/+ cells. In contrast, activity in swil-, 2-, 3-, and 5- mutants was between 28% and 93% of that observed in wild-type cells. Our results differ in one respect from those of Breeden and Nasmyth (1987): they reported a requirement for SW/3 in addition to SW14 and SW16 in the CCB-UAS activity. Although we found the levels of fi-galactosidase recovered from transformants of swil-, 2, and 3- mutants to be slightly reduced relative to wild type, the CCB sequence retained efficient activating function in these strains (between 30% and 50% of wild type for the swi3- mutant). This reduction in B-galactosidase activity in swil-, 2, and 3- strains is also seen for the parent CYC-/acZ fusion plasmid (PASS), which lacks a UAS, and for CYC-IacZ fusion plasmids with the UAS sequences from other genes (such as the RP39 gene; 8. A., data not shown). This reduction in pgalactosidase activity in swil-, 2, and 3 strains is another manifestation of the pleiotropy of the swi mutants, which result in poor general health and slow growth (Stern et al., 1984).

Cell 24

CCBF

c-

Figure SW16

4. The

CCBF-URS2

Complex

Is Dependent

free

on SW14 and

The reactions of lanes 1-6 illustrate the effect of SW14 and SW16 gene dosage on CCBF complex formation. The radiolabeled URSP fragment bearing three CCB elements was incubated with crude extract from SW/+ cells (lanes 1, 3) SWI+ cells bearing the high copy number plasmid pSWI4 (lanes 2, 4) swi4- cells (lane 5) and swi6 cells (lane 6). The reactions of lanes 1 and 2 contained equal amounts of extract (6 ug), as did the reactions of lanes 3, 4, 5, and 6 (16 ng). Reactions were electrophoresed on a nondenaturing gel to separate free and bound DNA. For the purpose of comparison to the mutant extracts, lanes 7-Q illustrate a competition analysis in which SW/+ extract was preincubated with the following competitors: lane 7, the URSP probe; lane 6, a synthetic CCB element; lane 9, a non-CC&containing fragment of comparable size.

The more pronounced effect on HO expression caused by a swi3- mutation observed by Breeden and Nasmyth (1987) may reflect some exacerbation of the nonspecific effect that we observe. We interpret the large difference in the requirement for SW/4 and SW16 in comparison with the other SW/ genes for the activation of transcription from the CCB sequences as indicating a specific requirement for SW/4 and SW16 in this process. CCB Complex Formation Requires SW14 and SW16 but Not Other SW Genes The results described above as well as previous studies (Breeden and Nasmyth, 1987) formally define the CCB sequences as targets for the SW14 and SW16 gene products. The SWl4, B-dependent behavior of the CCB sequences is consistent with the observation that HO promoter deletions that remove all of the CCB sequences are functionally independent of SW14 and SW16 (Breeden and Nasmyth, 1987). These promoter dissection and genetic experiments predict that the principal factor responsible

C$Cycle

Regulation

g ----

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r ; Q)

of HO Expression

cu ; (I)

56

I m ggf

76

2

9

g

-c 3 u)

11 12

13 14

’

10

--

for recognizing the CCB sequences in the HO promoter should be dependent on SW/4 and SW16 but not on the other SW/ genes. To examine the formation of the CCBF complex in various swi- mutants, gel binding assays were performed with extracts from mutant strains. Extracts prepared from swi4- or swi6 mutants did not support formation of the CCBF complex (Figure 4, lanes 5 and 6). Thus, the SW/4 and SW/6 gene products encode or control CCBF. More CCBF activity was recovered from a yeast transformant carrying a high copy number plasmid carrying the cloned SW/4 gene (lanes l-4, Figure 4) further emphasizing that the SW/4 gene product is either a component of CCBF itself or that it controls CCBF activity. In contrast to the lack of complex formation in swi4and SW& mutants, there was no detectable effect on the recovery of CCBF complex when extracts were prepared from swil-, 2-, 3, or 5 mutants (Figure 5). This biochemical survey of the swi mutants for CCBF complex formation supports the genetic evidence described above that SW/4 and SW/6 are the only SW/ gene products involved in recognition and activity of the CCB sequences within the URS2 region of the HO promoter. Discussion We present two sets of observations, one in vivo and one in vitro, that indicate the existence of a SWl4, SW/6dependent activity that recognizes the CCB sequences within the URS2 region of the HO promoter and is likely to be directly involved in cell-cycle regulation. The URSP region, which encompasses sequences between the far upstream activating region (URSl) and the TATA element, is involved in the cell-cycle regulation of HO transcription as indicated by the START-independence of URS2 deletions (Nasmyth, 1985b). The CCB sequences contained

Figure 5. CCBF Complex Formation Does Not Require SWI7, SW12, SW13 or SW15 The end-labeled URSP probe was incubated with crude extracts from six different veast strains. Bound and free DNA were separated by electrophoresis and visualized by autoradiography of the dried gel. The yeast strains carried different swi- mutations: lanes 1 and 2, SW/+; lanes 3 and 4, swiALEU2; lanes 5 and 6, swi2-7; lanes 7 and 8, swi3-I; lane 9, SW/+; lane 10, swi4-; lanes 11 and 12, swiSAEU2; lanes 13 and 14, SWP. The extracts used in the reactions of lanes l-10 were all prepared on the same day and electrophoresed on a single gel. The swi5- extract was prepared at a different time, and is shown together with a SWlt extract (lanes 13 and 14) prepared simultaneously. The arrow at the left of the figure illustrates the position of migration of the CCBF complex in lanes l-10, while the arrow at the right denotes the position of the CCBF complex in lanes 11-14. Total yeast protein in the reactions was approximately 5 ug in odd-numbered lanes and lane 10. All other reactions contained approximately 10 pg of total protein.

within URS2 are important targets for this cell-cycle control, since they can confer cell-cycle regulation upon a reporter gene (Breeden and Nasmyth, 1987). In Vivo Activity of the CCB Sequences: a W/4, SWIG-Dependent Activation Pathway Six different SW/ genes are necessary for transcription of the HO gene. Our in vivo analysis of the SW/ genes required for the UAS activity of the CCB sequences confirms the results of an earlier study (Breeden and Nasmyth, 1987) with one important exception. We find that SW/4 and SW/6 are uniquely required for activation via the CCB sequences. In contrast, Breeden and Nasmyth (1987) reported that CCB expression also requires SW/3. The reason for the difference between these studies is not clear. However, our results are consistent with previous genetic studies that have allowed grouping of the SW/ genes into related activities or pathways. Mutants defective in SWI7, 2, and 3 share all phenotypes (Stern et al., 1984, Sternberg et al., 1987), whereas mutants defective in SW14 and SW16 share a distinct set of phenotypes (Stern et al., 1984; Breeden and Nasmyth, 1987). This study, which shows that swil-, 2-, and 3 mutants exhibit identical phenotypes with regard to the activity of the CCB elements in vivo, supports the genetic data that suggest a distinct role for SW14 and SW/6 in HO gene expression. In Vitro Recognition of the CCB Sequences by a SW14, SWIG-Dependent Binding Activity Our in vitro search for factors that specifically interact with the CCB sequences provides additional support for the view that SW14 and SW16 play a role distinct from the other SW/ genes in HO expression. We have used gel retardation and footprinting techniques to identify a factor that binds to the CCB sequences within the URS2 region. The CCBF (cell-cycle box factor) activity is absent in extracts

Cell 26

CELL START

CYCLE SIGNALS

I7

-

7T7

cell-cycle regulated transcription

SIN1

Figure

6. Overview

of Regulation

at URSP

The URSP region, which contains ten CC6 sequences (black bars), is depicted. The CCBF, cell cycle box factor, described here binds to the CCB elements within this region. Competition and footprinting experiments suggest that CCSF interacts with the CCB elements dispersed over the entire URSP region. CCBF activity requires the SW14 and SW6 gene products. The activity of the CCSF-URS2 complex is regulated in two ways. First, it receives signals from regulators of the yeast cell cycle such as CDCPB in an as yet undetermined manner. In addition, CCBF must be sensitive to additional controls on the URS2 region including negative regulation mediated by the S/N1 gene product (see text for more discussion).

from SW& and swi6 mutants, but present in extracts from all other swi- mutants. Taken together, our in vivo and in vitro analyses suggest a key role for this factor in cell-cycle control of HO expression. The footprinting analysis shown in Figure 3 suggests that CCBF binds to all three CCB elements within the URSP probe. The elements were completely protected from nuclease digestion by CCBF binding. No intermediates consisting of CCBF bound to only one CCB were recovered. Intermediates were similarly not detected when crude extracts were diluted and assayed for CCBF activity (data not shown). The absence of detectable intermediates in the CCB binding reaction may indicate a cooperative interaction between CCBF proteins during the binding process. Competition experiments with various segments of URSP indicate that CCBF interacts with other CCB elements from the entire URS2 region (data not shown). CCBF cooperativity may be involved in facilitating the interaction of this factor with ten CCB sequences dispersed over a region of 600 bp, and presumably results in the URS2 region being in a fully active or fully inactive state. At this time, the actual composition of CCBF-in particular, whether the SW14 and SW16 gene products are present-is unknown. Biochemical fractionation of the crude extracts used in this study is being undertaken in order to address this question. SW14 and SW16 may form a heteromerit complex that binds to the CCB sequences. In this case, the absence of one component of the complex, as in swirl- or swi6 cells, is sufficient to interfere with CCBF activity. A heteromeric complex is known to be involved in the regulation of the yeast CYCl gene. The yeast HAP2

and HAP3 activators bind to a CCAAT-containing upstream sequence in the CyC7 gene promoter in an interdependent manner (Oleson et al., 1987; Hahn and Guarente, 1988). Likewise, mammalian CCAAT-box binding factor has been shown to consist of two subunits that are analogous to HAP2 and HAP3 (Chodosh et al., 1988). If SW14 and SW/6 function solely by such a pairwise interaction, then one might expect the phenotype of a swi4-swi6double mutant to be the same as the phenotype of either single mutant. However, a swi4-swid- double mutant is inviable (Breeden and Nasmyth, 1987) whereas the single mutants are viable, suggesting that the function of SW/4 and SW16 may be more complex. The observation that mixing extracts from swi4- and swi6- cells does not reconstitute CCBF binding activity (data not shown) is consistent with the view that the SW14 and SW16 gene products may not be actual components of CCBF, and instead may control its expression or activity. In this case, the genes encoding for the components of CCBF remain unidentified. We suggest that these genes may be essential and that mutants defective in these constituents would be less readily obtained than mutants defective in the known SW/ genes. Regulation of CCBF by the Cell Cycle The CCBF-URS2 complex must be regulated in two ways (Figure 6). The SW/4/SW/6 regulatory pathway, which controls CCBF activity, must include a mechanism for linking the activation of transcription with the execution of START In addition, the activity of CCBF must be integrated with other controls of HO expression (see below). The connection of CCBF to the cell cycle likely involves the CDC28 gene product, a protein kinase (Reed et al., 1985) that is required in the transition from the Gl to S phase of the cell cycle (at START, Hartwell, 1974). Both HO and CCBpromoted transcription are CDC28-dependent (Nasmyth, 1985b; Breeden and Nasmyth, 1987). Because the CCBFDNA complex involves the CCB sequences, it is likely that CCBF activity is responding to the CDC28 gene product. Cell cycle regulation might occur by phosphorylation of CCBF itself or of a factor that governs its activity. If CCBF itself is the target, modification could alter its DNA binding properties or alter the activity of consititutively bound CCBF. Both of these mechanisms have precedents in the activation of yeast (Sorger and Pelham, 1988) and mammalian heat shock genes (Zimarino and Wu, 1987; Larson et al., 1988). Our preliminary results suggest that CCBF remains bound to the CCB elements throughout the cell cycle (B. Andrews, unpublished data) favoring the view that an activity of CCBF distinct from DNA binding is modified in response to cell-cycle regulators such as C/X28. CCBF in the Context of the URS2 Region In addition to being regulated by the cell cycle, CCBF must interact with other factors that control HO expression. As described above, isolated CCB sequences confer UAS activity to a test promoter. However, the entire URS2 region, which contains multiple CCBs, does not function as an activating sequence (Nasmyth, 1985a; W. Kruger and B. Andrews, unpublished data). The inability of the CCB se-

Cell-Cycle 27

Regulation

of HO Expression

quences to function as activating sequences in their native context suggests the existence of negative control acting on the URS2 region. This negative control could be exerted on the CCB sequences themselves or upon surrounding sequences. Genetic studies from this lab identified the SIN6 gene, which was a candidate for a repressor acting through the CC6 sequences and a putative target for the SW14 gene product (Sternberg et al., 1987). Subsequent analysis has shown that SIN6 encodes a nonsense suppressing tRNA (6. Andrew% unpublished data). Thus, we have no genetic evidence to suggest that SW14 acts by antagonizing a repressor. Rather, we have evidence that the utilization of the CCB sequences within the URS2 region is blocked by a negative regulator encoded by SIN1 that acts through adjacent sequences (B. Andrews, W. Kruger, and I. Herskowitz, unpublished data). We suggested above that the cooperative binding of CCBF leads to full occupancy of the dispersed CC6 elements and therefore to the fully active state of the URSP region. The SIN1 gene product may be responsible for maintenance of an inactive state. The interaction of positive and negative regulators has been shown to be critical in the regulation of a number of genes, such as those that are controlled by sterols (Dawson et al., 1988) and in histone gene regulation (Barberis et al., 1987). The mechanism of cellcycle control of HO expression likely involves both the antagonist and synergistic interaction of factors such that the various controls over HO expression are coordinated. Having biochemically identified one activity responsible for regulation of HO, we should be in a position to determine how its activity is regulated to trigger cell-cycledependent expression. Experimental

8galactosidase activity. 8galactosidase activity was assessed as previously described (Sternberg et al., 1987) and by modification of a filter assay described by Breeden and Nasmyth (1985). To establish that the complementing plasmid indeed carried the cloned SW14 gene, a 7.4 kb Clal-Sal1 fragment containing the complementing activity was subcloned into the integrating vector YIPS (Struhl et al., 1979) and targeted to the yeast chromosome in diploid strain BY238 as described above. The resulting strain was sporulated, giving rise to URA+ SW/+ segregants presumably with URA3 linked to sW14. Two such URA+ segregants were crossed to a swi4-3 HO::/acZ ura3-52 strain. Tetrad analysis showed a 2 URA+ SWV: 2 ura- swi- pattern of segregation. Thus, the insertion is closely linked or allelic to the SW14 locus. Plasmid pBB1 (see above) carries a 2.2 kb BamHl fragment of SW14 cloned into Ylp5. This fragment is internal to the SW14 gene as determined by complementation experiments. To construct plasmid pBA128, a 113 bp Xmnl (-507) to Nrul (-394) fragment (using the numbering scheme of Russell et al., 1986) of the upstream region of HO was subcloned into the Smal site of pUC18Bgl (a derivative of pUC18 in which a Bglll linker was inserted at the EcoRl site, reconstituting the EcoRl site in the process). The HO upstream fragment was obtained from a subclone of plasmid pHO-lac-cl2 (Russell et al., 1986). Plasmids containing the CCB consensus sequence were constructed by insertion of a synthetic CC8 cassette. Two complementary oligomers with Xhol sticky ends, 5’TCGATCCACGAAAA3’and 5’TCGATTTTCGTGGA3’ (synthesized on an Applied Biosystems 380A DNA synthesizer by the Biomolecular Resource Centre at UCSF), were annealed and cloned into the Sal1 site of pUC18 or a derivative of pUCl8, pUC18Bg12, in which Bglll linkers were introduced at the Hindlll and EcoRl sites. Plasmid pBA74 contains a single copy of the CCB cassette; pBA167 carries three copies of the CCB sequence. Plasmids for in vivo analysis of the UAS activity of the CCB sequence were constructed by cloning of the annealed CCB oligomer into an Xhol site upstream of the CYCl TATA box in vector PASS. PASS is a derivative of pLGA312 (Guarente and Hoar, 1984) containing the cyC7 promoter fused to Cyc-lacZ but lacking UAS sequences (Sternberg et al., 1987). Plasmids pBA118, 120, and 161 contain two, three, and four copies of the CCB sequence, respectively. The relative orientations of the CCB sequences in these constructs was determined by dideoxy sequencing of the plasmid DNA using an end-labeled primer that hybridized 30 nucleotides TATA-proximal of the Xhol site.

Procedures

Yeast Strains Saccharomyces cerevisiae strains used in this study are derivatives of strain 1998 from our collection. Strain 1998 (MATa HO::/acZ ura3-52 /eu2-3 leu2-772 his- mer can7 HMRa), 1999 (MATa HO::/scZ swiALEU2) and 2001 (MATa HO::lacZ swi5ALEU2) comprise an isogenie series constructed by gene replacement. The swi7A and swi5A substitution alleles were constructed by Michael Stern (Stern, 1985) and are described by Sternberg et al., 1987. The HO::/acZfusion allele (Russell et al., 1986) was used to score the presence of swi- mutations Strains 2226 (MATa HO::lacZ swi3-7 /eu2-3 /eu2-772 ura3-52 his- met can7 aded /##?a) and 2228 (MATa HO::/scZ swi2-7 /eu2-3 /eu2-772 ura3-52 his- met csn7) are also related to strain 1998 (Stern, 1985). The swig strain (BY202, MATa HOdacZ45 swi6-399 ~~3-52 his- canl-700 ade2-7 trppl-7 leu2mer) was derived from strain BY142, which was kindly supplied by Linda Breeden (Breeden and Nasmyth, 1987). The swi4 disruption strain (BY233: swi4::pBB7, otherwise isogenic to 1998) was constructed by targeted integration (Orr-Weaver et al., 1983) of plasmid pBBl (see below) to the SW14 locus in a diploid (BY238: HO::lacZ SW/+, derived from 1996) strain (Jon Pollack, unpublished data). When these diploids were sporulated. they showed a 2 URA+ swi-:2 ura- SkVrC segregation pattern. BY205 was constructed by transformation of strain 1996 with a high copy SW14 plasmid (pswl4+). Plasmids A plasmid complementing the swi4-3 mutation was obtained from a gene bank constructed in the low-copy CEN vector YCp60 (supplied by Mark Rose and Gerald Fink). The complementing plasmid (B32A) was identified by transformation of a swi4-3 HO::lacZ strain (2008, Sternberg et al., 1987) with the bank and screening for restoration of

Media and General Methods YEPD medium, SD medium, and supplements are described by Hicks and Herskowitz (1976). All synthetic media are supplemented to 300 uglml of leucine to allow growth of some SW/- mutations (Stern et al., 1984). Yeast transformations were performed by a modification of the lithium acetate protocol of Ito et al. (1983). Extract Preparation Cells were grown in YEPD or SD medium to an ODm, value of 1.0-1.5 (approximately 3 x 1O’cells per ml), harvested by centrifugation, and washed with 1160 volume of extraction buffer (Buffer E: 100 mM Tris-HCI (pH 8) 1 mM EDTA, 10% glycerol, 400 mM [NH4]zS04. 10 mM MgCIz, 10 mM f3-mercaptoethanol, 1 mM PMSF, 2 mM benzamidine, 1 uglml of leupeptin, 1 uglml of pepstatin). The cell pellet was resuspended in extraction buffer (2.5 ml/g wet weight) and disrupted by agitation at 4% in the presence of 2/3 volume of glass beads (0.5 mm diameter) in a mini-beadbeater (BioSpec Products). After 5 min on ice, cellular debris was cleared from the lysate by a brief spin in the microfuge. The extract was centrifuged for 1 hr at 100,000 x g in a Beckman TLlOO centrifuge. The supernatant was collected, and proteins were precipitated by the addition of saturated (NH&S04 in 50 mM Tris-HCI (pH 8.0) 0.5 mM EDTA to 50% saturation. After a 10 min incubation on ice, the protein pellet was recovered by centrifugation and resuspended in a minimal volume (about l/1,000 of culture volume) of dialysis buffer (Buffer D: 50 mM Tris-HCI (pH 8) 20% glycerol, 50 mM NaCI, 0.1 mM PMSF, 0.2 mM EDTA, 10 mM 8-mercaptoethanol, 1 us/ml of leupeptin, 1 pglml of pepstatin.). The extracts were dialyzed against the same buffer and stored at -70% Protein concentrations were determined using the Bio-Rad assay. Pmbes for DNA Binding Assays URS2 probes containing CCB sequences

were isolated

from pBA126.

Cell 28

To obtain a URSP probe labeled in me coding strand, pBA128 digested with BamHl and end-labeled with reverse transcriptase [a-32P]-dATP as previously described (Andrews et al., 1985). The quely end-labeled fragment was released by digestion with Bglll purified by polyacrylamide gel electrophoresis and electroelution. noncoding strand probes, plasmid DNA was end-labeled at the site, and the probe was released by digestion with BarnHI.

Competitors

for Gel Electrophoresis

DNA Binding

was and uniand For Bglll

Assays

Unlabeled competitor fragment corresponding to the URS2 probe was prepared by digestion of pBA128 with EcoRl and BarnHI, and isolation of the 135 bp fragment by polyacrylamide gel electrophoresis and electroelution. A competitor containing a single copy of the CCB fragment was prepared by digestion of pBA74 with EcoRl and Hindlll and isolation of the 85 bp fragment. A fragment containing three copies of the CCB box was similarly prepared by digestion of pBA167 with EcoRl and Bglll and isolation of the 93 bp fragment. Recovery of competitor fragments was quantitated by electrophoresis on 4% NuSieve gels.

Gel Electrophoresis

DNA Binding

Assay

Binding reactions were carried out in 20 ul volumes containing 0.5x Buffer D, 3mM MgCIP, NaCl supplemented to 50 mM and 0.05 to 0.25 ug/td of poly(dl-dC).poly(dl-dC) (Pharmacia). After addition of protein extracts (typically 5-30 ug) to the buffer-nonspecific competitor mixture, the reactions were incubated on ice for 10 min. Approximately 0.02 picomoles of radiolabeled probe was added, and the reaction was incubated for an additional 15 min at room temperature. Protein-DNA complexes were resolved by loading the reactions directly onto 4% (35:l crosslinking or 8O:l crosslinking) polyacrylamide gels in 0.5x TBE buffer (45 mM Tris-borate (pH 8.3) 1 mM EDTA). Gels were prerun for l-2 hr at 180 V. After sample loading, gels were electrophoresed at 180 Vat room temperature until bromophenol blue tracking dye had reached the bottom of the gel. Gels were dried on Whatman 3MM paper and autoradiographed.

Cu-Phenanthrollne

Footprinting

The nuclease activity of copper-phenanthroline reagents was exploited in a footprinting assay as described by Kuwabara and Sigman (1987). The binding reaction described above was scaled up lo-fold, and complexes were resolved on nondenaturing gels. The entire gel was immersed in 200 ml of 10 mM Tris-HCI (pH 8) in a clean Pyrex dish. Solution A (20 ml) was added to the buffer solution (Solution A: 1 ml of 9mM CuSO,, 1 ml of 40 mM phenanthroline, 18 ml of HeO). In situ digestion of the protein-DNA complexes was initiated by the addition of 20 ml of Solution B (1:200 dilution of mercaptoproprionic acid). After 7-10 min at room temperature, the reaction was stopped by the addition of Solution C (0.116 g neocuprione in 20 ml of ETOH). Free and complexed DNA were visualized by autoradiographyof the wet gel. Labeled DNA was excised from the gel and recovered by electroelution using Stratagene electroelution columns. The purified DNA was resuspended in denaturing buffer, heated at 90% for 2 min and electrophoresed on 8% denaturing polyacrylamide gels. Sequencing standards for footprinting reactions were prepared by the method of Maxam and Gilbert (1980).

Acknowledgments We thank Shane Climie, Beth Shuster, Keith Yamamoto, and members of our laboratory for valuable diScussions and comments on the manuscript. We acknowledge the important contribution of Jon Pollack to this work while he was a rotation student in the lab. We are grateful to Linda Breeden for sending us the SW& strain. B. J. A. is a fellow of the Medical Research Council of Canada. This research was supported by a grant (Al 18738) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

November

18, 1988; revised

January

12, 1989.

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