JOHN MCKINNEY AND FRED CROSS CELL CYCLE

A switch-hitter at the Start of the cell cycle Budding yeast protein Swi6 is a component of two distinct

transcription factors which activate expression of two sets of genes after the cell cycle progresses past 'Start'.

In the yeast Saccharomyces cerevisiae, commitment to cell-cycle progression occurs at a point known as 'Start' in G1 phase, the period between one mitosis and the next round of DNA replication. A similar commitment step in late G1 phase has been described for mammalian cells. Execution of Start requires the CDC28 and CLN gene products - - p34 kinase and Gl-acting cyclins respectively - - and is closely followed by events that prepare the cell for S phase, the period when genomic DNA is repli- cated. For example, expression of a number of genes encoding DNA-synthesis enzymes - - such as DNA poly- merase, ribonucleotide reductase and thymidylate syn- thase - - is induced in late G1 phase, during or just after Start. The transcription factors that regulate the DNA-syn- thesis genes are potential substrates for the molecular machinery that drives the Start transition.

The identification of a gene whose product controls ex- pression of the DNA-synthesis genes is therefore of con- siderable interest. In two recent papers the groups of Leland Johnston and Kim Nasmyth independently report that the SWI6 protein is a component of a DNA-binding factor previously implicated in the regulation of DNA-syn- thesis gene expression [1,2]. Swi6 is already known as a regulator of transcription of the HO gene, which encodes the endonuclease responsible for mating-type switching in late G1 phase after Start.

Interestingly, although expression of HO and the DNA- synthesis genes is induced in parallel, the cis~acting pro- moter elements responsible for their induction are quite different. Cell-cycle control of HO transcription depends on the motif CACGAAAA, known as the Swi4/6-depen- dent cell-cycle box (SCB). Cell-cycle control of transcrip- tion of the DNA-synthesis genes, however, depends on the motif ACGCGTNA, known as the MluI cell-cycle box (MCB). Either of these sequence elements can Confer cell cycle-regulated transcription when placed upstream of an otherwise unregulated promoter, although the pattern of expression directed by isolated MCB elements differs somewhat from that of the DNA-synthesis genes [3].

Transcription of the HO gene and activity of isolated SCB elements have previously been shown to depend on the products of the SWI4 and SWI6 genes, which can bind as a complex to the SCB motif in v i t ro-- Swi4 and Swi6 thus constitute the activity known as SBF, for SCB-bind- ing factor. Before the new work no genes were known to act in trans to regulate expression of the DNA-synthesis genes, although yeast extracts had been shown to contain an activity, designated DSC1 (for DNA synthesis control), which could bind specifically to the MCB motif in vitro

[4]. Remarkably, the Nasmyth and Johnston groups have now both demonstrated that Swi6, but not Swi4, is a component of the DSCl activity (designated MBF, for MCB-binding factor, by the Nasmyth group) [1,2]. Fur- thermore, mutation of SW/6, but not SWI4, eliminates the DSC1/MBF activity and disrupts the normal transcription pattern of the genes for ribonucleotide reductase and thymidytate synthase during the cell cycle.

The DSCl DNA-binding activity had previously been re- ported to fluctuate during the cell cycle approximately in parallel with transcription of the DNA-synthesis genes [4]. Nasmyth's group, however, find that the MBF DNA- binding activity does not vary during the cell cycle [2]. It is most likely that MBF and DSC1 are same DNA-binding activity, so the reason for the discrepancy between the results of the two groups is not clear. The issue is an important one, as modulation of the DNA-binding activity of DSC1/MBF is one possible mechanism for regulation of the DNA-synthesis genes during the cell cycle.

Nasmyth's group describe a second possible compo- nent of MBF, designated p120 on the basis of its elec- trophoretic mobility,which can be UV-crosslinked in vitro to DNA containing the MCB element. Interestingly, under the same conditions, Swi6 is not crosslinked. The authors suggest that SWI6 itself does not directly contact DNA, but is tethered to the MCB element by another factor, which may be p120. They further suggest that SWI6 might similarly bind only indirectly to SCB elements in the 140 promoter, in this case by interacting with SWI4. This conjecture is supported by the recent observation that SWI4, but not Swi6, can by itself bind specifically to isolated SCB elements [5]; it would be interesting to know if p120 can bind specifically to isolated MCB elements in the absence of Swi6.

It is most likely that p120 is distinct from Swi4, as Swi4 migrates at a somewhat more retarded position during electrophoresis on the gels used to detect p120 [2]. It is still possible, however, that p120 is a minor variant form of Swi4 that cannot be detected with antibodies directed against Swi4 and that has a faster electrophoretic mobility than the major form of Swi4. Given this caveat, it would be interesting to known whether p120 can be detected in extracts of a swi4 mutant strain. The observation that SCB elements can compete with MCB elements for binding in vitro to DSCl/MBF may be relevant to this issue of whether p120 and Swi4 are distinct proteins or different forms of the same protein [2].

It has recently been suggested that Swi4 and Swi6 also control expression of CLN1 and CLN2, two of the

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budding yeast Gl-cyclin genes [6,7]. Transcription of C/dV1 and CLN2 is regulated during the cell cycle in par- allel with that of HO and the promoters of both genes contain sequence elements similar to the SCB motif [6,7]. The CLN1 promoter also contains a sequence that matches the MCB motif (K. Nasmyth, personal communi- cation). Interestingly, the Nasmyth and Johnston groups both find that .a swi6 mutation reduces, but does not completely abolish, cell-cycle regulation of CLN2 expres- sion [1,2]. The effect of the sw/6mutation on CLN1 ex- pression is less clear: the Johnston group find that in a swi6 strain the regulation of C/dV1 expression is re- duced and occurs with aberrant timing [1]; the Nasmyth group, however, report that a swi6 mutation completely abolishes regulation of CLN1 expression [2]. It would be interesting to know whether a swi4 mutation has similar effects on the regulation of CLN1 and CLN2 expression. If neither swi4 nor swi6 mutations abolish regulation of CLN2 expression, the question is raised of what is responsible for the SW/-independent regulation of CLN2.

Sta.

HO SCB

MCB DNA-synthesis genes

Fig. 1. Swi6 is a component of two distinct transcription factors that activate expression of two classes of genes as cells pass Start in G1 phase of the cell cycle. There is evidence that the complex that activates the HO gene (and possibly the CLN'/ and C/_N2 genes [6,7]) binds DNA via Swi4 [5] and it is suggested that the complex activating the DNA-synthesis genes may similarly bind DNA via p120 [2].

The results described above imply that Swi6 can partici- pate in two apparently distinct protein complexes to reg- ulate transcription by binding to different promoter ele- ments (Fig.l). In the case of the SCB-binding activity, the SW/6 requirement for HO expression can be bypassed by overexpression of SWI4 [8], indicating that Swi6 does not have an essential role in transcriptional activation via SCB elements (cell-cycle regulation of HO expression is, however, reduced in this situation). Cell-cycle regula- tion of gene expression via MCB elements is reduced or

abolished in the absence of Swi6. The constitutive transcription level in the absence of Swi6 is, how- ever, intermediate rather than low and probably MCB- independent, as isolated MCB elements are not ac- tive in a swi6 mutant [1,2]. This result suggests that Swi6 may have a negative regulatory role outside of the period in late G1 during which MCB-regulated genes are expressed. Thus, for both SCB- and MCB- dependent gene expression Swi6 appears not to be essential for transcription per se, but rather to be required for the correct modulation of transcription during the cell cycle.

The discovery that Swi6 is a component of a DNA-bind- ing complex implicated in the regulation of DNA-syn- thesis genes in budding yeast is likely to be of broad significance, as an analogous activity has recently been identified in the distantly-related fission yeast, Scbizosac- dmromyces pombe [9]. towndes et at report that the product of the S. pombe cdclO + gene, which has lim- ited sequence similarity to Swi6, is a component of an DSC1/MBF-like factor that can bind in vitro to MCB elements [9]. MCB-like sequence elements are present in the promoter of the S. pombe cdc22 + gene, which encodes a subunit of ribonucleotide reductase and is maximally expressed during the G1/S phase transition. Although it is not known whether the MCB elements in the cdc22 + promoter are necessary for cell cycle- regulated expression, it was shown that MCB elements in isolation can direct cell cycle-regulated transcription in S. pombe in parallel with cdc22 + expression [9]. Fur- thermore, cdc22 + transcription is low in cells arrested in G1 by a temperature-sensitive cdclO mutation [9]. These results suggest that cdclO + may be the S. pombe homo- log of the S. cerevisiae SW/6gene; cdclO + cannot, how- ever, functionally substitute for SWI6when expressed in S.cerevisiae, nor can expression of SWI6 in S. pombe complement a cdclO mutation ([10,11]; see [3,5] for further discussion of these findings).

The findings we have discussed leave many important is- sues to be resolved. For example, although Swi4 is almost certainly the DNA-binding component of SBF [5], it is not yet clear whether p120 is the analogous component of DSC1/MBF [2]. The activation of DSC1/MBF and SBF in late G1 must be linked in some way to passage of a cell through 'Start', but the molecular nature of this linkage is not known. Given that an MCB-binding activity is found in two distantly related yeasts, it is reasonable to ask whether a similar activity exists in other eukary- otes. An intriguing parallel can be drawn between yeast DSC1/MBF activity and the mammalian E2F DNA-binding factor, which is also thought to regulate DNA-synthesis genes. During the cell cycle E2F engages in a complex series of interactions with various cell-cycle regulatory proteins, including the retinoblastoma protein p l l 0 RB, cyclin A and the cdk2 protein kinase [12-14]. How these protein-protein interactions affect the activity of E2F dur- ing the cell cycle, however, is not yet known. It is also not known whether any of the molecular components of E2F are related to Swi6, Swi4 or p120.

422 © 1992 Current Bio logy

References 1. LOWNDES NF, JOHNSON AL, BREEDEN L, JOHNSTON LH: SWI6 is

the common element in control of periodic gene expression in late G1 in budding yeast. Nature, 357:505-508.

2. DIRICK L, MOLL T, AUER K, NASMY~ K:" A central role for SWI6 in modulating cell cycle START specific transcription in yeast. Nature, 357:508-513.

3. ANDREWS BJ: Dialogue with the cell cycle. Nature 1992, 355:393-394.

4. LOWNDES NF, JOHNSON AL, JOHNSTON LH: Coordination of ex- pression of DNA synthesis genes in budding yeast by a cell- cycle regulated t rans factor. Nature 1991, 350:247-250.

5. PRIMIG M, SOCKANATHAN S, AUER H, NASMYTH K: The anatomy of a transcription factor necessary for the start of the cell cycle in the yeast Saccharomyces cerevisiae. Nature, in press.

6. NASMYTH K, DImCK L: The role of SWI4 and SWI6 in the activity of G1 cyclins in yeast. Cell 1991, 66:995-1013.

7. OGAS J, ANDREWS BJ, HERSKOWITZ I: Transcriptional activation of CLN1, CLN2 and a putative new G1 cyclin (HCS26) by SWI4, a positive regulator of Gl-specific transcription. Cell 1991, 66:1015-1026.

8. BREEDEN I, MIKESELL GE: Cell cycle-specific expression of the SWI4 transcription factor is required for the cell cycle reg- ulation of HO transcription. Genes Dev 1991, 5:1183-1190.

9. LOWNDES NF, MCINERNY CJ, JOHNSON AL, FANTES PA, JOHNSTON LH: Control of DNA synthesis genes in fission yeast by the cell cycle gene cdclO. Nature 1992, 355:449-453.

10. BREEDEN L, NASMYTH K: S'mailarity be tween cell-cycle genes of budding yeast and fission yeast and the Notch gene of Drosophila. Nature 1987, 329:651~54.

11. FANTES P& Cell cycle controls. In Molecular Biology of the Fission Yeast. Edited by Nasim A, Young P, Johnson B.F. San Diego: Academic Press; 1989: 127-204.

12. DEVOTO SH, MUDRYJ M, PINES J, HUNTER T, NEV1NS JR: A cyclin A-protein kinase complex possesses sequence-specific DNA binding activity: p33cdk2 is a component of the E2F--cyclin A complex. Cell 1992, 68:167-176.

13. PAGANO M, DRAETrA G, JANSEN-DURR P: Association of cdk2 kinase with the transcription factor E2F during S phase. Science 1992, 255:1144-1147.

14. SHIRODKAR S, EWEN M, DECAPPaC10 JA, MORGAN J, LIVINGSTONE DM, CHITI~NDEN T: The transcription factor E2F interacts wi th the retinoblastoma product and a p l07-cycl ln A com- plex in a cell cycle-regulated manner. Cell 1992, 68:157-166.

John McKinney and Fred Cross, The Rockefeller Univer- sity, 1230 York Avenue, NewYork, NewYork 10021, USA

COMING UP IN CURRENT O P I N I O N IN GENETICS A N D DEVELOPMENT

The October 1992 issue of Current Opinion in Genetics and Development, edited by Ursula Goodenough and Terry Platt, will contain the following reviews on Prokaryotes and Lower Eukaryotes:

Genetic analysis o f ion channels in prokaryotes and lower eukaryotes by RR Preston, Y Saimi, B Martinac and C Kung

The nuclear envelope and nuclear targeting in yeast by MA Bossie and PA Silver Genetic analysis o f DNA transposition in lower eukaryotes by SB Sandmeyer

Genetics of secretion in lower eukaryotes by MKY Fung, HB Skinner and VA Bankaitis Genetics o f the cell cycle in prokaryotes and lower eukaryotes by WD Donachie

Translational control in prokaryotes and lower eukaryotes by L Lindahl and A Hinnebusch Genetic analysis o f photosynthes is in prokaryotes and lower eukaryotes by J-D Rochaix

Genetic rearrangement o f antigenictty in prokaryotes and lower eukaryotes by J Swanson Genetic analysis o f flagella in prokaryotes and lower eukaryotes by SK Dutcher and DF Blair

Bacteriophage lambda requirements for host cell proteins during its life cycle by DI Friedman Genetic analysis o f DNA transposition in prokaryotes by DB Haniford and G Chaconas

mRNA turnover in prokaryotes and lower eukaryotes CF Higgins, SW Peitz and A Jacobson Genetic analysis o f recombination in lower eukaryotes by JN Strathem

Sporulation in prokaryotes and lower eukaryotes by MA Strauch and JA Hoch Genetic analysis o f recombinat ion in prokaryotes by RG Lloyd and GJ Sharpies

RNA splicing in prokaryotes and lower eukaryotes by J Woolford Heat shock proteins and stress tolerance by S Lindquist

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