Overexpression of SIS2 Increases the Expression of SW4, CLNl and CLN2 in sit4 mutants

Copyright 0 1995 by the Genetics Society of America

Overexpression of SIS2, Which Contains an Extremely Acidic Region, Increases the Expression of SW4, CLNl and CLN2 in sit4 mutants Charles J. Di Como,*'tRon Bose"" and Kim T. Amdt" *Cold Spring Harbor LaboratoT, Cold Spring Harbor, New York 11 724-2212 and tGraduate Program in Genetics, State University of New York, Stony Brook, New York 11 794

Manuscript received July 5 , 1994 Accepted for publication September 26, 1994 ABSTRACT The Saccharomyces cermisiaeSZS2 gene was identified by its ability, when present ona high copy number plasmid, to increase dramatically the growth rate of sit4 mutants. SIT4 encodes a type 2A-related protein phosphatase that is required in late G1 for normal G1 cyclin expression and for bud initiation. Overexpression of SIS2, which contains an extremely acidic carboxyl terminal region, stimulated the rate of C L N I , C W 2 , SWI4 and CLB5 expression in sit4 mutants. Also, overexpression of SIS2 in a CLNl cln2 cln3 strain stimulated the growth rate and the rate of C L N l and CLB5 RNA accumulation during late G1. The SIS2 protein fractionated with nuclei and was released from the nuclear fraction by treatment with either DNase I or micrococcal nuclease, but notby RNase A. This result, combined with the finding that overexpression of SIS2 is extremely toxic to a strain containing lower than normal levels of histones H2A and H2B, suggests that SIS2 might function to stimulate transcription via an interaction with chromatin.

T

HE Saccharomyces cermisiae SIT4 gene encodes proa

tein with homology to the catalytic subunit of mammalian type 1 and type 2A serine/threonine protein phosphatases (ARNDT et al. 1989).All known conditional sit4 strains arrest as unbudded cells in the G1 phase of the cell cycle (SUTTON et al. 1991). Moreover, cultures ofslow-growing sit4 mutantsare highly enriched in unbudded cells. SIT4 is required for the execution of Start, which is the commitment point for entry into the cell division cycle. The requirement for SIT4 for the execution of Start is due to the requirement for SIT4 for the normal expression of the CLNl and CLN2 G1 cyclin genes (FERNANDEZ-SARABIA et al. 1992).

The RNA levels of CLNl and CLN2 increase dramatically during late G1 and then decrease during S phase.

A critical level of G1 cyclin activity is required for the execution of Start (CROSS and TINKELENBERG 1991;DIRICK and NASMWH 1991). However, very little is known about the mechanisms that regulate the expression of the CLNl and CLNZ genes during late G1. Both CLN function(either CLNl,CLN2 or CLN3) and CDC28 function (which encodes the catalytic subunit of a protein kinase that interacts withCLN proteins) are required for CLNl and CLN2 expression during late G1 (CROSSand TINKELENBERG 1991; DIRICKand NASMWH 1991).The transcription of CLNl and CLNZis activated, at least in part, bySWI4 and SWIG, which bind as a complex to sites in the CLNl and C W 2 promoters Corresponding author: Kim T. Amdt, Cold Spring Harbor LaboraP.O. Box 100, Cold Spring Harbor, NY 11724-2212.

Present address: Graduate Program, Rockefeller University, 600

tory,

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York Ave., New York, NY 10021.

Genetics 1 3 9 95-107 (January, 3995)

(OGASet al. 1991). S W 4 RNA itself undergoes a 10-fold cell cycle-dependent variation in levels (BREEDEN and MIKESELL1991) where the S W 4 RNA levelspeak at or just before those of CLNl and CLNZ (FERNANDEZSARABIA et al. 1992). The normal expression of S W 4 may require MCB promoter elements (MluI cell cycle box elements), which alsoare containedin the promoters of many genes required for DNA synthesis (FOSTER et al. 1993). The major in vitro binding activity to MCB elements is due to a MBPl/SWIGcontaining complex (KOCHet al. 1993). The requirement for SIT4 for CLNl and CLN2 expression is at least partly due to the requirement for SZT4 for the accumulation of S W 4 RNA during late G1 (FERNANDEZ-SARABIA et al. 1992).

In addition to the role of SIT4 in CLNl and CLN2 expression, SIT4 also is required for another late G1 process. If CLN2 is expressed at low levels from a SZT4 independentpromoter,the cells can replicate their DNA in the absence of SIT4 function (FERNANDEZ-SARABIA et al. 1992). However, the cells still are blocked for bud initiation. Additional evidence for a role of SIT4 for bud initiation is that sit4 mutations are synthetically lethal in combination with a bem2 mutation (K. T. ARNDT, unpublished results). The BEM2 gene encodes a protein with similarity to Rho GTPase activating proteins and seems to function for bud emergence (BENDER and PRINGLE 1991; ZHENC et al. 1993).

Where could SIT4 function in the budding process?

Once a visible bud has formed, SIT4 is no longer required until late G1 of the next cell cycle. Therefore, although bud initiation requires SIT4, SIT4 is not required for continuedgrowth of the bud.LEWand REED (1993) haveshown that polarization of cortical actin

96

C. J. Di Como, R. Bose and K. Arndt

patches occurs after Start and does not require de novo protein synthesis (other than CLN2). Although blocking protein synthesis does not prevent actin polarization to the site of future bud emergence, it does prevent budemergence (LEWand REED 1993). This finding raises the possibility that some essential factor(s) required for bud emergence does not preexist at Start, but must be expressed during late G1 at some time post-Start. The CLN-independent requirement for SIT4 for bud formation possibly may be due to a requirement for SIT4 for the late G1 expression of such a gene(s) required for bud initiation.

Tobetterunderstand howSIT4 functions for G1 cyclin RNA accumulation and for bud initiation, we isolated genes that in high copy number suppress the slow growth ratephenotype of sit4 mutants (which spend most of their time in G1 due to the late G1 delay). From this screen, we isolated four genes, termed SISl through SIS4. Here, we report on the SIS2 gene.

Overexpression of SIS2 stimulated the rateof CLNl and CLN2 RNA accumulation under two different conditions that result in slow G1 cyclin RNA accumulation.

Additional results suggest a mechanism where SIS2 might function via an interaction with chromatin.

M A T E W S AND METHODS Yeast strains and media: Yeast strains are shown in Table 1. Yeast cultures were grown as indicated, on either YEPD medium (1% yeast extract, 2% Bacto-peptone, 2% glucose) or synthetic complete (SC) medium(SHERMANet al. 1989) containing all amino acids and uracil at 0.1 g/liter (except leucine at 0.2 g/liter) and adenine at 0.075 g/liter. For plasmid selection, the appropriate aminoacid or uracil was omitted.

Isolation of SZS2: Two sit4 strains (CY410 = sit4-?6 and CY422 = sit4-?7) were transformed with a high copy number (YEp24) library containing yeast genomic DNA inserts (CARG SON and BOTSTEIN 1982). After 3 days at 30", the library plasmid was recovered from 84 fast-growing colonies out of a total of 50,000 transformants. About 50 of these plasmids contained the wild-type SZ'T4 gene, butthese plasmids were recovered from colonies that grew faster than the other fast-growing colonies. In addition to SZT4,we obtained four different genes (SZSl through SZS4) that increased the growth rate of both sit4-36 and sit4-37mutants. All four of these genes also increased the growth rate of strain S/A258, which contains the sit4-258 allele. Each of the fourgenes (SZSI through SZS4) was independently isolated from at least three different fast-growing transformants.

DNA sequence analysis: The SZS2 gene was localized on the original SISZ-containing library plasmids by subcloning various restriction fragments into YEp24 and assaying for the ability to stimulate the growth rate of sit4 mutants. A 3.0-kb EcoRI/SacI subclone, in highcopy number, containedfull sit4 suppressor activity. This restriction fragment was sequenced et al. on both strands as described previously (TICE-BALDWIN 1989).

Geneticmapping of SZS2 To place SZS2 on the genetic map, a 4.8-kb SacII/SacI restriction fragment containing the SZSZ gene was cloned into theyeast Urn? integrating plasmid Wp5. This plasmid was digested with BamHI (cuts at nucleotide 760 of SZS2) and transformed into a wild type SZS2 strain.

Integration was shown to occur at the SZSZ locus by Southern analysis. Meiotic mapping shows that the distance between SZS2:URA?and met1 is 5 cM ( 5 tetratype, 50 parental ditype, 0 nonparental ditype tetrads).

Preparation of Asis2-1 and Asis2-2 A 4.8-kb SacII/SacI restriction fragment containing the SZS2 gene was placed into the SmaI site of pUC118. An oligonucleotidedirected deletion (KUNKEI. 1985) removed DNA sequences encoding amino acids 3-554 of SZS2 (of 562 total) and replaced them with a XhoI site. For Asis2-1 (::LEU2),a 2.2-kb SalI/XhoI DNA fragment containing theLEU2 gene was cloned into the XhoI site. The resulting plasmid was digested with XbaI Hind111 and transformed into Cy1226 (a diploid formed by mating CY94 and CYl85). For Asis2-2 (::TRPl),a 0.8-kb EcoRI/PstI DNA fragment containing theTRPl gene was cloned into the XhoI site. The resulting plasmid was digested with PstI and transformed into CYl85. Replacement of the SIS2 open reading frame with LEU2 or TRPl at theSZSZ locus was confirmed

by Southern analysis.

Epitopetagging of SZS2: Oligonucleotide-directedmutagenesis (KUNKEL 1985) was used to insert a 6-bp XbaI restriction site between the second and third codons of SISL. The added XbaI restriction site changes SIS2 from MTAV... to "ICRAV.. . Next, a duplex oligonucleotide encoding the hemagglutinin epitope (HA) was inserted into the XbaI site, changing the M E RAlr..., SIS2 amino acid sequence to MTCSYPYZIWDYACRAV ... . The epitopetagged SIS2 protein is functional because high copy number HA:SZS2 stimulated the growth rate of a sit4 mutant almost as well as high copy number wild-type SZS2.

Carboxyl terminal truncationof SZS2 To remove the acidic carboxyl terminalregionfrom the SIS2 protein, a duplex oligonucleotide encodingthe sequence NKIVMKL(stop)(stop) was inserted in the correct orientation into the unique HpaI site located at nucleotide +1443. The resulting gene (sis2ACOOH) encodes a SIS2 protein whose carboxyl terminal residue is amino acid 489. The sis2A COOH gene in high copy number is unable to stimulate the growth rate of a sit4 mutant.

Preparation of cellular extracts and Western immunoblots Cellular extracts and Western immunoblots were prepared as described previously ( S U ~ OetNal. 1991).Briefly, exponentially growing cells were harvested by centrifugation and washed with ice cold lysis buffer [lo0 mM Tris-HC1 (pH 7.5), 200 mM NaC1, 1 mM EDTA, 5% glycerol, 0.5 mM dithiothreitol]. Cells were resuspended in 300 mloflysis buffer [containing 1 mM phenylmethylsulfonyl fluoride and 1.2 pg each of leupeptin, antipain, chymostatin and pepstatin per ml (Sigma)] and lysed by vortexing four times for 15 sec in the presence of glass beads. An additional 350ml of lysis buffer containing protease inhibitors was added, and thecells were vortexed again for 15 sec. The liquid was pipetted from the glass beads and centrifuged at 16,000 X g for 8 min to remove cell debris. An equal volume of 2 X gel sample buffer (SILHAVY et al. 1984) was added to the extracts, which were then heated for 5 min at 95", centrifuged for 3 min at 16,000 X g, and electrophoresed through an 8% SDSpolyacrylamide gel. The separatedproteins were analyzed by Western immunoblotting.

Subcellularfractionation of SIS2: Subcellular fractionation was performed as describedin LUE and KORNBERG (1987). Briefly, cells were harvested at an O D ~ of ~O 0.6 by centrifugation, resuspended in 50 mM Tris-HC1 (pH 7.5) and 30 mM dithiothreitol and shaken slowly at 30" for 15 min.

Next, the cells were digested with Zymolyase 1OOT (1.67 mg/ g cells). The spheroplasts were recovered by centrifugation, washed with 1.0 M sorbitol, and lysed in IO ml of a buffer consisting of 18% (w/v) Ficoll (Pharmacia), 10 mM Tris-HC1

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SIS2 Can Suppress sit4 Mutations

97

TABLE 1 Yeast strains

Strain CY94 cn 85 CY248 CY410 CY422 CY591

cn 266 Cy1298 Cy1402 m779 cn 898 cn 899 CY2125 CY2221 CY2297 CY2921 CY2922 CY3169 CY3237 CY3238 CY3527

Genotype

MATa ssdl-dl ura3 leu2 his3 t q l ade2-101 lys2-801 MATa SSDl-v ura3-52 leu2AI (S288C derived) MATa Asit4-2(::HIS3)SSDI-VI ura3 leu2-3 his3 MATa sit4-36 gcn4-2 basl-2 bas2-2 ura3-52 SSDI-v2 MATa sit4-37 gcn4-2 basl-2 bas2-2 ura3-52 SSDI-v2 MATO Asit4-2(::HIS3)ssdl-dl (sit4-102 on YCp50) ura3 leu2 his3 trpl ade2 lys2-801 cad-100 MATa pGALSIT4(::HIS3) Assdl(::LEU2)ura3-52 leu2-3 his3A200 lys2 MATa Asis2-l(::LEU2) SSDl-u (HASIS2 on YCp50) ura3 leu2 his3 trpl MATa Asis2-l(::LEU2) SSDl-v {HA:SIS2on YEp241 ura3 leu2 his3 trpl MATa Asis2-I(::LEU2)SSDl-v ura3 leu2 his3 t q l ade2 MATa Asis2-l(::LEU2) SSDI-v (HA:SIS2on YEp241 ura3 leu2 his3 t q l MATa Asis2-l(::LEU2) SSDl-v (HAsis2ACOOH on YEp24) ura3 leu2 his3 trp1 MATa Aclnl-ll(::HIS3) Assdl(::LEU2) ura3-1 leu2-3,112 his3-11,15 trpl-I ade2-1 canl-100 MATa Asis2-2( :: TRPl) SSDl-v ura3 leu2 t q I MATa Aclnl-ll(::HIS3) Acln2-lI(::TRPl)SSDl-v ura3 leu2 his3 t q l MATa Asit4-2(::HIS3) SsDl-vl (YEp24)ura3-52 leu2-3 his3 MATa Asit4-2(::HIS3)SSDI-vl (SIS2 on YEp24) ura3-52 leu2-3 his3 MATa Ahtal Ahtbl(::URA3) ura3-1 leu2-3,112 his3-11,15 t q l - 1 MATa Acln2-11(::TRPl) Acln,3(::HIS3) Asis2-l(::LEU2)SSDl-v (YEp241 ura3 leu2 his3 trpl MATa Acln2-lI(::TRPl) Acln3(::HIS3) AsisZ-I(::LEU2) SSDl-v (SIS2 on YEp24) ura3 leu2 his? trpl MATa Asit4-2(::HIS3) Asis2-2(::TRPl) SSDl-vl (sit4-102 on YCp50) ura3 leu2 his3 t q l lys2

(pH 7.5), 20 mM KCl, 5 mMMgC12, 3 mM dithiothreitol, 1 mM EDTA, 0.5 mM spermidine, 0.15 mM spermine, 1 mM phenylmethylsulfonyl fluoride and 1.2 pg each of leupeptin, antipain, chymostatin and pepstatin per ml (Sigma) using a Douncr homogenizer. Unlysed spheroplasts and cell debris were removed by four 5-n-min centrifugations at 3000 X g resulting ina uniform whitesupernatant. Thenuclei were recovered by centrifugation at 25,000 X gfor 30 min and suspended in 2.5 ml of lysis buffer. An equal volume of 2X gel sample buffer was added to the nuclear and cytoplasmic fractions.

The samples were electrophoresed on an 8% SDSpolyacrylamide gel. The separated proteins were analyzed by Western immunoblotting.

For DNase, RNase, micrococcal nuclease anddetergent treatments, nuclei were isolated as described above, with one modification. Just before the final centrifugation step, the crude nuclear fraction was divided in two and then centrifuged at 25,000 X gfor 30 min. For the RNase digestion (37O, 20 min) and detergent treatments (4", 10 min), the nuclear pellet was resuspended in 18%Ficoll buffer. The 18% Ficoll buffer inhibited the enzymatic activity of DNase I and micrococcal nuclease. Therefore, for the DNase I (37", 20 min) and micrococcal nuclease (37", 10 min) digestions, the nuclear pellet was resuspended in SPCP buffer [1.1 M sorbitol, 0.02 M PIPES (pH 6.3), 1 mM phenylmethylsulfonyl fluoride, minus the CaClp]. Then, 0.1-ml aliquots of the nuclear fraction were treated as described previously (ALLEN and DOUGLAS 1989) using DNase I (Worthington),RNase A (Sigma),micrococcal nuclease (Boehringer), TritonX-100 (Bio-Rad) or Digitonin (Sigma). All samples were centrifuged at 16,000 X g for 30 min, the pellet resuspended in 0.1 ml of 1X gel sample buffer, heated at95" for 5 min, centrifuged for 3 min at 16,000

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X g and electrophoresed through an8% SDSpolyacrylamide gel. The separated proteins were analyzed by Western immunoblotting.

Northern analysis and probes: Total RNA was loaded onto a 1% agarose gel containing 6% formaldehyde, 0.02 M morpholinopropane sulfonic acid, 0.005 M sodium acetate, and 0.001 M EDTA (final pH of 7.0). The gels were blotted onto BioTrans nylon membranes. The probes were the 1.4kb ClaINot1 fragment of CLNI, the 0.75-kb XhoI-Hind111 fragment of CLN2, the 0.6-kb AluI fragment of ACTl, the 2.2-kb BamHI fragment of SW4, and the 0.5-kb EcoRI fragment of CLB5.

The blots were washed twice (15 min each) at 24" using 2X SSC, 0.1 % SDS, followed by twice (15 min each) at 65" using 0.1X SSC, 0.1% SDS.

RESULTS

The SZS2 gene, when present on ahigh copy number plasmid, stimulates the growth rate of sit4 mutant strains: Yeast strains containing the sit4-36 or the sit437 mutation have a very slow growth rate (ARNDT et al.

1989). To identify genes whose products are substrates of SIT4 (or act downstream of SIT4 in the SIT4 pathway), regulate SIT4 activity or can substitute partially for SIT4, we isolated genes that, when present on a high copy number plasmid(YEp24),dramatically increase the growth rate of a sit4-36 or sit4-37 mutant strain. From a large-scale screen (see LUKEet al. 1991 for the isolation procedure), we obtained four genes,

C.J. Di Como, R. Rose and K. Arndt

98

Y Ep24

HA' :ssz

YEp24

HA:SISZ.COOH

FIGURE I.-High copy number SIS2 stimulates the growth rate of sit4 mutants. Strain CY410 ( d 4 - 3 4 was transformed with either YEp24, p<;R972 (.TIS2 on YEp24), pCR126.7 (HA:SIS2on W,p24) or pCR144.7 (HA:sis2A(:OOHon W,p24).

Transformantswere streakedonto SC minus uracil plates and incubated at 30" for 4 days.

which we term S Z S I through SfS4 (for si14 suppressor).

S f S I encodes an essential DnaJhomologue (LL~KE PI nl. 1991) that is required for the normal initiation of translation (ZI-IONGand ARNDT 1993).SfS4, renamed P P H h , encodes a type 2A protein phosphatase homologue (SNEDDON d nl. 1990; S v - r r o ~d nl. 1991) that, when overexpressed, can possibly substitute for some of the functionsof the SIT4 phosphatase. In this report, we present an analysis of the SLY2 gene. The SIS2 gene, when present on a high copy number plasmid, greatly stimulates the growth rate of si/4-36, sit4-37 and s i t 4 258 strains (Figure 1 and data not shown). Therefore, the stimulation of the growth defect of si14 mutants by high copy number SfS2 is not specific to the particular sit4 allele. Of the SISI through SfS4 genes, the SfS2 gene in high copy number is the best suppressor of the growth defect of sir4 mutant strains.

SZS2 encodes a protein with an extremely acidic carboxyl terminal region: The SfS2 gene is located on a 3.0-kb E c o N / S n d DNA fragment (see MATERIAIS A N D METHODS). Conceptualtranslation of the DNA sequence of the EmRI/SncI DNA fragment shows that it contains only one large open reading frame (nucleotides 1- 1686, Figure 2). This open reading frame encodes SIS2 because a frameshift mutation at nucleotide 760 (created by digesting with BnmHI, filling in using Klenow enzyme and ligating) completely eliminatedthe ability of the EcoN/SnrI DNA fragment on YEp24 to stimulatethe growthrate of si14 mutants(datanot shown).

The predicted SIS2 protein has 562 amino acid residues and a calculated molecular mass of 62,481. The carboxyl terminal region of the predicted SIS2 protein

is extremely acidic (Figure 2). Within a stretch of 58 amino acid residues (from 496 to 553), 51 residues are either glutamate (E) or aspartate (D). We wanted to make certain that the extremely acidic region of the predicted SIS2 protein is expressed as part of the SIS2 protein in 71i710 (this region of the SIS2 gene was somcwhat difficult to sequence due to its toxicity, in bklwn'rhin roli, of some of the ordered deletions of SIS2).

For this analysis, we first prepared a version of the SfS2 gene that encodes a S I 3 protein tagged at the amino terminal end with a nine amino acid hemagglutinin (HA) epitope (see MATERIAIS ANI) METHODS). The full length HA-SIS2 protein hasa calculated molecular mass of 64 kD and migrates on SDSPAGE gels as a protein of 70 kD (Figure 3 ) . Next, we introduced a Stop codon in place of the codon for amino acid 489 (the acidic regionstartsat amino acid 496, see MATERIAI.~ AND METHODS). This truncated HA-SIS2 protein has a calculated molecular mass of -56 kD and migrates on SDS PAGE gels as a 58-kD protein (Figure 3). Therefore, the acidic region of SIS2 is present on the matureSIS2 protein in 7 ~ i 7 1 0 .Moreover, the acidic carboxyl terminal region of SIS2 is required for high copy number SLY2 to stimulate the growth rate of a siI4-36 strain (Figure 1).

Searches o f the data bases with the predicted SIS2 protein showed that SIS2 has significant similarity to only one protein, the predicted S. rmmisinr M(L088w protein, which was identified during the sequencing of chromosome X I (DL!]oN PI nl. 1994). The carboxyl terminal half of SIS2is -38% identical, over a 267 amino acid region,to the carboxyl terminal half of M(L088w (Figure 4). Both SIS2 and YKL088w have very acidic regions at the very carboxyl terminus. The amino terminal regions of SIS2 and YKL088w are not significantly similar. In addition, bothSIS2 and YKL088w have potential purine binding motifs (starting at aminoacid 467 for S1S2) of the type G,,G,G,,,,,,,IV,K (FRYPI nl.

1986). The data base searches also showed that the carboxyl terminal acidic region of SIS2 showed weak similarity to the acidic regions of some other acidic proteins,such as SPT8, nucleolin and UBF2 (SRIVASTAI'A PI nl. 1990; O'MAHONY andROTHRIXM 1991; EISENMANN PI nl. 1994).

To place the SfS2 gene on the yeast physical map, RILES probed the Riles and Olson collection of A phage containing yeast genomic inserts (OISONPI nl. 1986) with a labeled SfS2 DNA fragment. SfS2 hybridized to contig 397 (defining clone 3867) on the right arm of chromosome XI. Meiotic mapping shows that S I S Z is located -5 cM from mPI1 on chromosome (see MATER I A I S AND METI-IOIX).

Strains containing a deletion of SZS2 are viable: Chromosomal deletion alleles ofSIS2 were prepared that replace SIS2 DNA sequences from 7- 1662 (removes amino acids 3-554 of 562 total) with either the IXU2 o r TRPI gene (see MATERIALS AND METHODS). The Asis2

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FIGURE2.-The predicted SIS2 protein has an extremely acidic carboxyl terminal region. The nucleotide (accession#U01878) and predicted aminoacid sequence of SZS2 are shown. An arrow indicates the siteof the SIS2 carboxyl terminal truncation (see MATERIALS AND METHODS). The SZS2 gene corresponds to open reading frame YKR072c on the recently completed S. cerevisim chromosome Hsequence (DUJONet al. 1994).

strains have a normal growth rate (on YPD or synthetic complete media),arenottemperature sensitive for growth (at 37" or 38"), accumulatenormal levelsof glycogen, have no obvious mating defect, have a normal cell size and grow normally using glycerol+ethanol, galactose or raffinose as carbon sources (data notshown).

One possibility is that the function performed by SIS2 is not required for a normal growth rate. Alternatively, if the type of function performed by SIS2 is important or essential for normal growth, Asis2 cells would have to contain an activity (such as that provided by YKL088w or other proteins) that performs a function similar to that of SISS. Geneticinteractions of SZS2 with SIT4 and SSDlu: The SZS2 gene was isolated by its ability, when present on a high copy number plasmid, to stimulate the growth rate of strains containing transcriptional suppressor alleles (sit4-36 and sit4-37) of SIT4. We also investigated if overexpression of the SISS protein could

suppress other sit4 mutants. The phenotype of sit4 mutants depends on the unlinked polymorphic SsDl locus (SUTTONet al. 1991).In ssdl-d or Assdl strains, deletion of SZT4 is lethal and the sit4-102 allele results in a temperature-sensitive phenotype. By contrast, in SsDlv strains, deletion of SZT4 results in viability (but with avery slow growth rate) and thesit4-102allele does not result in a temperature-sensitive phenotype. Although high copy number SZS2 does not rescue the lethality of a Asit4 ssdl-d strain (data not shown), high copy number SZS2 is able to suppress partially the temperature sensitive phenotype of a sit4-102 ssdl-d strain (Figure 5A).

High copy number SZS2 also is able to greatly stimulate the growth rate of a Asit4 SsDl-vl strain (Figure 5B). A single extra copy of SIS2 on a centromere plasmid also stimulated the growth rate of a Asit4 SsDl-VI strain (Figure 5C). That higher than normal levels of SIS2 increased the growth rate of a strain completely

~

C.J. Di Como, R. Rose and K. Arndt

100

HA :SISZ Full length

\

HA:SEZ ACOOH

200

100

71

FIGURE3.-Immunohlot analysis of full length and carhoxyl terminal truncated SIS2. Strains CYI898 [Asi.r2 .%7T)I-71 (HA:SIS2 on YEp24)I and CYI899 [Asis2 SSDI-TI (HA:sis2ACOOH o n YEp24)] were grown in SC minus uracil medium at 30". At an OD,,,,, of -0.2, the cells were harvested and an extract prepared hy glass head lysis. Total protein ( 1 00 mg) was loaded in each lane of an 8% SDSpolyacrylamide gel.

The proteins were analyzed

by Western immunohlotting using as a prohethe 12CA5 ascites (FII.:I.I>r / nl. 1988) directed againstthehemagghltinin(HA)epitope.The 45-kD band present in hoth lanes is not specific to SIS2 hut is due to a protein that cross-reacts with the 1 2 C X antibody. The levels of HA-SIS2 protein do not vary in the cell cycle (data not shown).

SIS2 KL088w SISZ KL088w

lacking SIT4 indicates either that SIS2 functions downstream of SIT4 within the SIT4 pathway or that SIS2 functions in a pathway parallel to the SIT4 pathwav.

Deletion of SIS2 causes no easily detectable growth defects in an otherwise wild type strain. Also, a Asi14 SSDI-v strain is viable (but with a slow growth rate).

However, when a Asit4:HlS3 SSll1-vI strain (CY279) was crossed to a Asis2:IJXJ2.%'~I)I-?J] strain ( c " 7 7 9 ) , we failed to recover any His' Leu' (Asi14 Asis2 S S I l I 7 1 1 ) haploid progeny in 40 tetrads. When the diploid from this cross was transformed with the S I S 2 gene on the URA3 containing YCp50 plasmid, we obtained His' Leu+ haploid progeny, but they always contained the SIS2 gene on YCp50 (they were Ura'). The Asit4 Asis2 . S S I l 1 - ~ ~(.SI.S2/YCp50) 1 haploid strains could not grow in the absence of the SIS2 plasmid (data not shown).

Therefore, the SIS2 gene is essential for viability in the absence of SIT4. If the function of YKL088w overlaps with that of SIS2, the activity of W 0 8 8 w is not suffcient for viability in a Asi14 Asis2 SSDI-71 strain.

If SIS2 flmctions in a pathway that is parallel to the SIT4 pathway, one possibility is that SIS2 might function in the SSDl-v pathway. If true, high copy number SIS2 would stimulate the SSD1-v pathway (making a Asif4 SSDI-711 strain grow faster) and deletion of SIS2 would inactivate the SSDl-v pathway (making a Asi14 SSDI-VI strain inviable). However, high copy number SlS2 can stimulate the growth rate of strains defective for SIT4 in the complete absence of SSDI.A pGAI,:SIT4 Assdl strain grows witha wild-type growth rate on YP-galactose plates (where SIT4 is transcribed) but was not able to grow on YP-glucose plates (where transcription of SIT4 is repressed, giving rise to extremely low levels ofSIT4).

However, a pGAL:SIT4 Assdl strain containing theSIS2 gene ona high copy number plasmid (YEp24) was able to grow very well on YP-glucose plates (Figure 5D). This SIS2-stimulated growth on glucose containing medium is not due to an increase in the expression of SIT4 from the pGAL promoter (as determined by Northern

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FIGLIKE 4.-SISZ has a region ofsimilarity with the YKL088w protein. The similarity of S1S2 to the S c~r~r~i.rinr YK1,088w protein (DU~ONet nl. 1994; accession #228088) is shown. 1, identical amino acids; :, highly conserved amino acids; *, thepotentialpurine hinding motifs (FKYr/ nl. 1986).

SlS2 Can Suppress si14 Mutations

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FIGLIRE .?.-Genetic interactions of SLY2 with ,717'4 and .7SI)I-n (A) Strain Cl59l (Asi14 s.sdl-dl (sit4-102 on YCp.?OJ) was transformed with either YEp13 or pCB1442 (SLY2 on YEpl3). Transformants were patched onto S C minus uracil, minus leucine plates. After 1 . 5 days at 24", the cells were replica plated onto S C minus uracil, minus Irrlcine plates antl incubated at 37" for I day. (B and C) Strain CY248 (Asi14 SSDI-7)) was transformed with either Wp24, pCB97!! (SIS2 on YEp24), YCp.50 or p(X967 (SIS2 on YCp.50). Transformants were streaked onto SC minus uracil plates and incuhated at 30" for 3 days. (D) Strain CM266 (pGAL:S17'4A s s d l ) was transformed with either YEp24 or pCB972 (SIS2 on YEp24) antl plated onto SC minus uracil/2% galactose plates (SIT4 is expressed from the GAL promoter). Transformants were streaked onto S C minus uracil/2% glucose plates (which represses the CAI. promoter) and grown at 30" for 2 days. " h e n streaked from galactose t o glucose medium, the pG4I,:S17'4 A s s d l cells give rise to tiny colonies most likely because the levels of' ,717'4 RNA and/or protein reqnire tlilrltion (by cell growth and division) before the cells arrest growth. The cells in these tiny colonies on thc first SC minus uracil/2% glucose plates do not divide if streaked onto a second SC minus 11racil/2% glucose plate. (E) A A r I n I A h 2 .Ya7Dl-nstrain (Cy2297) was crossed to either a Asls2 . ~ . S I I ~ -strain V (CM779) or a Arlnl Assdl strain (CW12.5). These deletion alleles of CI.Nl and CXN2 remove almost the entire open reading frames. The diploids were sporulated and tetrads ( 2 4 0 for each cross) were dissected. The spores were germinated for 2 4 days at 30". Shown are colonies of representative haploid progeny on YEPD plates grown at 30" for 2..? davs.

analysis, data not shown). Therefore, in the complete absence of SSDl, overexpression of SISP can stimulate the growth rate of a strain surviving on very low levels of the SIT4 protein.

In addition, we have other tests for a functional SSDIv pathway and SIS2 is neither required for nor can stimulate these functionsof the SSDl-v pathway. The growth rate of a Aclnl A h 2 CLN3 SSIII-v strain (which has a growth rate slightly slower than a wild type strain) was reduced severely when the SSD1 locus was changed to a ssdl-dallele (CVKCKOVA and NASMYI-H 1993) or a Assdl allele (Figure 5E). However, a Aclnl A h 2 CI,N3 S S D I v Asis2 strain grew at the same rate as a Arlnl A h 2 CIdN3SSDI-1) strain (Figure 5E).Also, high copy number SIS2 did not stimulate the growth rate of a Aclnl Acln2 CLN3 SSDI-v strain (data not shown). These re-

sults suggest that SIS2 is not required for the function of SSDI-v that stimulates the growth rate of a Arlnl Acln2 C I A 3 strain. The SSIII-I)gene also is able to s u p press partially the temperature-sensitive phenotypes of ~p-31(MOSRIN P/ nl. 1990), c / k l (LEE and GK~XNI.E~\F 1991), hql (TODA r/ nl. 1987) and ins1 strains (M'lt.Sos rf nl. 1991). Deletion of SIS2or high copy number SIS2 does not alter theability of SSDI-IJto suppress partially the temperature-sensitive phenotypes of these mutant strains (data not shown). These results suggest that SIS2 does not ftunction in the SSDI-v pathwav. If SISP functions in a pathway that is parallel to the SIT4 pathway, overexpression of SIS2 would stimulate the SIS2 pathway so that si14 mutant cells (defective for the SIT4 pathway) have an increased growth rate.

SZS2 can stimulateG1 cyclin transcription:Because

C . J . Di Como, R. Rose and K. Arndt

102

A. '10cel Is

with bud:

B. YEp24

Y Ep24/SISZ

I

0 I 2 2 2 3 4 8 I5 21 I 3 3 5 12 2 58 1

60 80 94

CLNI FIGURI:.6.-Overexpression of SIS2 increases the rate and levels at which S W 4 , CI,Nl, CLN2 and C I I 5 RNAs accumulate in a si14 mutant. Strains CY2921 [A, Asit4 SSDl-ul IYEp24]] and CY2922 [B,Asit4 SSDI-III ( S I S 2 on YEp24]] were grown exponentially at 30" in SC minus uracil medium. At an ODoM,of 0.2, a-factor was added to 0.006 m u . After growing 3 3 hr, the cultures were filtered in parallel. The cells then were washed and resuspended in fresh SC minus uracil mediumand grown at 50". Samples were collected at the indicated times after resuspension of the cellsin fresh medium. Total RNA was prepared . i mg of' which was loaded into each lane. Theprobes for Northern analysis are listed in M,\TERI~\ISANDM E T I I C ~ S .

CLN2

t r w o w r w u ~ ~ u r r r u ~ w a O w o timeafter 0 8 16 243240485664 72 0 8 16 24 3240485664 72 release from min min Nfactor

SIT4 is required for the normal rate of increase and for the normal levels of CLNl, CLN2, HCS26, SW4 and C I A 5 RNAs during late G1 (FERNANDEZ-SARARIA et nl.

1992), we examined if high copy number US2 could stimulate G1 cyclin expression in a sit4 mutant. To test this possibility, we arrested YEp24- or SIS2/YEp24containing Asit4 SSDI-v cells in G1with a-factor [which represses CILN1and CIA2 expression (WITTENBERG et nl. 1990)]. The a-factor was washed away and the cells were inoculated into fresh medium. Because of the absence of SIT4, the YEp24containing Asit4 SSDl-v cells had a very slow gradual increase in the levels of CIAl, CIiV2, SW4 and CLR5 RNAs (Figure 6 A also see FERNANDEZ-SARARIA et nl. 1992). The levels of these RNAs were still increasing at 72 min after a-factor release, at which time only 21% of the cells had formed a visible bud (bud formation is dependent on the execution of Start, which in turn is dependent on G1 cyclin expression).

In contrast, isogenic SIS2/YEp2kontaining Asit4 SSDI-v cells increased the levels of CLNI, CIA2, SW4 and CI,R5 RNAs much more rapidly, reaching a peak at 32 min after a-factor release (Figure 6B). In addition, by 72 min, almost all (94%) of the cells had formed a visible bud. Therefore, in a AsM mutant, overexpression of SIS2 stimulated CWVI and CLN2 expression. By contrast, overexpression of SIS2in a Asrui4 mutant resulted in extremelyslow growth that was most

likely due to a defect in CLNl and CLN2 expression (heterologous expression of CLN2 in the As7ui4 strain cured the SIS2-induced toxicity, C. J. DI COMO and K. T. ARNDT, unpublished observations). These findings suggest that the SWI4independent expression of CLNl and CLN2 is probably repressed by overexpression of SIS2. Therefore, in a sit4 mutant, SIS2 either stimulates the expression of SW4 (which in turn induces CIAl and CIA2 expression) or SIS2 stimulates the SWI4-dependent expression of the CLNI and CIA2 promoters.

We also examined if overexpression ofSIS2 could stimulate G1 cyclin expression in a CLN1 Acln2 A c h 3 strain, which has a moderate slow growth phenotype that is directly due to a lower rate of G1 cyclin expression. Because CLNI expression requires CIA function (CROSSand TINKELENBERG 1991; DIRICK and NASMYTH 1991), the absence of CLN2 and CLN3 causes not only a loss of the CLN2 and CLNS proteins, but also a defect in the rateof CLNl RNA accumulation. High copy number SIS2 increased the growth rate of a CIA1 Acln2 Acln3 strain whereas deletion of SIS2 made a CIAl Acln.2 Acln3 strain grow even more slowly (data not shown). As a result, high copy number SIS2, compared with a deletion of SIS2, significantly increased the growth rate of a CLNl Acln.2 Acln3 strain (Figure 7A).

These findings suggest either that SIS2 can stimulate C I N I expression or thatSIS2 can allow a strain to grow

SrlppressSIS2 Can

si14 Mutations

IO3

A.

B. %cells with bud:

2

7

YEp24 8 15 18 29

0

YEp24/SIS2 II 15 39 71 97

CLNI

CLBS

FIGC'KEi.-Overexpression of SIS2 stimulates Cl.A~I and U . B 5 RNA ;Irrrlmulation in a Cl.h'l Ar1n2 Arln3 strain. (A) Strain CY2473 (Arln2 A h 3 Asis2 SSl11-1r) was transformed with either YEp24 o r pCR972 ( S I S 2 on YEp24). Transformants were streaked onto S C minus uracil plates and incubated at30" for 3 days. (E) Strain CW237 [Arln2 Arln3Asis2 S.Sl)l-v (YEp24)Jand (XZ238 [ A h 2 Arln3 Asis2 S S I ~ I - I I{.$IS2on W,p24)] were grown exponentially at 30' in SC minus uracil medium. Cultures were treated as in Figure fi except that 8 mg oftotal RNA was loaded into each lane.

The probes used for Northern analysis are listed in MATERIAIS ANI) !4l~.TllOl>S.

rRNA

ACT/ 1 time after 0 release from o( factor

20 40 60 80 1 0 0 0 min

20 40 60 80 100 min

faster even though it has lower than normal GI cyclin levels. To distinguish between these two possibilities, we examined the rate of increase in U.NI RNA levels after release from an a-factor arrest (Figure 7R). The CI-NI A h 2 A h 3 Asis2 (YEp24)cells had a very slow rate of increase in 0'1,NI RNA levels (with a peak at -80 min after a-factor release). Even at 100 min after a-factor release, only 29% of the C1,NI Acln2 Acln3 Asis2 (yEp24) cells had formed a visible bud. In contrast, overexpression of SIS2 in the C I f l I Arln2 A h 3 cells resrllted in a much more rapid rate of increase in the levels of U , N I RNA (Figure 7R). Moreover, by 100 min after a-factor release, nearly all (97%) of the cells formed a visible bud. The transcription of CIB5 has beenproposedtobedependenton C1.N function (SCHWOS and NASMWH1993) and the rate of increase of CLB5 RNAisslowin the CLNI A h 2 Acln3 Asis2 cells after release from the a-factor arrest (Figure 7R).

Overexpression of SIS2 also resulted in a much more rapid increase in CLB5 RNA levels (Figure 7B).

Cells arrest in G1 in the absence of both SIT4 and SISZ function: In ssdl-d o r Assdl genetic backgrounds, the sil4-102 allele results in a temperature sensitive

phenotype and deletion of SIT4 results in lethality. By contrast, Asi14 S S I I I - ~ Icells are viable (at either 24 o r 37.5") but with a slow growth rate phenotype. When shifted to 37.5', si14-102 SSDI-v SLY2 cells were viable and continued to divide: after 4 hr, the culture had almost equal numbers of In and 2n DNA content cells and 56% of the cells were budded (Figure 8, middle panels). Rv contrast, a si14- 102 .s.yDI-v Asis2 strain was temperature sensitive and, after a shift to 37.5", became enriched in unbudded cells and cells with a In DNA content (Figure 8, left panels). Therefore, .S.Sl)l-v cells lacking both SIT4 and SIS2 function are blocked mostly in GI. Interestingly, low-level heterologous expression of CLN2 inthe si14- I02 kY.YD1-7~ Asis2 cells did not cure the G1 arrest(Figure 8, right panels). Therefore. although SSDl-71 cells lacking both SIT4 and SIS2 function may have a defect in G1 cyclin expression, they also have some additional defect(s) that blocks both DNA synthesis and bud formation. This additional defect(s) might be the reduced expression of some genes, in addition to CLNI and CLN2, that are required for DNA synthesis and/or bud formation.

SISZ is found in the nuclear fraction and may have

C. J. Di Como, R. Bose and K. Arndt

104 ~it4-102

I

24%

37.5%

1

+ Control

SSDI-VI I

+us2

4 . p . ADâ&#x201A;Źâ&#x201A;Ź:CLN2

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FIGURE %-In the absence of both SIT4 and SIS2 function, the cells arrest in G1. FACS analysisof propidium iodide stained cells to determine DNA content. The vertical axis is the number of cells; the horizontal axis is fluorescence intensity. Cells were prepared for FACS as described by NASHet al.

(1988) exceptthat after RNase treatment,the cells were treated with 0.5 mg/ml of proteinase K (Boehringer Mannheim) for 30 min at 50". The budding index (upper right corner) is the percent of cells with a visible bud (counting 2100 cells). All three strains are CY3527 [Asit# Asis2 SSDIu (sit#-102 on YCp50)I transformed with either a LEU2 centromere plasmid (pAB484), pCB974 (SIS2 on pAE5484) or pCBl311 ( S . pombe ADH CLN2 on pAB484). Asynchronous cultures were grown at 24". Half of each culture was shifted to 37.5", and samples were collected at 4.0 hr.

a role in normal chromatin function: Overexpression ofSIS2 increases the rate of CLNI, CLN2, S W 4 and CLB5 RNA accumulation during late G1 under two different conditions where the expression of these genes is defective. If SIS2 functions directly in the transcriptional process or fornormalchromatinfunction, it should fractionate with nuclei [SIS2 does not have an obvious basicdomain nuclear targeting sequence(ROBBINS et al. 1991)l. Yeast cells containing HA-SIS2 protein as the only source of SIS2 were used to prepare nuclear and cytoplasmic fractions by the procedure of LUE and KORNBERC(1987). This procedure commonly is used to prepare a nuclear fraction for in vitro transcription assays. Western analysis of the nuclear and cytoplasmic fractions shows that the HA-SIS2 protein fractionates almost completely with thenuclear fraction (Figure 9A). It is possible that the acidic carboxyl terminus of SIS2 might interact with the basic histone proteinspresent in the nucleus. However, such an interaction can not be the sole reason that SIS2 fractionates inthe nuclear fraction because a truncated SIS2 protein without the carboxyl terminal acidic region still is found almost exclusively in the nuclear fraction (Figure 9A).

Both the full length SIS2 and the truncated SIS2 protein (without the carboxyl terminal acidic region) are released from thenuclear fraction with 0.3 M NaCl (data not shown).

In vitro, HA epitope-tagged SIS2 bound quantitatively to a histone agarose column (equilibrated in 0.075 M KC1 buffer) and elutedat -0.3 M NaCl (datanot shown). Also, HA epitope-tagged SIS2 missing the carboxyl terminal acidic region bound weakly to the histone agarose column(data not shown). The in vitro binding of SIS2 to the histone agarose column may be because of the fact that histones are basic and SIS2 is acidic. To explore apossible in vivo interaction between SIS2 and chromatin, we determined if SIS2 can be released from a nuclear fraction by treatments that digest the DNA within chromatin. Either DNase I treatment or micrococcal nuclease treatment could release much of theSIS2 from the nuclearfraction (Figure 9B). Much of the 70-kD subunit of the replication factor RFA (BRILLand STILLMAN 1991) also was released from the nuclear fraction by DNase I or micrococcal nuclease treatment (Figure 9B). Interestingly, compared with SIS2, the 70-kD subunit of RFA was released from the nuclear fraction under conditions of more limited DNA digestion. In contrast to SIS2 and the 70-kD subunit of RFA and as shownpreviously for DNase I treatment (CAPLAN and DOUGLAS 1991; LUKEet al. 1991), neither SISl nor YDJlwas released from the nuclear fraction

by DNase I or micrococcal nuclease treatment (Figure 9B). Treatment of the nuclear fraction with RNase I did not release SIS2 (or RFA or YDJ1) from the nuclear fraction. RNase I treatment did release a portion of SISl from the nuclear fraction (Figure 9B), as shown previously in LUKEet al. (1991). In addition, treatment of the nuclear fraction with Triton X-100 or Digitonin released almost allofSIS2 and RFA (but not YDJ1) from the nuclear fraction (Figure 9B).

To explore a possible functional interaction between SIS2 and chromatin, we determined the effect of overexpression ofSIS2 in a strain containing lower than normal levels of histone H2A and H2B. Histone H2A is encoded by HTAl and HTA2 whereas histone H2B is encoded by HTBl and HlB2 (NORRIS and OSLEY 1987). The HTAl and HTBl genes are adjacent and are transcribed divergently. Likewise, the HTA2 and HTB2 genes areadjacent and are transcribed divergently (HEREFORD et al. 1979). Cells containing a deletion of the HTAl and HTBl gene pair are viable, but have moderate a slow growth rate. Interestingly, A h t a l A h t b l cells are extremely sensitive to increased levels of the SIS2 protein. When A h t a l A h t b l cells contained the SIS2/YEp13 plasmid, theygrew extremely slowly (Figure 9C). For the SZS2/YEp19containing Ahtal A h t b l strain, only -15% of the cells streaked onto the plate gave rise to the very small colonies (visible in Figure 9C). Most of the SIS2/YEp13 A h t d A h t b l cells streaked onto the plate either failed to divide or gave rise to only microcolonies (not visible in Figure 9C). Also, even an extra copy of SIS2 (on a ZJW2 centromere plasmid) caused Ahtal A h t b l cells to grow slower (Figure 9C). By contrast, when wild-type cellscontained

the XS2gene ona high copy number plasmid (yEpl3), they had a normal growth rate (data not shown).

where the slow rate of CIA,VlRNA accumulation is clue solely to a defect in U,,V activity. Therefore, overexpression o f SIS2 can stimulate the expression of certain genes that are activated periodically during late G1.

DISCUSSION " h y does high copy ntunher SIS2 stimulate the growth rate o f sit4 muvants? si14 mutants have a greatlv The SIT4 phosphatase is required in late GI for the expanded GI phase of the cell cycle and a defect in normal accumulation of SW14, CLNI, CLN2 and C1J5 and CI.,V2 RNA accumulationdtlringlate GI. RNAs ( F E R N ~ ~ ~ d~01.~1992). ~ - SOverexpression ~ ~ ~ ~ R I ~ CI-NI ~ However, the growth rate of ;I A.si/4 *vc51)1-v strain is not of SIS2 stimulates the rate of SW14, CLN1, USN2 and stimulated by either lowor high-level expression of CLB5 RNA accumulation in si14 mutants. I n addition, CLN2 from a SI7'4-indepentlent promoter (FI:.KNANI>)I.:%overexpression of SIS2 stimulates the rate o f CI.NI and SARARIA p/ nl. 1992) or lowlevel expression of SW14 from CLB5 RNA accumulation in CLNI A h 2 A hstrains, 3 a SI7'4independent promoter (C.J. Di Como and K. T. Arndt, unpublished observations). Also, although lowhlg h copy # A. high copy # low copy # level heterologous expression of CI,N2 in a si/4-102 HA:S/SZ HA:S/SZ HA:SISZdCOOH ssdl-d strain allows the cells to replicate their DNA at N C N C N C 37.5", the cells still are blocked for bud initiation and have a temperature-sensitivephenotype (FERSANDFZSIS2 0 0 L SAR4RIA PI nl. 1992). Theseresults indicate that, in addition to a defect in CLNI and CLN2 expression, si14 NOPl 0 mutants have some other defect(s) that blocks or delays other processes that normally occur during late G1.

This other defect(s)is most likely a defect in the expresRFA m a" sion of some gene(s) required (directly or indirectly) for bud initiation. Therefore, although high copy number SIS2 stimulates C I N I and CI.N2 RNA accumulation PGKI in si/4 mutants, high copy number SIS2 also must be HAC

-

-

0

FIC;~.KI:.

9.--Physical antl f~lnctionalinteraction of SIS2w i t h chromatin. (X) Fractionation of isogenic strains <:M402 [Ask2 SSlll-~f(If,4:.SI.S2 on \1Ep24)], (;M298 [Asis2 S S I I I - v { IfA:SIS2 011 Y(;p.50]] ;lntl CM 899 [A.(i~2 S S D I - V (lfA:~i~2ACOOH on Wp24]] w a s carried out a s descrihetl i n \llhTI.:RlhlS XSII \lI:.TIIOI~Sto obtain a nuclear pellet antl a cytosolic supernatant. The samples \\we c~lectrophoresedon an 8% SDS polyacrylamide gel and analyzed hy Western immunohlotting.

The antibodies used were the 1 2 ( X ascites [directed against the hemagglutinin (HA) epitope], anti-NOPI monoclonal antihotly A66 (XKISand B1,ol%I:.[.,1988), anti-RFA p i 0 (directed against the if)-kD suhrlnit of RFA), anti-PGK1 polyclonal antibody and anti-YDJI polyclonal antihody ACl I O ( ( ~ I ' I A S d (11.

1991). NOPl is ;I nlICle;lr marker antl PGKl is a cytoplasmic marker. HAC is a cytoplasmic protein (as determined hy imInunofluorescencc) that cross reacts with the l X A 3 antihody.

(R) Aliqrlots (0.1 ml) o f the nuclear fraction isolatrd from strain CM402 [Asis2 SSll!-71[IfA:SI.S2on YEp24)]wcrc treated separately w i t h DNase 1, micrococcal nuclease (M.Nase), RNase A, 0.1% Digitonin (D), or 1% Triton X-IO0 (T) as descrihetl hy AI.I.KX and D O ~ W A S(1989, also see \ l ~ \ l l ~ R l A l - 5 ASII ~rr11ol1s).

The samples were centrifuged and thepellets resuspended in 0.1 ml gel sample huffer. The samples were boiled lor 5. min and separated on an 8% SDS-polyacl~lamide gel. Western immunohlotting was performed as in (A). (C) Overexprcssion of SISL!is toxic to a strain with lower histone H2A and H2B gene dosage. Strain CY3169 ( A h / n l Ah/hI)was transformed w i t h either \iEplS (a 2p IJ,X2pl;~smitl),pCB96.5 ( S I S 2 on W.pl3) or pCR974 (SIS2 o n a I1W2 centromere plasmid). Transformants were streaked onto SC minus leucine plates and incubated at SOo for 4 days. The growth rate of CY3169 transformed with the IJ;lr2 centromcrc plasmid without SIS2was the same as CY3169 containing W,plS (data n o t shown).

106

C. J. Di Como, R. Bose and K Arndt

able to suppress the other late G1 defects of sit4 mutants. We suggest that high copy number SZS2 stimulates the late G1 expression of some other gene(s) required (directly or indirectly) for bud initiation and whose expression is defective in sit4 mutants. The identity of such a gene(s) is currently not known.

Asit4 SSDl-v cells are viable, but with a slow growth rate due to a greatly expanded G1 phase. Although Asis2 strains have no readily detectable phenotypes, Asit4 SSDl-vAsis2 strains are inviable and sit4-102 SSDI-v Asis2 strains arrest at 37.5" primarily as unbudded G1 cells. Moreover, low-level expression of CZX2 from a SZT4independent promoter does not allow the cells to replicate their DNA in the absence of both SIT4 and SZS2function. This finding differs from thatof wildtypeSIS2 cells lacking both SIT4 and SSDl function.

When shifted to 37.5", sit4-102 ssdl-dl SZS2 cells arrest as unbudded G1 cells with verylow levels of CLNl and CLN2 RNA (FERNANDEZ-SARABIA et al. 1992). However, when shifted to 37.5", sit4-102 ssdl-dl SZS2 cells containing CLN2 expressed from a low-level heterologous promoter arrest with replicated DNA (a 2n arrest), but the cells are still blocked for bud formation (FERNANDEZ-SARABIA et al. 1992).

We interpret these findings as follows: SIT4and SIS2 have overlapping functions that are required for the expression ofCLNl and CLN2, for the expression of some additional gene ( s ) required for DNA replication and for some gene(s) required for bud initiation. Lowlevel expression of CLN2 from a SZT4independent promoter is not able to cure the DNA replication block and is not able to cure the bud initiation block that occur in the absence of both SIT4 and SIS2 function.

Like the SIT4 and SIS2 overlapping functions, SIT4 and SSD1-v have overlapping functions that are required for the expression of CLNl and CLN2, for DNA replication and for the expression of some gene(s) required for bud initiation. Although low-level expression of CLN2 from a SIT4independent promoter is not able to cure the bud initiation block, it does cure the DNA replication block that occurs in the absence of both SIT4 and SSDI-v function. Therefore,unlike the DNA replication block that occurs in the absence of both SIT4 and SIS2 function, the DNA replication block that occurs in the absence of both SIT4 and SSD1-v function is due solely to low levels of G1 cyclin expression.

SIT4, SIS2, and SSD1-v all seem to be either required for, or are able to stimulate, the expression of certain genes that are expressed periodically during late G1 (e.g., SW4, CLNl, CLN2, HCS26 and CLB5). SIS2 possibly functions in a pathway that is parallel to the SIT4 pathway (and we have not beenable to detect any physical interaction, by coimmunoprecipitation, between SIT4 and SIS2). Also, SIS2 does not seem to function in the SSD1-v pathway (and we have not been able to detect any physical interaction, by coimmunoprecipitation, between SSD1-v and SIS2). One possibility is that

SIS2 might modulate chromatin function.

SIS2 fractionates with nuclei and is released from the nuclear fraction by 0.3 M NaCl and by treatments that digest DNA, but not by treatments that digest RNA. Also, a strain containing lowerlevelsof histones H2A and H2B (a Ahtal Ahtbl strain) is extremely sensitive to increased levels of SIS2.Perhaps this effect is due to an interaction of the very acidic carboxyl terminus of SIS2 with the basic histones (alternatively, overexpression ofSIS2 might reduce transcription of the HTA2-HTB2 locus, which would cause a further reduction in histone H2A and H2B levels). Modulation of histone function by depletion (HANet al. 1988; KIM et al. 1988) or by amino terminal truncation (ROTH et al. 1992) and modulation of chromatin function by factors such as SNF2, SNF5 and SNF6 (HIRSCHHORN et al. 1992) have now become recognized mechanisms for the normal regulation of transcription. Also, it has been shown that the periodic expression of SW4, TMPl and POL1 during late G1 is due notonly to activation during late G1, but repression at other stages of the cellcycle (FOSTERet al. 1993; KOCHet al. 1993). Perhaps a normal chromatin function is involved in such periodic expression. The mechanism by which SIS2 might modulate chromatin functionand stimulate the expression of genes periodically expressed during late G1 will be the subject of future studies.

We thank KAREN FIENfor the anti-p70 RFA antibody; AVROM CAPIANfor the anti-YDJ1 antibody;JOHN A R I S for anti-NOP1 autihody; JEREMYTIIORNERanti-PGK1 for antibody; members of the A R N ~ Y I ' and FUTCXIPR laboratories for discussion; JOSEPH C O I A S ~ ~HONG . ~ I , MA and A N N SUTTONfor comments 011 the manuscript. This research was supported by National Institute of General Medical Studies grant GM-45179 to K.T.A.

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Communicating editor: F. WINSroN