Vol. 12, No. 10

MOLECULAR AND CELLULAR BIOLOGY, Oct. 1992, p. 4503-4514

0270-7306/92/104503-12$02.00/0 Copyright © 1992, American Society for Microbiology

Involvement of the SIN4 Global Transcriptional Regulator in the Chromatin Structure of Saccharomyces cerevisiae YI WEI JIANG AND DAVID J. STILLMAN* Department of Cellular, Viral, and Molecular Biology, University of Utah Medical Center, Salt Lake City, Utah 84132 Received 12 May 1992/Returned for modification 19 June 1992/Accepted 23 July 1992

We have cloned and sequenced the SIN4 gene and determined that SIN4 is identical to TSF3, identified as a negative regulator of GAL] gene transcription (S. Chen, R. W. West, Jr., S. L. Johnson, H. Gans, and J. Ma, submitted for publication). Yeast strains bearing a sin4A null mutation have been constructed and are temperature sensitive for growth and display defects in both negative and positive regulation of transcription. Transcription of the CTSI gene is reduced in sin4A mutants, suggesting that Sin4 functions as a positive transcriptional regulator. Additionally, a Sin4-LexA fusion protein activates transcription from test promoters containing LexA binding sites. The sin4A mutant also shows phenotypes common to histone and spt mutants, including suppression of 8 insertion mutations in the HIS4 and LYS2 promoters, expression of promoters lacking upstream activation sequence elements, and decreased superhelical density of circular DNA molecules. These results suggest that the sin4A mutation may alter the structure of chromatin, and these changes in chromatin structure may affect transcriptional regulation.

transcription. The SINI gene, identical to SPT2 (72), encodes an HMG-like protein (45). SIN2 encodes histone H3 (45). SIN3, which is identical to SDI1, RPD1, and UME4, negatively regulates a number of yeast genes (59, 85, 93, 95) and encodes a 175-kDa protein that interacts with DNAbinding proteins (97). In this article, we report the cloning and DNA sequence of the SIN4 gene. Sequence comparison has revealed that SIN4 is identical to TSF3, a negative regulator of GALl transcription (1Sa). SIN4 was identified as a bypass suppressor which permits HO expression in the absence of the SWI5 transcriptional activator. We have constructed sin4 null mutations and found that they are pleiotropic. We have obtained evidence suggesting that the Sin4 protein functions as a transcriptional activator. This result is surprising because SIN4 was originally identified as a negative transcriptional regulator. Finally, we present evidence suggesting that the sin4 mutation alters chromatin structure.

Regulation of transcription by RNA polymerase II is a complicated process involving many gene products. Genetic analyses have been used to identify mutations in genes involved in transcriptional regulation. A number of transcriptional regulators have been identified in screens involving the Saccharomyces cerevisiae HO gene, which encodes an endonuclease which initiates mating type switching. The HO gene is subject to multiple forms of transcriptional control, including cell type (diploid cell repression), cell cycle (G1 expression), and asymmetric expression (mother cell only) following mitotic division (for reviews, see references 36, 37, and 58). This complex pattern of transcriptional regulation leads to a specific pedigree of mating type interconversion. Genetic analysis has identified six genes, SWIJ through SWI6, required for the transcriptional activation of HO. SWII, SWI2, and SWI3 are believed to encode general transcription factors (70). Strains with mutations in any of these genes have similar phenotypes and are defective in expression of a number of yeast genes, including ADHI, ADH2, GALI, GAL1O, HO, and SUC2. It has been recently determined that SWIJ is identical to ADR6, an activator of ADHI and ADH2 (66, 70) and that SWI2 is identical to SNF2, an activator of SUC2 (47, 54a, 70). The SWI4 and SWI6 gene products are involved in the cell cycle-regulated expression of HO and of the GI cyclins CLNI, CLN2, and HSC26 (8, 10, 56, 65). The Swi4 and Swi6 proteins are components of a cell cycle-regulated DNA-binding activity (3, 56). The SWI5 gene encodes a zinc finger DNA-binding protein which recognizes the HO promoter (86). SWIS is cell cycle regulated in two ways: transcription of the gene is expressed only in the G2 phase of the cell cycle, and the nuclear localization of the Swi5 protein is regulated during the cell cycle (55, 57). Negative regulators of HO, identified as suppressors of swi mutations, have been shown to encode regulators of *

MATERIALS AND METHODS Strains and medium. The strains of S. cerevisiae used are listed in Table 1. Strains FW518, FW842, and FY56 were obtained from Fred Winston. The swilA::LEU2 and swi2A::HIS3 alleles were obtained from Craig Peterson (70), and the swi4A194::LEU2 allele was obtained from Linda Breeden (8). The sin4::TRPI and sin4::URA3 alleles are described below. All gene disruptions were confirmed by Southern blot analysis. Standard genetic methods were used for strain construction and tetrad analysis (75, 79). Yeast cells were transformed by the lithium acetate method (40). Cells were grown at 30°C, unless otherwise specified, either in rich YEPD medium or in synthetic complete medium (78) supplemented with adenine, uracil, and amino acids, as appropriate, but lacking either histidine, leucine, tryptophan, or uracil, to select for plasmids. High- and lowphosphate media were prepared as described before (77). Cloning of SIN4. S. cerevisiae DY1038 (MATaHO::lacZ swiSA::LEU2 sin4 ura3) was transformed with a genomic

Corresponding author. 4503

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JIANG AND STILLMAN

MOL. CELL. BIOL. TABLE 1. S. cerevisiae strains

Strain

Relevant genotype

DY1038 ..A.. 4Ta HO::lacZ swiS::LEU2 sin4 ade2 ade6 his3 leu2 trpl ura3-52 DY131a..... MATa HO::lacZ ade2-1 ade6 canl-100 his3-11,15 leu2-3,112 trpl-1 ura3-52 DY1345a .... MATa HO::lacZ sin4A::TRPI ade2-1 ade6 canl-100 his3-11,15 leu2-3,112 trpl-1 ura3-52 DY1671a ..... A4Ta HO::lacZ sin4A::TRPI ade2-1 ade6 canl-100 his4 leu2-3,112 trpl-1 ura3-52 DY1675a .... A4Tt HO::lacZ sin4A::TRP1 ade2-1 ade6 canl-100 leu2-3,112 trpl-1 ura3-52 his3-11,15 DY143a....MA4Ta HO::lacZ swiSA::LEU2 ade2-1 ade6 canl-100 his3-11,15 leu2-3,112 trpl-1 ura3-52 DY1258W ....MA4Ta HO::lacZ swiSA::LEU2 sin4A::TRP1 ade2-1 ade6 canl-100 his3-11,15 leu2-3,112 trpl-1 ura3-52 MATa swi4Al94::LEU2 HO::lacZ ade2-1 ade6 canl-100 his3-11,15 leu2-3,112 trpl-1 ura3-52 DY1380a .... DY1431a ....MA4Ta HO::lacZ swi4Al94::LEU2 sin4A::TRPI ade2-1 ade6 canl-100 his3-11, 15 leu2-3, 112 trpl-l ura3-52 DY1741a....M AlTa HO::lacZ swi2A::HIS3 ade2 ade6 his3 leu2 trpl ura3-52 DY1825 a ....MA4Ta HO::lacZ swi2A&::HIS3 sin4A::TRPI ade2 his3 leu2 trpl ura3-52 ade6 DY150b.....MATa ade2-1 canl-100 his3-11,15 leu2-3,112 trpl-1 ura3 DY1702b .....MATa sin4A::TRPl ade2-1 canl-100 his3-11,15 leu2-3,112 trpl-l ura3 DY882C....MA4Ta ade2-101 his3-A200 leu2-A1 lys2-801 trpl-A63 ura3-52 DY1692C .... MATo swilA::LEU2 ade2-101 his3-A200 leu2-Al lys2-801 trpl-Al ura3-52 DY1773C ....MA.4Ta swilA::LEU2 sin4A::URA3 ade2-101 his3-A200 leu2-AlJ lys2-801 trplA ura3-52 DY1725C .... MATa sin4A::TRPI ade2-101 his3-A200 leu2-Al lys2-801 trpl-A63 ura3-52 DY1712C ....MA.4Ta sin4A::URA3 ade2-101 his3-A200 leu2-A1 lys2-801 trpl-A63 ura3-52 FW518 ..M..ATa his4-9128 ura3-52 CryR FW842 .. MATa his4-9128 ura3-52 lys2-173R2 spt2-150 FY56 .. MATa his4-9128 lys2-1288 ura3-52 a Isogenic strains in K1107 background (54). b Isogenic strains in W303 background (91). c Isogenic strains in S288C(YPH499/YPH500) background (84).

library (14) in the multicopy vector YEp24 by selection for uracil prototrophy. The sin4 mutation causes a clumpy phenotype, and a procedure was used that selects nonclumpy cells. The transformants (approx. 4,000) were suspended in synthetic complete medium lacking uracil and subjected to sedimentation at 1 x g until most of the cells had settled (10 min). The supernatant was removed, and the cells were plated on selective medium and subjected to a blue-white visual assay for lacZ expression with the chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-3-Dgalactopyranoside) and nitrocellulose filters (9). Among approx. 200,000 colonies, 101 white colonies were identified. These white colonies were rescreened in several ways. First, cells were allowed to lose the plasmid during growth without selection, and Ura- cells were selected on medium containing 5-fluoroorotic acid (6). These Ura- derivatives were screened to verify that they were phenotypically blue (i.e., sin4 mutants) in the absence of the plasmid. Second, the colonies were tested for their ability to mate, since a plasmid containing the MATa gene would lead to production of the al-a2 repressor, and this repressor prevents both HO expression and mating. Of 77 strains that survived the secondary screens, 16 were chosen for further analysis. DNA was prepared from these strains and transformed into Escherichia coli, and plasmid DNA was prepared and subjected to restriction analysis. Nine of the plasmids had an identical restriction map, and one of these plasmids, M1304, was retransformed into strain DY1038 and produced a SIN4+ phenotype. The seven plasmids that contained inserts different from that in M1304 failed to complement the sin4 defect when retested and were not analyzed further. Plasmid constructions. pTF63 is YEplacl95 (26) with the BluescriptII KS+ polylinker (Stratagene) and was constructed by Tim Formosa (52a). A 4.8-kb BamHI fragment from M1304 was cloned in both orientations into the BamHI site of pTF63, creating M1305 and M1306. M1387 contains the 4.8-kb BamHI fragment cloned into the pRS316 CEN vector (84). M1359 was created by cleaving M1306 with PstI and religating, joining the first PstI site in SIN4 to the PstI

site in the pTF63 polylinker. M1358 was created by cleaving M1305 with EcoRI and religating, joining the EcoRI site in SIN4 to the EcoRI site in the pTF63 polylinker. SBA117 and SBA120 were created with exonuclease III (ExolII) as described below and extend from the EcoRI site to nucleotide 2938 and 2767, respectively (where + 1 is the ATG codon). The sin4A::TRPI and sin4A::URA3 alleles were created by the y gene disruption method (84) with plasmids YIplac2O4 and YIplac211, respectively (26). SIN4 sequences from -64 to +2718 have been deleted; the endpoints were generated with ExoIll (described below). The Sin4(1-969)-LexA fusion protein was made in several steps. First, a 500-bp BamHI DNA fragment containing the lex.A DNA-binding domain (LexA87) from p6R-LexA87 (obtained from Joanne Kamens and Roger Brent) (27) was subcloned into the BamHI site of pTF63, generating M1504. Second, a three-part ligation was performed to join SIN4, LexA87, and the YEplacl81 vector (26). The SIN4 fragment was from plasmid SBA130 (an Exoll deletion clone) and had HindIII and BssHII ends (both restriction sites are from the polylinker). The LexA87 fragment was from M1504 and had BssHII and EcoRI ends. The YEplacl81 vector was restricted with HindIII and EcoRI. Finally, the clone resulting from this three-part ligation was cleaved with SacI and BssHII (polylinker sites between SIN4 and LexA87), and the ends were blunted with T4 DNA polymerase and deoxynucleoside triphosphates and religated. This last step puts LexA87 into the SIN4 reading frame. The junction was verified by sequencing from the C termini of the new gene fusion through the entire LexA87 region. The resulting clone, M1514, generates a Sin4-LexA fusion protein under control of the SIN4 promoter. The fusion is at the codon for amino acid 969 of SIN4 (nucleotide 2907). The Sin4(1-969)LexA fusion construct provides SIN4 function, since this plasmid complements a sin4 defect. The Sin4(1-59)-LexA plasmid was constructed in two steps. First, a three-part ligation was performed to join the SIN4 promoter, LexA87, and the YEplacl81 vector. The SIN4 fragment was from plasmid SBA104 (an ExolII dele-

VOL. 12, 1992

tion clone) and had HindIII and BssHII ends (both restriction sites are from the polylinker). The LexA87 fragment was from M1504 and had BssHII and EcoRI ends. The YEplacl81 vector was restricted with HindIII and EcoRI. Finally, the clone resulting from this three-part ligation was cleaved with BamHI and BssHII (polylinker sites between SIN4 and LexA87), and the ends were blunted with T4 DNA polymerase and deoxynucleoside triphosphates and religated. The resulting clone, M1543 [Sin4(1-59)-LexA], directs the expression of a Sin4-LexA fusion protein with the first 59 amino acids of Sin4 (out of 974) fused in frame to the 87 amino acids comprising the LexA DNA-binding domain. This LexA protein is expressed under the control of the SIN4 promoter and was designed as a control for expression of the Sin4-LexA plasmid. The YEp-CTS1::lacZ reporter plasmid M1591 is derived from the YEp353 vector (53) and contains 1.3 kb of the CTSI promoter driving expression of the lacZ gene (20a). Plasmids pLR1Al (98), p1840 (33), and pSH18-18 (33a) were obtained from Roger Brent's lab. Plasmid pCK30 was obtained from Cindy Keleher (43). Plasmid pLGA312S contains the CYCl promoter, including the upstream activation sequence (UAS), driving lacZ expression (28). The PHO5::lacZ reporter plasmid pMH313 contains the entire PH05 promoter driving lacZ expression (31). The pLL53 plasmid contains a HIS4::lacZ gene fusion under control of the his4-9128 promoter (with a Ty 8 insert) on a YCp vector (48a). The UAS-less HO::lacZ plasmid was constructed by replacing the XhoI-SacI fragment of pLGA-178 with a HindIII-SacI fragment from HO::lacZ46 (10) after converting the HindIII site to an Xhol site. This UAS-less HO::lacZ plasmid (M740) has the HO promoter without a UAS driving lacZ expression. The UAS-less CYCJ::lacZ plasmid pLGA-178 has the CYCl promoter without a UAS driving lacZ expression (29). The UAS-less GAL1::lacZ plasmid pLRlAl has the GALl promoter without a UAS driving lacZ expression (98). The UAS-less PHO5::lacZ plasmid pMH324 has the PH05 promoter without a UAS driving lacZ expression (31). Sequencing of SIN4. Sets of overlapping, unidirectional deletions were made from M1306 (5' deletions) and M1358 (3' deletions) by the ExoIII-mung bean nuclease method of Henikoff (35). M1306 was cleaved with SacI and NotI before ExoIII cleavage, generating the SN deletion set. M1358 was cleaved with Sacl and BamHI before ExoIII cleavage, generating the SB deletion set. All deletion subclones retain the BssHII site present in the BluescriptIl polylinker (Stratagene), and these BssHII sites were used to excise the deletion clones for other constructions. The DNA sequence was determined by the dideoxy chain termination method with Sequenase 2.0 (U.S. Biochemical Corp.) from doublestranded templates. Both strands were completely sequenced. Chloroquine gel electrophoresis. Yeast DNA was prepared from isogenic wild-type (DY150) and sin4A (DY1702) strains as described before (1). Cells were grown under uracil selection to maintain the plasmid, either YCp5O or pTF63. For the one-dimensional gel, the DNA was electrophoresed on a 1% agarose gel containing 2.5 ,ug of chloroquine (Sigma) per ml as described before (30, 83). The two-dimensional electrophoresis was performed essentially as described elsewhere (69). Electrophoresis in the first dimension was done in 0.7% agarose with TBE buffer (89 mM Tris borate [pH 8.3], 25 mM EDTA) containing 0.8 ,ug of chloroquine per ml for 20 h at 40 V. The gel was soaked in TBE buffer containing chloroquine (4.0 ,ug/ml) for 5 h and then electrophoresed for 20 h at 40 V in TBE buffer containing chloroquine (4.0 ,g/ml)

SIN4 AFFECTS CHROMATIN STRUCTURE

4505

in the second dimension. The DNA in the one- and twodimensional gels was transferred to BioTrace HP membranes (Gelman Sciences) by a capillary transfer method (52), and a Stratagene UV transilluminator was used to cross-link the DNA to the membrane. The blots were probed with 32P-labeled YCp5O, pTF63 (BamHI cleaved), or YEp24 (the 2.2-kb EcoRI fragment as a probe for the 2,um circle) DNA, labeled with a Random Primers DNA labeling kit (BRL) and [a-32P]dATP (3,000 Ci/mmol; Amersham). Autoradiographs were obtained by exposing the blots to preflashed X-ray film with the aid of an intensifying screen. The autoradiograph of the one-dimensional gel was scanned with a Bio-Rad densitometer, and film densities were plotted with 1-D Analyst Macintosh software (Bio-Rad). Other methods. Quantitative determinations of P-galactosidase activity were performed in triplicate as described before (10). Units of P-galactosidase activity were calculated as OD420 t-1 v-l p-1 x 1,000, where OD420 is the optical density of 420 nm, as determined in the ONPG (o-nitrophenyl-13-D-galactopyranoside) assay, t represents the time of the reaction (in minutes), v represents the volume of supernatant used (in milliliters), andp is the protein concentration (in milligrams per milliliter), as determined by Bradford assays (7). RNA blot hybridization was performed as described before (20). The enzymes used for molecular cloning were obtained from Bethesda Research Laboratories or Stratagene and were used according to the manufacturer's specifications. Nucleotide sequence accession number. The GenBank accession number for SIN4 is M93050.

RESULTS Cloning and sequencing of the SIN4 gene. The SIN4 gene was identified previously as a suppressor mutation which permits expression of an HO::lacZ reporter gene in the absence of the SWIS transcriptional activator (85). The SIN4 gene was cloned by using a blue-white colony screen with a chromosomal HO::lacZ reporter (see Materials and Methods). Yeast colonies with this reporter gene are white on X-Gal if the genotype is swiS but blue if the genotype is swiS sin4. A plasmid (M1304) was isolated which complemented the sin4 defect, and the restriction map of M1304 is shown in Fig. 1. Evidence that M1304 contains the SIN4 gene was obtained by demonstrating linkage of the cloned DNA to the sin4 locus. Specifically, the TRPJ gene was inserted into the cloned DNA (the sin4A::TRPI disruption described below), and this construction was integrated into the yeast chromosome. This sin4A::TRPJ swiSA::LEU2 HO::lacZ strain (blue phenotype) was crossed with a sin4 swiSA::LEU2 HO::lacZ strain (blue phenotype), and the resulting diploids were subjected to tetrad analysis. (Since the sin4 homozygous diploid is deficient in sporulation, the diploid was transformed with a plasmid containing the SIN4 gene. After tetrad dissection, cells were cured of the plasmid before SIN4 phenotype was determined.) Of 103 tetrads, 100 had four blue colonies and are therefore parental ditypes. A1though three tetrads had one white colony, each of these white colonies was actually a diploid cell, and HO expression is blocked in diploids. Thus, no recombinants were obtained from this cross, and we conclude that M1304 contains the SIN4 gene. The location of the SIN4 gene on M1304 was deduced by constructing deletion derivatives and testing their ability to complement a sin4 defect. As shown in Fig. 1, a 4.8-kb

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JIANG AND STILLMAN

MOL. CELL. BIOL. SIN/ Open Reading Frame

1OOObp EcoRI Hindlll

I

M1304

I

BamHl Sail EcoRI

I I I

Krnl

17

Krni

BamHl EcoRI PstlPstl XbalPstI Bglll Pstl

11 11~~

BamHl

M1305

SIN4 Function

71 +

Pstl

BamHI

EcoRI

M1358

stI

BamHl

BamHI

M1 359

I

+

I

EcoRI

SBA 117

+

EcoRI

SBA1 20

SIN4A::TRP1

II I

SIN4A::URA3

. I., . TRPI

1..

I

URA3 I_

FIG. 1. Map of the SIN4 gene. Plasmid subclones tested for complementation of a sin4 mutant are indicated. The endpoints of the SBA117 and SBA120 clones were generated with ExollI. The entire SIN4 open reading frame was deleted in the sin4A::TRP1 and sin4A::URA3 gene disruptions, using ExoIII-generated deletion endpoints.

BamHI fragment complements sin4 (on either a YCp singlecopy or a YEp multicopy plasmid), whereas the 3.4-kb BamHI-PstI fragment does not, suggesting that the SIN4 gene traverses the PstI site. A 4.4-kb EcoRI-BamHI fragment also complemented the sin4 defect, and this restriction fragment containing SIN4 was used for further studies. Figure 2 shows the sequence of the 4.4-kb EcoRI-BamHI fragment containing the SIN4 gene and the predicted amino acid sequence of the Sin4 protein. There is an open reading frame of 2,922 bp that could encode a 111,226-Da protein (974 amino acids). A search of the GenBank data base revealed no significant homologies. A computer search of a private data base (performed by Mark Goebl, University of Indiana) revealed that the SIN4 gene is identical to the TSF3

(1Sa). Disruption of the SIN4 gene. To determine the phenotype of a null mutation at the SIN4 locus, we disrupted the chromosomal copy of SIN4. A sin4A disruption was created by removing a 2.78-kb fragment of SIN4 and replacing it with TRP1 or URA3 sequences from the integrating vectors YIplac2O4 and YIplac211, respectively. These gene disruptions entirely remove SIN4 coding sequences. The wild-type chromosomal SIN4 gene in three strains (K1107, S288C, and W303 strain backgrounds) was replaced with one of these gene disruption constructions, and the structure of the sin4A allele was confirmed by Southern blotting. The fact that haploid yeast cells bearing a sin4 null mutation are viable allows us to conclude that SIN4 is not essential for growth at 30°C. However, the sin4 null mutation is lethal at 37°C. The sin4 null mutants are temperature sensitive for growth in all three strain backgrounds. All experiments that follow in this article were conducted at 30°C. Mutations in swi genes are suppressed by sin4. The sin4 mutation was originally identified as a suppressor of swiS, allowing expression of a chromosomal HO::lacZ reporter in the absence of the SWI5 activator. As shown in Table 2, the swiS mutation reduces HO::lacZ expression to less than 1% of that in the wild-type SWIv control. The sin4A null mutation overcomes the swiS defect, restoring HO::lacZ gene

expression to 180% of the wild-type level. Interestingly, the sin4A null mutation causes a fourfold increase in HO::lacZ expression in a SWI' strain. The sin4A null mutation also suppresses mutations in swi4 and swi2 (Table 2). SWI4 and SWI6 are involved in the cell cycle regulation of HO and the G1 cyclins of S. cerevisiae and are components of a cell cycle-regulated DNA-binding activity (3, 56, 65). SWI2, along with SWIJ and SWI3, is thought to be a component of the general transcriptional machinery (70). The swi4 suppression is significant, causing expression to rise from 0.1 to 66% of the wild-type level. However, the swi2 suppression is modest, raising expression from 1.0 to nearly 12% of the wild-type level. These results differ from those of Stemnberg et al. (85), who reported that sin4 suppressed swiS but not other swi mutations. This apparent discrepancy may be explained by the fact that Stemnberg et al. (85) did not use null alleles of sin4, swi4, and swi2, whereas the present work does. SIN4 was also identified in a screen for mutations that suppress a swi6 defect in expression of HO::lacZ (70a). SIN4 can function as a transcriptional activator. Microscopic examination of sin4A mutants revealed a slightly clumpy phenotype (data not shown). Since swiSA mutants are slightly clumpy (20), we also examined sin4A swiSA double mutants. The clumpiness is much more severe in the double mutant, suggesting that SIN4 and SWIS act in different pathways that affect cell separation and morphology. The clumpiness of the sin4 swiS double mutant strains led us to ask whether CTS1 expression is altered by the sin4A mutation. The CTSJ gene encodes chitinase, which is needed to degrade the chitin septum separating mother and daughter cells, and strains with a null mutation in ctsl show a clumpy phenotype (46). We have shown previously that swiS ace2 double mutants show an additive increase in clumpiness and that CTSI expression is defective in ace2 mutants (20). It therefore seemed possible that CTSJ transcription could be regulated by SIN4. Figure 3 shows the results of RNA blot hybridization analysis of CTS1 transcription in isogenic strains differing at

SIN4 AFFECTS CHROMATIN STRUCTURE

VOL. 12, 1992

CAATTCTACCTATACTACTTCAMAGCGAAA

-481

ACAAGCATTCCTTCGATCATACGAACTTG,AACAGGAAAACAATAACGTGACCTTTGAGCACx ATAIACACAACGAAGATCTGGATGAAG ATACCACTCCTTACGTGAAT -361 AGTGGCCCAACTAAIIMATTAAACTTTAACTCCATCCTACACCCTCTTGTAGAAGCATTCTAAOITATTTTTTATAATACTCTAAATGCGGACTGATAACTTTGC -241 -~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~~~~~-6 ACACTTTGTATTCAAATTGTTTGCATAAATATATAAAAATTCTTGCC_TTTGClCGCTCATGATGATAAATCTCGCTA -121

NGTAAATATTAGT

-1

M M L G E H L M 8 W S K T G I I A Y S D S Q S S N A N I C L T F L E S I N G I N jATAGTGAACTTATGAGCT _z __CTA_GCTGTTACATACAGTGIACTITCCAATGCCAATATGCCACATTTTGAACAAAGAATAAAC

40 120

TTTM

R

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F

H

L

T

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Q

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P

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TCCAAAGAAAAGAAACTAGCAG YSDSAASGG

Q

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Y

Q

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8

S

T

L

S

T H

S

T

T

T

S

V

N

G

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TGGCGATTTCATACGCCGCAAAAATATGTATTACACCCGCAATTGCACGAAGTTCAATACCAAGAAAGTAGTAGTACTCTAAGCACTCACTCGACAACAACTTCTGTCAATGGATCAACT T A G V ACA

G S T

P N F C G

N S N K 8

P P Q F F Y N I

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CAACAGCAACAAGTCTCCTCCACAGTTCTTT1TATAATATATCTTCCATTCATTGGAACAACTGGTTTAGTTTACCAGGTGACATG

N

I

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I

120

3360

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160 48 0

L E F R W L T S S

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200 60 0

MT N L I T C Q R P D R A T T Y E K L T N V F Q D N V Y L A V C D E L C N 1 TTGGCTGTATGTGACGAATTAG GTAACATGACCATGTTlTATGCiAGCCITGATCGGGCCACTACATACGAAAAGTTAGAGTATTTCAAGsbA

N H V X P L K P V D K L K P K AACCACGTTAT

80 240

K

G__zACAAGAATACAATACATCATCTGAATTTCGCTGCTCACCTCTTCAAArA

.ATTCTGCOCATTTGATAGTATCTAAATACAACCAACACAGCAATCCTCCATATGGTGTGTACCATCACTTAAATATGCGTGCTTG

240 720

A

I R K N G Q I D F W Y Q F S N S R D H K K I T L Q L L D T S N Q R F K D L Q W GCAATAAGGAAAAACOGCAATG =TTTGTACCAATTTTCAAACTCAAAGGATCATAAAAAAATAACCTTACACCTGCTGGACACTTCAAATCAAAGATTCAAGGATCTTCAATGG

280 84 0

L E F A R I T P M N D D Q C X L I T T Y S K L S X N I S F Y K L H V N W N L N A CTTGAATTTGCTAGAATTACACCTATGAAMTTATGATAT GTTGATAACCACATATTCAAAATTATCTAAAAATATrCTTTTTTACAAACTACACGTTAATTGGAACCTTAATGCC

320 9 60

L S T T L D P T D D E G H V L K L E N L H V V S CACTACTTTA GATCCAACGG ATGATGAAG GGCATGTsTGTTGATTAGAGAACCTGCATGTTCTGTCT

360 1080

V L Y N V C D T S K S L V K R Y R L A P T Q L S A E Y TCTGCGAAATATAGACTAGCTCCAACACAACTTTCAGCCTGATAT GTTTGCGATACATCAAAA

400 1200

S V I V S TcscTAATCG

K

T

P

N

V

Q F C A F D S S

L

N

D

P

S

S

L

K

I Q C

ACCAAACAAGCTAGCCCAAA K S S I E K D P S AA _ACATGAAAATCCCTCC

P

E

I

L

N

I

D

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N N

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F

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Y

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L

TTGGTGATATTGAAACCAGATTTAAATATCGATCGAAATAATTCTACGAATCAAATATTCCAATCGCGCCGTTACAACCTTCGTCGCCACAGTGATATTCTCGACAAAAGTACC

440 1320

L I T S 8 M F D A F V S F Y F E D G T I E S Y N Q N D W K L E T E R L I S Q S Q CTTCGAGACT GAACGACTTATAAGTCAGAGCCAA TTGATA rTAATTCGrGTACCATCGAAT CCTACAATCAAAATGATTG GAA

480 144 0

V

L

L

G

I

K

L

F

K

P

K N

D

I

L

I

A

K Q

Y

N

K R

W

P

L

8

A

G

T

F

N

N

I

Q

Y

G

F

K

Q S

L

P

R

R

L

P

Y

P

N

S

L

V

R

E

R

W

H

X

S

D

I

K V S

V

P

L

S

D

N

K

C

K

G

V

V

T

I

520

ACCATACCAGATTTAATTATGGGAAGmTOCTACCTCCATTCACTTGGATGAAGGTTTCACCATCTATCTCTCGGGTTATC 156 0

TTAGGAAAATTCAAAATT V

8

8

P

Q F Y A A V Q R N Y A D P E X D S

I

N

A

T A

L A

F

G

Y

V

K

S

L

560

G=AAATATAATAAATGCCACAGTATCTGTTCAAACAAATCCTGAGAAGGATTCTATAAATGCTACTTTGATTGATACGTAAAAGTTTA 1680 R K R A K E F I T T L L K S L Y ATAAAAAO _CA_AAAT_TTTACGCTTTAAAAGCCTATAC

600 1800

S F F N I 8 P D A P R E I N D K I I T S R P L Q K I M L L Q L E L G S C F S Q E TCGTTTTTCs\AACAC TCCCTAAAGACATTATGGATAAAATAATAACACA £CACTTCAAAAATTATGCTATT CCAGCTGGAACTTG GCA7GTTCGCAAA

640 192 0

Q I S N N S N Q Q AACAAATAACTAATATCACACAA

680 2 04 0

H K Q I S A 8 D L T CATAAGCAAATATCTGCCC

N

I

E

E M A R

V

I

L

I

A

A

K

T

H

I

L

R

I

Y

L

K N

V

L

F

A

S

F

L

A

C

F

N

G

D G

V

A

R N

F

H

F

A

I

AAT_ATGAGTATGGGGATACTCTACTTGAAAAATGTTTTm CTTGCTTCATAATCTTaGCGAGAAATTTTCATTTCGTTG Q N P K L F Q T I

F

S

K

Q D L

I

H

S

L

I

P

V

A

K W

F

V

K

F

I

E

T

Y

L

T

Q E

I

L

I

L

720

CCTGTTGCTAAATGGTTTmCAATATCACTTATTTGACCAAGAATTTAATTTA 2160

CAAAACCCGAA

I N D P T N K E Y T L V H G I F C A K X S R T L I L S I L N E I K K V T Q I V A CTTTG.ATATTGTCTATTCTTAA CGAAAT CAAGAAATCTACCCAAATAGTGGCc ATAAATGATCCcAC ACAGAATACACACTAGTTAGATATCACAAAATGT CCAGGA

760 2 28 0

N

MlmGACTGGATTTCGAAAAATTTGAAACGTTTTTACTTCATGTAAACAAC

800 2 40 0

K F I A L C E Q Q P S Q E R E F S L L V K A E I P P E Y A K V G D F L L Q Y A N AAATTATCGCGCTATGCGAACAACAGCCATAAGAMACGTATTTTCGTITITTGGTGAAAGCTGAAATCCCCCCAGAATATGCCAAAGTGGGAGATT:TCCTCCTCAGTATGCTAAT

840 2 52 0

N

A V I S H A N A A A V Y F A D T S G L K I S N S E F F N P E I F H L L Q P L E AATGCAGTGATTTCCCATGC GAATGCGGCGCTATTTGCAGTATACATmGCCTTAAAATATCTAATTCAGACTTAATTCCCAATATTATTTATTGCAACCATTAGAA

880 2 64 0

G L I I D T D K L P I K N R T S K S F S K L L Y D D V T C D K L S V S E I S D TCTGT ACAAACTGAC TGTATCAGAAATATCCGAT AACAACTAAAAAAACCTCCAAATCTTTTAGCAAATTACTGTACGACGATGTAAC GAGGATATTGACAGAT

920 2 76 0

F

R

P

E

T

S

Y

P

I

L

N

E

AAAm=GASACAACAmA

S

S

T

F

L

K

L

V

L

S

E

S

P

V

D

F

E

K

F

E

T

F

L

V

D

V

N

E G

R

L

K

R

C

S

R

C

G

S

V

T

R A

C

N

I

I

S

S

D

K

T

I

V

P

T

S

I

Q T K

R

W

P

T

M

Y

T

GGGAAACTTAAGAGATGCAGTAGCTGCGGTTCCCGTCACAAGAGCAQGCAAATATAATATCGAGTGATAAGACAATTGTACCATCCCATCAAAGCCAAGATGGCCAACCATCTATACA R

L

C

I

C

8

C

M

L

F

E M

D

C

AnGTAGAsTGAAAGAGrPTCATTCTTAAGATACTTTTTTAATTTTAAACATTTCACTTCTCGACTTAATAAAAAACAAATCA A

960 2 88 0 974 3 00 0

ACCAACWTACGCATTTCTCTCCTACGGAATTTTCTGTGCAGATGTCCCGATCTACGTGGCAGATAGTGAAAACTCCGTAGCCAA 3120

GAGTGACAGM

AAGGGCCATCGGAAAAACCTCTTCAAAAAAAAAGGTTTTTAGGCTCGAACGAGCCGGAGCAATAATGTTCCTTTGAAAAATAGCAATCGTTATGCAACCATTTTATATGAGTCTGCCCCT 3240 CAAGGGACTCGCCACCGr.ACCGCTGCACTTGTTTGCATTCATCCACATATAACCTGCTTGTGTATGACAGGAGAAAAAATACTTCATTCGCACTTGTTAACCAACAGCGACATGTCTACC 3360 TCCACTCGATAATTTGAAAATAAACGAGAGACCTGGCTCTCATGATCAT

GGG

TCCATGAC,AACTCAMTGT

3480

CAATCTATTTCACGTTCCCAACCTACTCTCTACCAGCTCAACATAGATCCTOOTGACTCCCCGAATACA 3600

CAACGATTCGACTTTAAAGTTAATGCAAGAGATCTGCTMACTTCGAATGTCCTCGGATATCCTCCTCAGGGAGTATTTAACACCTAAAGAATTAAAAr.TTTTTCAAGCACTTCTGTAT 3720 TCCAATAAACA

ATTTCCAAAACTATTGACCCTACTGCCAGGCCACGTAAAACAAAACAAAGGGACAATGAT 3840 3902

FIG. 2. Nucleotide sequence and deduced amino acid sequence of the SIN4 gene.

4507

MOL. CELL. BIOL.

JIANG AND STILLMAN

4508

TABLE 2. Suppression of swi mutations by sin4a % Of

~~Relevant genotype

13-Galactosidase activity (U)

wild-type activity

DY131 (wild type) DY1671

SWI+ SIN4+ SWI7 sin4A

337.9 ± 43.9

1,417.6 + 108.3

100 420

DY143 DY1258

swiSA SIN4+ swi5A sin4A

2.9 ± 1.4 609.5 + 35.4

1 180

DY1380 DY1431

swi4A SIN4+ swi4A sin4A

0.4 + 0.1 223.7 ± 6.5

Involvement of the SIN4 global transcriptional regulator in the chromatin structure of Saccharomyces cerevisiae.

We have cloned and sequenced the SIN4 gene and determined that SIN4 is identical to TSF3, identified as a negative regulator of GAL1 gene transcriptio...
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