expression of dna replication genes in the yeast cell cycle

Mutation Research, 289 (1993) 61-72 61 © 1993 Elsevier Science Publishers B.V. All rights reserved 0027-5107/93/$06.00

MUT 00108

Expression of D N A replication genes in the yeast cell cycle

Evan M. Mclntosh Department of Biology, York University, Toronto, M3J 1P3, Canada

(Received 6 July 1992) (Revision received 17 September 1992)

(Accepted 17 September 1992)

Keywords: Cell cycle; Yeast; DNA replication; Transcription

Summary

In recent years, numerous studies using a wide variety of systems have clearly established some of the fundamental components of eukaryotic cell-division control. These include p34 °at2 protein kinases (henceforth referred to as p34) and closely related proteins (p33~c2), and the members of the cyclin gene family which, through interaction with the p34 (and p33) kinases, regulate transitions from one stage of the cell cycle to the next. The function of these proteins in the cell cycle has been conserved to the extent that p34 protein kinase and cyclin genes are, in some cases, interchangeable between organisms. Despite the tremendous insight that studies on p34 and the cyclins have provided, many questions remain about the details of the molecular events which allow these proteins to govern cell division. One question of particular interest concerns the means by which p34 interaction with G1 phase cyclins promotes G1 to S phase transition in the cell cycle. This is of primary importance since entry into the cell cycle is regulated, for most cells, by passage from G1 (or G 0) into S phase. Recent findings in the yeast Saccharomyces cerevisiae point to a potential link between the p34/G1 cyclin protein kinase complex and the regulation of DNA replication genes during the cell cycle. This paper reviews studies dealing with the transcriptional control of DNA replication genes in yeast and also briefly discusses the potential role of G1 cyclins in this process. A similar review of this subject has also been given by Johnston and Lowndes (1992),

dNTP metabolism, DNA replication genes and cell-cycle control

One approach to investigating cell-division control is to assume that it results as a conse- quence of differential gene expression. If this

Correspondence: Dr. E.M. Mclntosh, Biology Department, York University, 4700 Keele Street, North York, Ont. M3J 1P3 (Canada). Tel. (416) 736-2100 (ext. 33553); Fax (416) 736-5779.

assumption is correct, then identifying and study- ing genes that exhibit cell-cycle-dependent expression may illuminate the molecular events which ultimately regulate cell division. Since G1-S phase transition signals entry into the cell cycle, many researchers focused on this particular period to search for differentially regulated genes. Among the earliest proteins found to fluctuate during this interval were enzymes involved in dNTP metabolism such as dihydrofolate reductase, thymidine kinase and thymidylate synthase. This pattern of expression was reported for a

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number of these enzymes in a variety of organisms ranging from yeast and algae to mammalian systems (refs. cited in Mclntosh et al., 1986). Collectively, these observations suggested that genes encoding dNTP metabolizing enzymes in particular might generally be subject to cell-cycle control in all eukaryotes. Furthermore, the al- most co-incident pattern of activity displayed by these enzymes in various systems also suggested that their cell-cycle-dependent expression might be regulated by a coordinating mechanism.

Much of the early, pioneering molecular biology in this field involved studies of a few genes (e.g. dihydrofolate reductase, thymidine kinase) in various mammalian cell lines. However, it was of interest to isolate and examine the expression of several dNTP metabolism genes in one system. In this way, the possibility that these genes might be co-regulated within the cell cycle could be carefully explored. The budding yeast Saccha- romyces cerevisiae was an ideal system for this kind of study primarily because the molecular biology and genetics of this organism were well advanced. However, an additional advantage of this system was the extensive collection of temperature-sensitive cell-division cycle (cdc) mutants characterized by Hartwell and coworkers. These mutants provided a potential means of isolating genes involved in or affecting dNTP metabolism and DNA replication.

The first of the dNTP metabolism genes to be cloned from S. cerevisiae was thymidylate synthase (TMP1 or CDC21) (Taylor et al., 1982). Shortly thereafter, Storms et al. (1984) found that the amounts of both thymidylate synthase mRNA and enzyme fluctuated through the cell-cycle peaking during G1 to S phase transition. Subse- quent to this study, several o ther dNTP metabolism genes from S. cerevisiae were cloned and similarly analysed. Contrary to the results for TMP1, the levels of dCMP deaminase (DCD1), dihydrofolate reductase (DFR1) and dUTP py- rophosphatase (DUT1) mRNAs did not exhibit cell-cycle control (Mclntosh and Haynes, 1986; Mclntosh et al., 1986). However, for these particular genes, possible posttranslational forms of regulation were not investigated. Following these studies, White et al. (1987) reported thymidylate kinase (CDC8) and DNA ligase (CDC9) tran-

scripts to vary during the cell cycle in a manner essentially the same as TMP1. It was also shown in this study, that the cell-cycle-dependent (or "periodic") expression of TMP1, CDC8 and CDC9 differed from that of the yeast histone genes in both timing and regulation. Histone H2A transcripts were induced and peaked later in the cell cycle and this process was shown to be dependent on a functional CDC4 gene product. Conversely, the earlier and coincident induction of TMP1, CDC8 and CDC9 was found to be independent of CDC4. Thus, it became clear that some yeast dNTP metabolism genes exhibited cell-cycle regulation, at least at the level of mRNA abundance. The discovery that DNA ligase was subject to cell-cycle control indicated that this type of regulation was not unique to dNTP metabolism genes and therefore might be common to many genes involved in DNA replication in S. cerevisiae.

Evidence of a cell-cycle-dependent transcriptional mechanism operating on these periodically regulated genes, and preliminary identification of the common cis-acting element involved, came from studies of the TMP1 gene (Mclntosh et al., 1988; Ord et al., 1988). Using a CYC1-TMP1 reporter construct, it was shown that replacing the 5' nontranscribed region of TMP1 with the CYC1 UAS (upstream activation sequence) elements resulted in constitutive production of TMP1 mRNA during the cell cycle (Mclntosh et al., 1988). This observation argued against differential changes in mRNA stability as a mechanism for cell-cycle regulation of TMP1 transcript levels. Complementary analyses with TMP1 and TMPI-lacZ gene fusions confirmed that the periodic regulation of TMP1 mRNA levels resulted from transcriptional control. Deletion of a 37-bp segment between two MluI restriction sites present in the 5' nontranscribed region of TMP1 caused a severe reduction in transcription of this gene in asynchronous cultures (Mclntosh et al., 1988). The same deletion made in a TMP1-LacZ reporter similarly reduced expression and also resulted in the loss of periodic regulation of /3- galactosidase (Mclntosh et al., 1988; Ord et al., 1988). These results indicated that a periodic UAS element either lay between, or encom- passed, one or both of the MluI sites present in

the TMP1 upstream region. In the study of Mcln- tosh et al. (1988), it was also shown that TMP1 transcription remained under ceil-cycle control even when the gene was amplified on a high copy number vector. This result in particular, established that S. cerevisiae had the intrinsic capacity to coordinately regulate many genes at the transcriptional level in the same manner as TMP1 during G1 to S phase transition.

During and shortly after these studies, several other genes encoding proteins involved in DNA replication in S. cerevisiae were cloned and sequenced. Inspection of the 5' noncoding regions of these genes revealed that they all contained MluI sites (sequence 5'-ACGCGT-3') or sequences that closely resembled this site. The re- suits described above implicated these sites in the periodic transcription of TMP1. Studies by several groups then established that MluI sites are indeed involved in the periodic transcription of at least some of these genes. First, using site-directed mutagenesis of the native gene, and sub- cloning experiments with reporter constructs, Mclntosh et al. (1991) found that the 9-bp sequence ACGCGTFAA, containing the MluI site most distal to the TMP1 coding region, was sufficient to direct cell-cycle-dependent transcription. The smaller 6-bp ACGCGT sequence also worked as a periodic UAS element but appeared to be less effective than the larger 9-bp sequence. This indicated that sequences flanking the distal MluI site contributed to complete UAS activity. By comparison of sequences adjacent to MluI sites of seven cell-cycle-regulated DNA replication genes, Mclntosh et al. (1991) suggested a slightly larger consensus sequence of ACGCGTnA. Pre- sumably, the additional A residue plays an important role in recognition of this site by a positive transcription factor. Since the complete consensus ACGCGTnA is also present at the proximal MluI site on the TMP1 antisense strand, it was surprising that this site exhibited only weak UAS activity in vivo. However, this particular site lies close to a potential TATA box and steric hinder- ance of a transcription factor by TATA box binding factors could also account for the low UAS activity of this element.

Another important result from this study was the identification of particular bases within the

63

distal TMP1 MluI site that were either critical or dispensible for UAS function. Using site-directed mutagenesis, it was shown that two independent point mutations (ACGCGT ~ A C T C G T ; ACGCGT ~ ACGCAT) in the distal site severely reduced transcription of this gene in vivo. Substi- tutions at the 5'A or 3'T of this site had little effect on TMP1 transcription in asynchronous cultures. Furthermore, disruption of this MluI site by insertion of 4 bp (CGCG) did not ad- versely affect transcription, even when the proximal MluI site was destroyed. These results demonstrated that certain bases within the MluI site are critical for UAS activity. However, they also showed that the complete hexameric ACGCGT sequence is not absolutely necessary for this function.

Concurrent studies from other groups also demonstrated the role of MluI sites in the periodic transcription of these genes. Using a CDC9-lacZ reporter construct, Lowndes et al. (1991) found that deletion of a region encompassing the MluI site in the CDC9 upstream region resulted in the loss of cell cycle regulation. How- ever, they also found that two or more repeats of the MluI hexamer were necessary to activate periodic expression of a UAS-deficient CYC1- lacZ reporter. A mutated version of the MluI sequence (ACGCGT ~ ACTAGT), even when present in multiple copies, was inactive as a UAS element. Using gel shift assays, it was also shown that a factor (termed "DSCI") present in yeast crude cell extracts bound to probes containing two or more copies of the hexamer. The binding of this factor to labelled probes was sensitive to competition by unlabelled probes containing multiple ACGCGT sequences but was insensitive to competitors containing only one copy of this sequence or the mutant version described above. Evidence presented in this study also indicated that DSC1 activity is cell-cycle-dependent, however, other reports have contradicted this finding (Dirick et al., 1992; Marini and Reed, 1992).

At the same time, Gordon and Campbell (1991) reported that MluI sites are also essential for the periodic transcription of the POLl (DNA polymerase a) gene. Either one of the two MluI sites in the 5' noncoding region of this gene was sufficient for proper cell-cycle regulation. Dele-

64

tion of both sites caused a severe reduction in expression and also resulted in the loss of periodic regulation of a POLI- lacZ reporter gene. The 10-bp sequence 5 ' -ACGCGTCGCG-3 ' encompassing the more ATG-proximal of the two POLl MluI sites was sufficient to act as a periodic UAS element for a lacZ reporter. However, since deletion of the proximal MluI site from POLl did not eliminate all UAS activity, the remaining MluI site must contribute indepen- dently to a UAS function. The sequence encompassing the distal POLl MluI site (ACGCGT- TAA) matches the effective UAS element defined for TMP1. In another recent study of POLl, Pizzagalli et al. (1992) reported similar results. Consistent with findings for TMP1 and CDC9, it was shown in this study that the A C G C G T hexamer alone exhibited only weak UAS activity.

Collectively, the studies described above established that the A C G C G T hexamer, or some sub- sequence, is a critical component of a cell-cycle- dependent UAS element in S. cerevisiae. How- ever, they also indicated that sequences flanking this site are important for complete activity. Dirick et al. (1992) have recently reported that a par- tially purified complex containing the SWI6 gene product and a 120-kDa protein (p120) binds to the TMP1 MluI sites in vitro. The results of methylation and carbethoxylation interference analyses indicate that this complex binds to a slightly larger sequence present at both sites in- cluding the AC GCGTnA consensus. Cross-lin- king experiments also revealed that the p120 protein, but not Swi6, is in close physical contact with the DNA. Lowndes et al. (1992a) have demonstrated that Swi6 is a component of the DSC1 complex and so it is likely that the complex described by Dirick et al. (1992) is actually DSC1.

Apart from these reports only one other group has characterized an activity from yeast that is likely to interact with the MCB element in vivo. Verma et al. (1991) have purified to near homo- geneity a 17-kDa protein (henceforth referred to as p17) that binds specifically to probes containing one or more A C G C G T hexamers. DNAase I footprinting analysis demonstrated that this protein recognizes both MluI sites of the POLl gene in vitro. Using gel shift assays, it was also shown that the protein bound to probes containing MCB

elements from TMP1 and two other periodically regulated genes (CDC2 and CDC6). Evidence that the p17 protein represents a factor that binds the MCB element in vivo comes from the finding that oligonucleotides containing the same MluI point mutations which were found to severely inhibit TMP1 transcription in vivo (ACTCGT, A C G C A T ) fail as effective competitors in the gel shift assay. This suggests that p17 acts as a positive factor at these sites in vivo. Based on this work and that of Dirick et al. (1992), it appears that two distinct factors, p120 and p17, recognize MCB elements in S. cerevisiae. The relation between these two proteins, if any, is currently unknown.

To date, more than 20 genes from S. cerevisiae that are involved in dNTP metabolism and DNA replication have been cloned and sequenced. In most cases, these genes exhibit periodic regulation as evident from fluctuations in steady-state mRNA levels during the cell cycle. Each of these genes contain in their 5'-noncoding regions at least one MluI site or a slight variation of this sequence (Table 1). Comparison of the sequences encompassing these sites reveals that the A CG CG T hexamer, and in particular the 4-bp CGCG core, is well conserved. Simple inspection reveals little in common among the sequences flanking the MluI motifs other than a clear bias for a 3' A residue in the A CG CG Tn A consensus. However, a more careful analysis indicates that certain bases at particular flanking sites are slightly favored (50-117%) over random produc- ing a larger consensus of 5 ' -GTGACGCGT- nAnnT-3'. In most cases, at least one match to this consensus is found in sequences both 5' and 3' to the A C G C G T hexameric core. This larger sequence would be consistent with the results described above, indicating that the A CG CG T hexamer alone is insufficient for optimal activity of the UAS element. It could also account for the lack of the 3' A flanking MluI sites in some of these genes or why, in some cases, multiple A C G C G T hexamers are needed to observe UAS activity. For example, the presence of bases matching the 5' flanking GTG, or the 3' T of this larger consensus may compensate for absence of the conserved A residue. Thus, the minimal UAS defined for POLl, even though it lacks the termi-

nal A residue, is a reasonable fit with this sequence (5 '-ACGCGTCGCG-3 ', or on the antisense strand, 5'-CGCGACGCGT-3'). Although this consensus is derived by comparison of sequences only from genes proved to display cell- cycle control, it is not known if all of the sequences listed in Table 1 are functional as UAS elements. However, based on the results described above, it is likely that most of these genes are regulated coordinately in the cell cycle at the transcriptional level by a factor (or factors) operating through sequences encompassing MluI (or MluI-like) sites. The term MCB (MluI cell cycle box) element has been adopted to collectively describe these UAS sequences.

In addition to DNA replication genes, three genes involved in various aspects of DNA repair (UNG1, RAD51, RAD54) and one gene encoding a putative nucleoskeletal protein (NUF1) also contain potential MCB elements within their 5' noncoding regions. It has been reported (but data not presented) that UNG1 (Impellizzeri et al., 1991) and RAD54 (Marini and Reed, 1992) are cell-cycle regulated. If this is also true for RAD51 and NUF1, the MCB element may be an integral component of a major regulatory pathway that controls not only dNTP metabolism, but DNA replication, some aspects of DNA repair, and possibly, nuclear replication in general. Since the genes listed in Table 1 probably represent only a fraction of those required for these processes, the total number of genes in S. cerev/siae that are regulated by the MCB element is likely to be far more extensive than that shown here.

Apart from DNA replication genes, the HO endonuclease gene is also transcribed in a cell- cycle-dependent manner (in mother cells) during G1-S phase transition. A c/s-acting sequence termed the SCB element (consensus 5'- CACGAAA-3'), repeated 10 times in the URS2 region of the gene, acts cooperatively as a periodic UAS element (Breeden and Nasmyth, 1987; Andrews and Herskowitz, 1989). The products of both the SWI4 and SWI6 genes operate through this element to promote cell-cycle-dependent transcription of HO (Breeden and Nasmyth, 1987; Andrews and Herskowitz, 1989; Taba et ai.,1991). Because HO and the DNA replication genes display similar patterns of regulation in the cell

65

cycle, it is tempting to speculate that SWI4 and SWI6 coordinate periodic transcription for all of these genes. Contrary to this, however, the recent reports by Lowndes et al. (1992a) and Dirick et al. (1992) indicate that Swi6 operates at the MCB elements in the absence of Swi4. Gel shift assays have shown that anti-Swi6 antibodies supershift complexes bound to probes containing MCB elements, whereas anti-Swi4 antibodies do not. Fur- thermore, DSC1 complexes are active in extracts prepared from swi4- mutants but not from swi6- strains. Since the expression of the HO gene requires both Swi4 and Swi6 proteins, it is clear that the mechanisms of periodic transcription directed by MCB and SCB elements are distinct. To account for these findings it has been suggested that Swi6 acts as a modulator of gene expression through interaction with different DNA binding proteins (Dirick et al., 1992). Thus, Swi4/Swi6 "and Swi6/pl20 complexes bind to and regulate SCB and MCB elements respec- tively. However, since the Swi6/pl20 DNA binding activity may be cell-cycle-independent, Dirick et al. (1992) have also proposed that this complex acts as a repressor outside of late G1 and early S phase. Phosphorylation of Swi6, possibly by the Cdc28 protein kinase, might convert the repressor to an activator of transcription.

Although this is an interesting hypothesis, it seems inconsistent with the fact that SWI6 is a nonessential gene. It is also surprizing that the levels of transcripts from MCB-controlled DNA replication genes are not largely affected in asynchronous cultures by swi6- mutations (Lowndes et al., 1992a; Dirick et al., 1992). This suggests that SWI6 may be an essential component of repression at MCB elements, but a non-essential component of transcription activation in vivo. This alternative hypothesis does not preclude an essential role for SWI6 in the activation of transcription through SCB elements since this process is dictated by a different (Swi4/Swi6) protein interaction. However, it could account for the apparent existence in S. cerevisiae, of two different proteins (p120 and p17) which bind MCB elements. Thus, it is possible that Swi6/pl20 acts as a repressor complex at these sites and is dis- placed by p17 at the time of transcription activation during late G1. If p17 is also a component of

f 6

T A B L E 1

S E Q U E N C E S E N C O M P A S S I N G M C B E L E M E N T S IN S. cerevisiae G E N E S

G e n e P r o d u c t / f u n c t i o n S e q u e n c e Cell cycle Ref.

TMP1 thymidylate synthase gGTGACG¢GTtAaaT + a atTGACGCGTttccT

CDC8 t h y m i d y l a t e k ina se t t T G A C G C G T t A g g c + a

CDC9 D N A l igase ctTaACGCGaaAacg g a a a A C G C G T g A a a g

POLl D N A p o l y m e r a s e I t caaACGCGTtAaaa + a

t aaaACGt6TcgcgT cGCGtCGCGTgtatc

POLl2 D N A p o l y m e r a s e I g t a G A C G C G T a A t t T + b

f l - subun i t c GTGACGCGTc t c a c

POL2 D N A p o l y m e r a s e H c a g a A C G C G T a A g t T + c

DPB2 D N A p o l y m e r a s e H t G T G A C G C G T t A t t T + d

7 9 - k D a l s u b u n i t atTttC6CGTgAcaT

DPB3 D N A p o l y m e r a s e H t a T t A C G C G a a A t t a + e

30-, 3 4 - k D a s u b u n i t s

POL3 D N A p o l y m e r a s e H I t aTtACGCGTaActa + c

POL30 P C N A ACGCGTaAc tT + a

TOP2 D N A t o p o i s o m e r a s e II aaaGAC6CGcagt ac ? f

tagGACGCGTctttT

TOP3 D N A t o p o i s o m e r a s e I l l g t g a A C G C G a a A a t c ? g

PRI1 D N A p r i m a s e a6TatC6CGatcaaT + c

4 8 - k D a s u b u n i t t c c t A C 6 t GTgAa t g

aaTGAt6CGTgAgtT caTaACGCcaaAaag

PRI2 D N A p r i m a s e aaTtACGCGTcgcgg + a

5 8 - k D a s u b u n i t cGcGtCGCGgggaaT

gGaatCGCGTaAaac RFA1 D N A r e p l i c a t i o n f a c t o r A tGTaACGCGTaAaaa + h

6 9 - k D a s u b u n i t aaaGACGCGTgAacT

RFA2 D N A r e p l i c a t i o n f a c t o r A cGa aAC6CGTtAgga + h

3 6 - k D a s u b u n i t gGa aACGCGT t c t t T

RFA3 D N a r e p l i c a t i o n f a c t o r A atTGACGCGccAaaT + h

1 3 - k D a s u b u n i t caatAgGCGatAttT

CDC6 u n k n o w n f u n c t i o n gccGACGCGggtaag + c gccGACGgGaggccT ccTaACGCGTcggag

DBF4 D N A synthes i s tcgaACGCCTaAgtT + i

g c a a A C G C G T c t t a g

DNA43 D N A synthes i s t t T c A C G C G g t t t c T ? j

UNG1 u] acil D N A glycosy lase c G c a A C G C G T a A t t c + k

RAD51 D N A r e p a i r r ecA- l i ke t G c t A C G C G T c A t t T + I p r o t e i n c a g t A C G C 6 T g g t g g

RAD54 D N A r e p a i r ttTtAC6CGTtAccc + a a6T t tC6C6caAaaa

NUF1 n u c l e o s k e l e t a l a a T t A C 6 C 6 T t A t a a ? m

c o m p o n e n t a t g t g C G C G g a t a t c

C o n s e n s u s cGTGACGCGTnAnnT

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the CDC28 positive feedback loop, the potential interaction with Swi4 and Swi6 dependent processes becomes more complex. Clearly, the exact roles of these proteins at MCB elements requires further study.

From the results described above it is likely that the MCB element represents a major point of control over the initiation of DNA replication and possibly also G1 to S phase transition in S. cerevisiae. A recent report by Lowndes et al. (1992b) indicates that MCB elements are also involved in the periodic expression of the Schizosaccharomyces pombe cdc22 ÷ gene which encodes the large subunit of ribonucleotide reductase. An exact match to the ACGCGTnA motif and several other MCB-like sequences are present in the 5' nontranscribed region of this gene. A DSCl-like MCB binding activity is present in crude extracts of S. pombe and competition experiments indicate that this activity is specific for MCB elements. It was also shown that this activity was either defective or temperature sensitive in extracts prepared from cdclO- mutants indicating that the cdclO + gene product is a component of the binding complex. Based on these results, it is likely that S. cerevisiae and S. pombe utilize the same transcriptional control sequence, the MCB element, for periodic transcription of DNA replication genes. MCB-like elements are present upstream of the S. pombe DNA polymerase I (POLl) gene and also the Candida albicans thymidylate synthase gene (Ta- ble 2). Since S. cerevisiae and S. pombe are only distant relatives, these observations suggest the MCB element has been conserved to some extent in nature. This would not be surprising because other elements fundamental to cell-division control (p34, cyclins) have also, to some degree, been conserved throughout evolution.

NOTES TO T A B L E 1

A question arising from these findings is whether or not the MCB element, and its atten- dant transcription factors, are conserved in higher eukaryotic systems. Two obvious candidates as such homologues are the E2F and HIP1 transcription factors, both of which have been implicated in the cell-cycle-dependent expression of mammalian dihydrofolate reductases (Blake and Azizkhan, 1989; Means et al., 1992). Although on the basis of molecular weight, E2F (54 kDa) and HIP1 (180 kDa) are different proteins, they recognize very similar sequences (TTTCGCGC for E2F; TTCGCGCCA for HIP1). Contained within these sequences is the 4-bp CGCG motif that is the highly conserved core of the yeast MCB element. Site-directed mutagenesis of the HIP1 site (Means et al., 1992) and methylation interference analysis for E2F (Blake and Azizkhan, 1989) suggest that the G residues within these sites may be critical for" protein-DNA interaction. As described above, mutagenesis of the S. cerevisiae TMP1 MCB element has shown that the highly conserved G residues are also important for optimal UAS activity. Whether E2F or HIP1 homologues exist in yeast remains to be determined. The sizes of the MCB-binding proteins characterized by Verma et al. (1991), and Dirick et al. (1992) suggest that neither of these is similar to HIP1 or E2F. Nevertheless, it is remarkable that the 4-bp CGCG core sequence is common to at least some of the elements that influence cell- cycle-dependent transcription of various DNA replication genes in both lower and higher eukaryotic systems.

G1 cyclins and the MCB element

It is increasingly clear that interaction of G1 cyclins with the p34 (or p33) protein kinase is

References for sequences shown are given in a Mclntosh et al. (1991); or c Pizzagalli et al. (1992). Other references are as follows; b D. Hinckle, personal communication; d Araki et al. (1991a); e Araki et al. (1991b); f Giaever et al., (1986); g Wallis et al. (1989); h Brill and Stillman (1991); i Kitada et al. (1992); J Solomon et al. (1992); k Percival et al. (1989); I Sinohara et al. (1992); m Mirzayan et al. (1992). Genes that have been shown to exhibit cell cycle regulation are indicated (+ ) . Other genes that may be under cell-cycle control but that have not yet been shown to exhibit this regulation are also indicated (?). The consensus was derived by analysis of sequences from the genes listed in Table 1 that display cell-cycle control. The percentage over random expectation for bases at sites outside of the A C G C G T core is as follows: 5 ' -G (63) T (50) G (63) A C G C G T n A (117) nnT (50). Highlighted, uppercase bases indicate matches to the consensus sequence. Note that these sequences are taken only from the sense strand for each gene.

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largely responsible for G1-S phase transition and entry into the cell cycle in eukaryotes. Presum- ably, G1 cyclins activate and/or direct the p34 kinase to specific targets that promote cell-cycle phase transition. In the case of S. cereuisiae, a likely target for this kinase is the transcription factor(s) that operate through the MCB element. However, as discussed below, MCB elements may also contribute to the regulation of the p34/G1 cyclin complex.

Of the three known G1 cyclins of S. cerevisiae, two (CLN1, CLN2) exhibit periodic changes in mRNA levels during G1 to S phase transition (Wittenberg et al., 1990). CLN3 transcript levels are independent of cell-cycle stage, and regulation of the CLN3 gene product is likely achieved through posttranslational processes influenced by SW14 and SWI6 (Nasmyth and Dirick, 1991). The timing and pattern of CLN1 and CLN2 mRNA levels in the cell cycle are highly similar to those of CDC9 and HO (Marini and Reed, 1992). CLN1 or CLN2 overexpression can suppress the temperature-sensitive lethality of some swi4- strains, a fact suggesting that these cyclins influ-

ence SWI4 expression (Ogas et al., 1991). How- ever, swi4- and swi6- mutants exhibit reduced (but detectable) CLN1 and CLN2 transcript levels, indicating that SWI4 and SWI6 are involved, to some degree, in the cell-cycle-dependent transcriptional regulation of these cyclin genes (Nasmyth and Dirick, 1991; Ogas et al., 1991). To account for these findings, it has been proposed that these SWI and CLN gene products partici- pate in a positive feedback loop such that SWI4 and SWI6 stimulate G1 cyclin expression, and this, in turn, through activation the p34 kinase, enhances further production of these SWI proteins (Dirick and Nasmyth, 1991). Consistent with this hypothesis is the observation that sequences similar to the HO SCB element are found in the 5' noncoding regions of CLN1, CLN2 and another putative cyclin, HCS26 (Ogas et al., 1991). However, an MluI motif is also present upstream of CLN1, and sequences similar to this motif which contain the conserved CGCG core are present in the 5' noncoding regions of CLN1, CLN2 and HCS26 (Table 2). This observation raises the possibility that the transcription of these

T A B L E 2

M C B - L I K E E L E M E N T S IN S. cerevisiae G I C Y C L I N S A N D D N A R E P L I C A T I O N G E N E S F R O M O T H E R F U N G I

G e n e P r o d u c t / ~ n c t i o n S e q u e n c e Cell ~ c l e Ref .

CLN1 G1 ~ c l i n a G a G A C 6 C 6 T t c a a g + a

g a a t t C G C 6 a t t t t a

c6cacCGC6TtAgtg

CLN2 G1 ~ c l i n tcatcCGCGctttac + a

c G a a A C G g G c c A a a a

HCS26 G I ~ c l i n g 6 c a g C G C G g t c a t T ? a t c c G g C G C G a a A t t T

ttTttCGC6gaggca

S. pombe cdc22 + r i b o n u c l e o t i d e t G g a c C G ¢ G T g t t t a + b

r e d u c t a s e a c a G t C G g 6 T c g c g T

cGcGt£GCGTtgcaa tGaGACGCGTaAata ttaat¢GcGrttttT

POLl D N A p o l y m e r a s e I t c g t A C G C G c t g t a g 9 c

ca t a ly t i c s u b u n i t g t g a c C G C G T a c t a T

C. aibicans t h y m i d y l a t e s y n t h a s e t c T G t C G C G T c t c a c .9 d

t G a t g C G C 6 T a A a t c

Bold , u p p e r case b a s e s c o r r e s p o n d to m a t c h e s o f t he M C B e l e m e n t c o n s e n s u s s e q u e n c e s h o w n in T a b l e 1. R e f e r e n c e s for t hese

s e q u e n c e s a r e as fol lows; a O g a s e t al . (1991); b L o w n d e s et al. (1992b); c D a m a g n e z e t al. (1991); d S inge r et al. (1989).

three cyclins in particular, involves the SWI4/ SWI6 dependent pathway and also the transcription factor(s) that operate through the MCB element. This could explain the recent observation that swi6- mutations reduce, but do not eliminate, cell-cycle regulation of CLN1 and CLN2 (Lowndes et al., 1992b; Dirick et al., 1992) suggesting that the periodic transcription of these genes involves another component which is independent of a Swi4/Swi6 complex.

If indeed an MCB element is involved in the periodic transcription of CLN1, CLN2 and HCS26, it is curious that two elements which produce the same effect would be required for the transcription of these genes. One reason for this may be that two separate signals are necessary to achieve a threshold level of CLN1, CLN2 (and perhaps also HCS26) expression sufficient to form enough active p34/Cln protein kinase complexes to promote G1-S phase transition. This would be consistent with the fact that at least two signals, cell size and nutrient availabil- ity, are required for S. cerevisiae to enter into the

69

cell cycle (reviewed in Carter et al., 1983). In this model, (Fig. 1) the SWI4/SWI6-dependent component of CLN1 and CLN2 transcription may respond to nutrient quality, whereas an MCB component, also participating in a positive feedback loop, may respond to cell size. It is possible that only in response to both pathways would a sufficient amount of Clnl and Cln2 protein be synthesized to overcome their rates of degradation in early G1. This is an attractive model because such a system would ensure that both conditions are met before the cell could commit to the division process. A careful analysis of the putative SCB and MCB elements within the CLN1 and CLN2 upstream regions could resolve this question.

Other mechanisms of regulation

From the results described above transcriptional changes evidently play a major role in the periodic regulation of many DNA replication genes in S. cerevisiae. However, other processes

nutrients

cell size

HO transcription I

j" Swi4/Swi6 • CLN3

/ MCBTFs - p17, p120/Swi6

DNA and nuclear replication I (G1-S transition) I

e L . l , . Ip3,c0c2S/eL.1,2 ,31 SWI4, HCS26

Fig. 1. The possible role of an MCB element transcription factors (collectively referred to as MCBTFs) in the G1 cyclin positive feedback loop. In this model, which is a modified version of that proposed by Dirick and Nasmyth (1991), small amounts of Swi4/Swi6 complex are activated in response to signals conveying nutritional quality of the medium. In turn, this enhances expression of three G1 eyclins (CLN1, CLN2, CLN3) through SCB elements and also CLN3 expression. Similarly, small amounts of MCBTFs becomes activated (positive factors) or inactivated (negative factors) in response to cell size, and this also stimulates cyclin (CLN1, CLN2, HCS26) transcription through MCB elements. Only in response to both of these signals is a sufficient level of Cln production reached to overcome the rate of eyclin proteolysis prior to START. This leads to the formation of active p34CDC2S/Cln protein kinase complexes. Presumably through phosphorylation, these complexes then feedback to activate considerably more Swi4/Swi6 ultimately resulting in more Cln production and enhanced transcription of HO (through Swi4/Swi6). Similarly, p34 kinase activity also enhances positive regulation through MCBTFs and MCB elements to promote transcription of G1 cyclins and also the nuclear replication genes. The expression of the replication genes leads to the initiation of DNA synthesis and thus, the

transition from G1 to S phase.

70

are also involved in achieving cell-cycle regulation for some of these genes in both yeast and higher systems. For example, the S. cerevisiae thymidylate synthase is an unstable enzyme relative to the bulk of cellular protein (Greenwood et al., 1986), indicating that protein turnover may contribute to its cell-cycle regulation. Consistent with this notion, the human thymidine kinase is regulated in part by cell-cycle stage specific prote- olytic degradation of the enzyme (Kaufmann and Kelly, 1991). In this case, transcriptional and translational changes also contribute to cell cycle control (Sherley and Kelly, 1988). Apart from these forms of regulation, a recent study suggests that p34-dependent phosphorylation of human DNA polymerase a may govern its activity in the cell cycle (Nasheuer et al., 1991). This may also be true of replication factor A (RF-A) in both yeast and humans (Din et al., 1990). However, in yeast, transcriptional controls operating through the MCB element are also likely involved in the periodic expression of the RF-A subunit genes (Brill and Stillman, 1991).

From these few examples, it is clear that multiple forms of regulation can contribute to cell-cycle control during G1 to S phase transition in eukaryotic ceils. It may be that posttranscriptional or posttranslational processes constitute the major form of regulation for DNA replication genes in some systems, or that complete regulation results from two or more of these processes acting in concert. Whether or not all of these regulatory mechanisms are governed by p34 or related kinases remains to be determined. Further analysis of the S. cerecisiae MCB element and its atten- dant transcription factor(s) should prove to be rewarding in understanding the role that differential transcription plays in G1/S phase transition in this organism. However, it cannot be em- phasized enough that elucidating the molecular basis of all forms of cell-cycle-dependent gene expression will likely be necessary before a complete understanding of eukaryotic cell-division control is achieved.

Acknowledgement

I wish to thank Dr. Robert H. Haynes for his kind support and encouragement over the years

and also for comments on this manuscript. I also thank Dr. Reg Storms for many stimulating con- versations on the subject of this paper and an enjoyable research collaboration. The author is supported by a grant from the Natural Sciences and Engineering Research Council of Canada to Dr. Robert H. Haynes.

References

Andrews, B.J., and I. Herskowitz (1989) Identification of a DNA binding factor involved in cell-cycle control of the yeast HO gene, Cell, 57, 21-29.

Araki, H., R.K. Hamatake, L.H. Johnston and A. Sugino (1991a) DPB2, the gene encoding DNA polymerase II subunit B, is required for chromosome replication in Sac- charomyces cerevisiae, Proc. Natl. Acad. Sci. (U.S.A.), 88, 4601-4605.

Araki, H., R.K. Hamatake, A. Morrison, A.L. Johnson, L.H. Johnston and A. Sugino (1991b) Cloning DPB3, the gene encoding the third subunit of DNA polymerase II of Saccharornyces cerevisiae, Nucl. Acids Res., 19, 4867-4872.

Blake, M.C., and J.C. Azizkhan (1989) Transcription factor E2F is required for efficient expression of the hamster dihydrofolate reductase gene in vitro and in vivo, Mol. Cell. Biol., 9, 4994-5002.

Breeden, L., and K. Nasmyth (1987) Cell cycle control of the yeast HO gene: cis- and trans-acting regulators, Cell, 48, 389-397.

Brill, S.J., and B. Stillman (1991) Replication factor-A from Saccharomyces cerevisiae is encoded by three essential genes coordinately expressed at S phase, Genes Develop., 5, 1589-1600.

Carter, B.L.A., J.R. Piggot and E.F. Walton (1983) Genetic control of proliferation, in: J.T.F. Spencer, D.M. Spencer and A.R.W. Smith (Eds.), Yeast Genetics, Fundamental and Applied Aspects, Springer Series in Molecular Biol- ogy, Springer, New York, pp. 1-28.

Damagnez V., J. Tillit, A. de Recondo and G. Baldacci (1991) The POLl gene from fission yeast, Schizosaccharomyces pombe, shows conserved amino acid blocks specific for eukaryotic DNA polymerases alpha, Mol. Gen. Genet., 226, 182-189.

Din, S., S.J. Brill, M.P. Fairman and B. Stillman (1990) Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells, Genes Develop., 4, 968-977.

Dirick. L., and K. Nasmyth (1991) Positive feedback in the activation of G1 cyclins in yeast, Nature (London), 351, 754-757.

Dirick, L., T. Moll, H. Auer and K. Nasmyth (1992) A central role for SWI6 in modulating cell-cycle start-specific transcription in yeast, Nature (London), 357, 508-513.

Giaever, G., R. Lynn, T. Goto and J.C. Wang (1986) The complete nucleotide sequence of the structural gene TOP2 of yeast DNA topoisomerase II, J. Biol. Chem., 261, 12448 - 12454.

Gordon, C.B., and J.L. Campbell (1991) A cell cycle-respon- sive transcriptional control element in the gene encoding DNA polymerase a in Saccharomyces cerevisiae, Proc. Natl. Acad. Sci. (U.S.A.), 88, 6058-6062.

Greenwood, M.T., E.M. Calmels and R.K. Storms (1986) Growth-rate-dependent regulation of the expression and inactivation of thymidylate synthase in Saccharomyces cerevisiae, J. Bacteriol., 168, 1336-1342.

Impellizzeri, K.J., B. Anderson and P.M.J. Burgers (1991) The spectrum of spontaneous mutations in a Saccharomyces cerevisiae uraciI-DNA-glycosylase mutant limits the function of this enzyme to cytosine deamination repair, J. Bacteriol., 173, 6807-6810.

Johnston, L.H., and N.F. Lowndes (1992) Cell cycle control of DNA synthesis in budding yeast, Nucleic Acids Res., 20, 2403-2410.

Kaufmann, M.G., and T.J. Kelley (1991) Cell cycle regulation of thymidine kinase: residues near the carboxy terminus are essential for the specific degradation of the enzyme at mitosis, Mol. Cell. Biol., 11, 2538-2546.

Kitada, K., L.H. Johnston, T, Sugino and A. Sugino (1992) Temperature-sensitive cdc7 mutations of Saccharomyces cerevisiae are suppressed by the DBF4 gene, which is required for the G1/S cell cycle transition, Genetics, 131, 21-29.

Lowndes, N.F., A.L. Johnson and L.H. Johnston (1991) Coor- dination of expression of DNA synthesis genes in budding yeast by a cell-cycle regulated trans factor, Nature (London), 350, 247-250.

Lowndes, N.F., A.L. Johnson, L. Breeden and L.H. Johnston (1992a) SWI6 protein is required for the periodically expressed DNA synthesis genes in budding yeast, Nature (London), 357, 505-508.

Lowndes, N.F., C.J. McInerny, A.L. Johnson, P.A. Fantes and L.H. Johnston (1992b) Control of DNA synthesis genes in fission yeast by the cell-cycle gene cdclO +, Nature (London), 355, 449-453.

Marini, N.J., and S.I. Reed (1992) Direct induction of G1- specific transcripts following reactivation of the Cdc28 kinase in the absence of de novo protein synthesis, Genes Develop., 6, 557-567.

McIntosh, E.M., and R.H. Haynes (1986) Sequence and expression of the dCMP deaminase gene (DCD1) of Saccha- romyces cerevisiae, Mol. Cell. Biol., 6, 1711-1721.

McIntosh, E.M., M.H. Gadsden and R.H. Haynes (1986) Transcription of genes encoding enzymes involved in DNA synthesis during the cell cycle of Saccharomyces cerevisiae, Mol. Gen. Genet., 204, 363-366.

McIntosh, E.M., R.W. Ord and R.K. Storms (1988) Transcrip- tional regulation of the cell cycle-dependent thymidylate synthase gene of Saccharomyces cerevisiae, Mol. Cell. Biol., 8, 4616-4624.

McIntosh, E.M., T.A. Atkinson, R.K. Storms and M. Smith (1991) Characterization of a short, c/s-acting DNA sequence which conveys cell cycle stage-dependent transcription in Saccharomyces cerevisiae, Mol. Cell. Biol., 11, 329-337.

71

Means, A.L., J.E. Slansky, S.L. McMahon, M.W. Knuth and P.J. Farhnam (1992) The HIP1 binding site is required for growth regulation of the dihydrofolate reductase gene promoter, Mol. Cell. Biol., 12, 1054-1063.

Mirzayan, C., C.S. Copeland and M. Snyder (1992) The NUF1 gene encodes an essential coiled-coil related protein that is a potential component of the yeast nucleoskeleton, J. Cell. Biol., 116, 1319-1332.

Nasheuer, H., A. Moore, A.F. Wahl and T.S.-F. Wang (1991) Cell-cycle-dependent phosphorylation of human DNA polymerase a, J. Biol. Chem., 266, 7893-7903.

Nasmyth, K., and L. Dirick (1991) The role of SWI4 and SWI6 in the activity of G1 cyclins in yeast, Cell, 66, 995-1013.

Ogas, J., B.J. Andrews and I. Herskowitz (1991) Transcrip- tional activation of CLN1, CLN2, and a putative new G1 cyclin (HCS26) by SWI4, a positive regulator of Gl-specific transcription, Cell, 66, 1015-1026.

Ord, R.W., E.M. Mclntosh, L. Lee, P.P. Poon and R.K. Storms (1988) Multiple elements regulate expression of the cell-cycle-regulated thymidylate synthase gene of Sac- charomyces cerev/siae, Curr. Genet., 14, 363-373.

Percival, K.J., M.B. Klein and P.M.J. Burgers (1989) Molecu- lar cloning and primary structure of the uracil-DNA-glycosylase gene from Saccharomyces cerevisiae, J. Biol. Chem., 264, 2593-2598.

Pizzagalli, A., S. Piatti, D. Derossi, I. Gander, P. Plevani and G. Lucchini (1992) Positive c/s-acting regulatory sequences mediate proper control of POLl transcription in Saccha- romyces cerevisiae, Curr. Genet., 21, 183-189.

Sherley, J.L., and T.J. Kelly (1988) Regulation of human thymidine kinase during the cell cycle, J. Biol. Chem., 263, 8350-8358.

Singer, S.C., C.A. Richards, R. Ferone, D. Benedict and P. Ray (1989) Cloning, purification and properties of Can- dida albicans thymidylate synthase, J. Bacteriol., 171, 1372-1378.

Sinohara, A., H. Ogawa and T. Ogawa (1992) Rad51 protein involved in repair and recombination in S. cerevisiae is a recA-like protein, Cell, 69, 457-470.

Solomon, N.A., M.B. Wright, S. Chang, A.M. Buckley, L.B. Dumas and R.F. Gaber (1992) Genetic and molecular analysis of DNA43 and DNA52: two new cell-cycle genes in Saccharomyces cerev/siae, Yeast, 8, 273-289.

Storms, R.K., R.W. Ord, M.T. Greenwood, B. Miradammadi, F.K. Chu and M. Belfort (1984) Cell-cycle-dependent expression of thymidylate synthase in Saccharomyces cerevisiae, Mol. Cell. Biol., 4, 2858-2864.

Taba, M.R.M., I. Muroff, D. Lydall, G. Tebb and K. Nasmyth (1991) Changes in a SWI4,6-DNA-binding complex occur at the time of HO gene activation in yeast, Genes De- velop., 5, 2000-2013.

Taylor, G.R., B.J, Barclay, R.K. Storms, J.D. Freisen and R.H. Haynes (1982) Isolation of the thymidylate synthase gene (TMP1) by complementation in Saccharomyces cerevisiae, Mol. Cell. Biol., 2, 437-442.

Verma, R., A. Patapoutian, C. Gordon and J.L. Campbell

72

(1991) Identification and purification of a factor that binds to the MluI cell cycle box of yeast DNA replication genes, Proc. Natl. Acad. Sci. (U.S.A.), 88, 7155-7159.

Wallis, J.W., G. Chrebet, G. Brodsky, M. Rolfe and R. Rothstein (1989) A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase, Cell, 58, 409-419.

White, J.H.M., S.R. Green, D.G. Barker, L.B. Dumas and

L.H. Johnston (1987) The CDC8 transcript is cell cycle regulated in yeast and is expressed coordinately with CDC9 and CDC2I at a point preceding histone transcription, Exp. Cell Res., 171,223-231.

Wittenberg, C., K. Sugimoto and S. Reed (1990) Gl-specific cyclins of S. cerevisiae: cell cycle periodicity, regulation by mating pheromone, and association with the p34 cD¢2s protein kinase, Cell, 62, 225-237.

expression of dna replication genes in the yeast cell cycle

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