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Molecular and Cellular Biology, August 2000, p. 5960-5973, Vol. 20, No. 16
Department of Molecular and Cell Biology,
University of California, Berkeley, California 94720-3202
Received 3 February 2000/Returned for modification 8 March
2000/Accepted 16 May 2000
The eukaryotic transcript elongation factor TFIIS enables RNA
polymerase II to read through blocks to elongation in vitro and
interacts genetically with a variety of components of the transcription
machinery in vivo. In Saccharomyces cerevisiae, the gene
encoding TFIIS (PPR2) is not essential, and disruption strains exhibit only mild phenotypes and an increased sensitivity to
6-azauracil. The nonessential nature of TFIIS encouraged
the use of a synthetic lethal screen to elucidate the in vivo roles of
TFIIS as well as provide more information on other factors involved in
the regulation of transcript elongation. Several genes were identified
that are necessary for either cell survival or robust growth when the
gene encoding TFIIS has been disrupted. These include UBP3,
KEX2, STT4, and SWI2/SNF2. SWI1 and
SNF5 disruptions were also synthetically lethal with
ppr2 TFIIS promotes the readthrough of
blocks to elongation by RNA polymerase II by first stimulating the
polymerase to cleave its nascent transcript and then to read through
the block (62). In addition to intrinsic blocks determined
by the DNA sequence, nucleic acid binding proteins can also stall the
polymerase (15, 52). In the eukaryotic nucleus, the template
DNA is associated with many DNA binding proteins important in both
chromosome structure and regulation of gene expression. Indeed, in
vitro, chromatin severely inhibits transcript elongation, and factors
that allow efficient transcription of nucleosomal templates are just
being identified. These include FACT, Elongator, and HMG14, all
identified in vitro (18, 55-57). In addition, Spt4, Spt5,
and Spt6 have been genetically associated with chromatin and
transcription (5, 31, 50, 68, 69, 77). In vitro, DSIF, a
human complex with Spt4 and Spt5 homologs, also can alter transcription
on pure DNA templates (73). Its activity in vitro is
affected by both pTEFb and a protein complex termed NELF
(79). However, TFIIS itself does not facilitate efficient
transcription on chromatin templates in vitro (38).
A genetic approach was taken to learn about factors required for
efficient transcript elongation and the specific roles and requirements
for TFIIS in vivo. To do this, synthetic lethal genetic interactions
were investigated with a deletion of PPR2, the gene encoding
transcript elongation factor TFIIS from Saccharomyces cerevisiae (14, 35, 40, 41). PPR2 is not an
essential gene, and the disruption of the gene confers only mild
phenotypes. Both the viability of the ppr2 Several distinct complexes capable of remodeling chromatin have been
identified (reviewed in reference 42). It might be expected that such activities could participate in regulating transcript elongation. However, only for the Swi-Snf complex is there
evidence that it plays a role subsequent to the establishment of
preinitiation complexes at the promoter (3, 6, 67). The
potential involvement of the Swi-Snf complex during elongation is
supported further by results presented here demonstrating synthetic lethal genetic interactions between transcript elongation factor TFIIS
and several components of the Swi-Snf complex.
Strains, genetic methods, and media.
The S. cerevisiae strains used in this study are listed in Table
1. Strains were derived from CH1305
(45), YPH499 and YPH500 (64), W303
MATa and W303 MAT
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Genetic Interactions between TFIIS and the Swi-Snf
Chromatin-Remodeling Complex
and
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
, suggesting that the reduced ability to remodel
chromatin confers the synthetic phenotype. The synthetic phenotypes
show marked osmosensitivity and cytoskeletal defects, including a
terminal hyperelongated bud phenotype with the Swi-Snf complex. These
results suggest that genes important in osmoregulation, cell membrane
synthesis and integrity, and cell division may require the Swi-Snf
complex and TFIIS for efficient transcription. The detection of these
genetic interactions provides another functional link between the
Swi-Snf complex and the elongation machinery.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
strain and the
presence of a TFIIS gene family in the mouse (32, 37, 39)
and human (71, 72, 74) genomes suggested that TFIIS might
have more than one homolog in S. cerevisiae related by
sequence or function. The complete sequencing of the yeast genome
(24) revealed that PPR2 is the only full TFIIS
sequence homolog present in S. cerevisiae. However, the
possibility that PPR2 might have a functionally overlapping protein unrelated by primary sequence was still reasonable. The possibility of a functional homolog of PPR2 was also
supported by the presence of two functionally similar bacterial
proteins in Escherichia coli, GreA and GreB. GreA and GreB
are functionally similar to TFIIS but are unrelated to TFIIS by
sequence or structure (44, 54, 65).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
, (70), and
N222 and Z321 (78). Both Escherichia coli
calcium-manganese-based transformations and electroporation
transformations were used (29) with E. coli strain DH10B (recA1 hsdR mcrA mcrB mrr deoR)
(25). Yeast cells were transformed using lithium acetate
(23). Standard yeast methods and media were used
(27).
TABLE 1.
S. cerevisiae strains used
Plasmids.
All plasmids used and their cloning strategies are
listed in Table 2. The plasmid for the
synthetic lethal screen was based on pDS1, derived from pDK221
(43) by replacement of the LEU2 gene with
Ampr. Vent polymerase (NEB) was used for all
PCR-generated subclones.
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Yeast whole-colony PCR. A procedure for direct whole-colony PCR was modified for this work (36). The reaction buffer contained 12.5 µl of 10 mM Tris-HCl (pH 8.3)-50 mM KCl-2.5 mM MgCl2-170 µg of bovine serum albumin (BSA) per ml-200 µM each of the four deoxynucleoside triphosphates-1.0 µM each primer-50 U of AmpliTaq (Perkin-Elmer) per ml. A yeast colony was added to a 12.5-µl reaction, and the reaction mix was overlaid with mineral oil. The PCR was initiated with a 5-min 92°C denaturation step, followed by 35 cycles of 92°C for 1 min, primer annealing at the appropriate temperature for 2 min, and 72°C for 2 min. All primer annealing temperatures were above 50°C.
Synthetic lethal screen.
The red-white colony sectoring
assay has been described previously (43) and subsequently
applied to a synthetic lethal approach (45). An
ADE3 plasmid (confers a red color when placed in an ade2 ade3 background) containing a nonessential gene is lost
at a high frequency in the absence of selective pressure due to a mutant ARS/CEN sequence, leading to the appearance of
sectored colonies. An ade2 ade3 ppr2 strain (CMKy4) was
transformed with this ADE3 plasmid also containing
PPR2 and URA3 (pJD4).
Cloning of synthetic lethal genes. A LEU2 CEN/ARS genomic library (22) was used for the complementation screens. High-efficiency transformation conditions were optimized for each mutant. Between 5,000 and 10,000 transformants were screened for each mutant to ensure genomic coverage, and plasmids were recovered from transformants that sectored reproducibly. Each plasmid was used to transform the original mutant to confirm that it restored sectoring. Sequencing identified the genomic fragment responsible for restoring sectoring, and the specific gene was isolated after subclones of each library plasmid were tested.
Gene disruptions.
Disruptions of PPR2 were made
by replacing codons 40 to 284 of PPR2 with the
hisG-URA3-hisG cassette (2). The disruption was
confirmed by Southern blot analysis on
6-azauracil-sensitive transformants. These strains were
plated onto 5-fluoroorotic acid medium to select for Ura
recombinants (2).
, kex2,
snf2, swi1, and snf5 were
made individually in a ppr2/PPR2+ diploid
strain (CMKy35) by replacing one copy of the entire target gene with
the kanr gene (26). A
snf2 mutation was also made in CMKy48
(PPR2/PPR2). Transformants were selected on G418 plates and
replica plated to new G418 plates after 48 h. Correct integrants
were identified by PCR analysis. PCR primers containing ~45 bp of
homology to the target gene on the 3' end and 18 bp to the
kanr gene on the 5' end generated a fragment
containing the kanr gene flanked by the upstream
and downstream regions of the target gene.
The snf2 mutation in CMKy45
(ppr2::URA3/ppr2::hisG) was constructed by
kanr cassette insertion and confirmed as
described above. This heterozygous snf2 mutation resulted
in a strain with severe morphological defects noted after several days.
Thus, the transformants, once confirmed for the disruption, were
immediately stored at 
80°C in 15% glycerol-1 M sorbitol-YPD
medium. The snf2 alleles were created in the heterozygous
PPR2/ppr2::URA3 diploid strains CMKy76 (YPH499) and
CMKy78 (W303) by replacing the entire SNF2 gene with the
kanr gene as in CH1305.
The snf2Hind mutation was made by replacing only the
500-bp HindIII fragment within SNF2
(1) with kanr in CMKy35 and was
confirmed by PCR analysis.
The hta1/htb1::LEU2 allele was made as previously
described (33). The locus was disrupted in the diploid
CMKy73, and upon sporulation, there was 2:2 survival. The viable spores
were leucine auxotrophs. Several attempts to recover disruptions in the
haploid synthetic lethal mutant 28A were not successful.
Allele rescue. The synthetic mutant allele of SWI2-SNF2 was recovered by PCR (Vent polymerase [NEB]). The promoter region (480 bp) and the terminator region (360 bp) were also recovered. Due to the large size of SNF2 (5.1 kb), the mutant allele was recovered in three PCR fragments, each of which was subcloned into pRS314 and sequenced.
Linkage analysis for UBP3, KEX2, and
STT4.
Mutant 70B [MATa ura3 leu2 lys2
ppr2::his G (pJD4)] and a MAT ura3 leu2 lys2
ubp3::LYS2 strain (derived from CMKy27) were mated, and
Ura+ Lys+ diploids were selected. The diploid
was sporulated, and 10 complete tetrads were examined. Growth on lysine
was used to identify the ubp3 segregants. As the
ppr2 allele did not contain an auxotrophic gene, the
ppr2 allele was determined by PCR analysis on each segregant. Red segregants carried pJD4. As we have observed previously, pJD4 segregated very poorly, presumably due to the mutant
CEN/ARS.
::hisG(pJD4)] was mated to a MAT ura3 leu2
lys2 kex2::Kanr(pKEX2) strain (derived from
CMKy39). Plasmid pKEX2 is a LEU2 library plasmid that
contained KEX2. Ura+ Leu+ diploids
were selected. The presence of both PPR2 and KEX2
on plasmids would complicate the analysis, so the diploid was grown in
YPD for several generations and plated. A Ura
Leu diploid was sporulated, and 10 complete tetrads were
analyzed. The ppr2 allele was determined by PCR analysis.
Kanr was used to identify the kex2 allele.
The presence of pJD4 was indicated by a red color.
The mutant [56B MATa ura3 leu2 lys2
ppr2::hisG(pJD4)] was mated to CMKy22 (MAT ura3
leu2 lys2 ppr2::hisG trp1::hisG). Zygotes were
selected, and diploids were confirmed by the inability to mate to
tester strains. A diploid was transformed with a PCR-derived cassette
designed to disrupt STT4 (same strategy presented above). PCR analysis was used to identify five diploids heterozygous for the
stt4 mutation. The diploids were sporulated, and 10 complete tetrads were analyzed for each.
Recovery of swi-snf strains.
The initial work
with the snf2 mutation in CH1305 showed poor growth and
poor viability of spores. A modified method for strain recovery was
used in all the tetrad analyses of swi-snf strains in order
to unambiguously identify the genotype present at each spore position.
Fresh, moist YPD plates were used for tetrad dissection. Plates were
warmed to room temperature before use. After tetrads were dissected,
each plate (plate A) was grown for 7 days and microscopically examined
to determine the approximate number of cells at each spore position.
Small colonies were streaked in patches on a fresh YPD plate (plate B).
Plate A was then replica plated to both SC-Ura and SC-Trp media. Plate
B was grown for 7 days. All viable patches were transferred to separate
G418, SC-Ura, and SC-Trp plates.
Microscopic characterization of phenotypes. Yeast cells were resuspended in H2O to a concentration of approximately 7 × 107 cells/ml, and cellular morphology was observed on a Zeiss photomicroscope equipped with Nomarski differential interference contrast optics (Carl Zeiss, Thornwood, N.Y.). To monitor nuclear morphology, yeast cells were harvested from an overnight culture, rinsed in cold methanol for 10 min to fix the cells, and resuspended in 1× phosphate-buffered saline. Cells were stained with 30 ng of 4',6'-diamidine-2-phenylindole dihydrochloride (DAPI) per ml for 5 min. Stained cells (5 µl) were applied to a glass slide and examined using a Zeiss Axioskop fluorescence microscope and a 100× Neoflor objective (Carl Zeiss).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Rationale of screen. S. cerevisiae contains a single copy of the gene encoding TFIIS (PPR2), and gene disruption strains have only modest phenotypes (76). Thus, to investigate the function of TFIIS in vivo, a synthetic lethal screen was initiated. Synthetic lethal interactions might uncover proteins whose function overlaps that of TFIIS or proteins that share regulatory or functional roles with TFIIS.
Results of synthetic lethal screen. The synthetic lethal screen utilized the color phenotypes associated with the adenine biosynthetic pathway (43, 45). Approximately 75,000 mutagenized colonies were screened for a nonsectoring phenotype, and 187 mutants were identified that reproducibly produced nonsectoring colonies. Of these, 185 mutants were plasmid dependent. Eighty-nine mutants were recessive, and the survival of 55 of these depended upon the presence of PPR2. Of these, 49 mutants showed 2:2 segregation of the nonsectoring phenotype, indicating that each mutant resided in either one gene or a set of closely linked genes. Ten complementation groups were defined among these 49 mutants, and all were tested for growth on sucrose, raffinose, 6-azauracil, and medium lacking inositol and for growth at 37°C. The relevant phenotypes of the mutants are presented below.
A CEN/ARS genomic library was used to identify the genes that complemented the nonsectoring phenotype for each of the 10 complementation groups. Inserts from library plasmids that restored sectoring were end sequenced and identified from the genomic database. As the genomic library fragments were approximately 10 kb in length, subclones were used to define the gene that restored sectoring (Table 3). Four of the 10 complementation groups were analyzed in this way. For one complementation group, SNF2 was identified (Table 3), and allele rescue of the mutant locus allowed identification of the specific sequence change that led to the synthetic lethal phenotype. For the other three complementation groups (Table 4), phenotypic analysis suggested complementation rather than suppression of a mutation in a separate locus. To confirm complementation, each synthetic lethal mutant strain was crossed to a strain in which the suspected complementing gene had been disrupted (as described in Materials and Methods). Allelism was confirmed by the 2:2 segregation of synthetic lethal phenotypes following sporulation and tetrad dissection.
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strain has
a mild growth defect, and the ubp3 ppr2 strain,
while viable, has a more severe growth defect (Table 4). The synthetic
lethal phenotype of 70B segregated 2:2 with the severe growth defect of
the ubp3 ppr2 strain. While confirming allelism or
very tight linkage with UBP3, this result also indicated
that the synthetic lethality of mutation 70B in UBP3 with
ppr2 was allele specific.
The synthetic lethal mutant 44B was rescued by KEX2,
encoding a prohormone protease located in the Golgi. Mutant 44B also exhibited phenotypes characteristic of kex2 strains: slow
growth, -specific sterility, and altered temperature tolerance
(51). As both the synthetic lethal mutant 44B and the
kex2 strain were sterile, the MATa
synthetic lethal mutant was mated to a MAT
kex2 strain transformed with a KEX2-containing
library plasmid. To simplify the analysis, a diploid which had lost
both the library plasmid (KEX2 LEU2) and pJD4 (PPR2
ADE3 URA3) was used. Following sporulation and tetrad dissection,
no segregants showed wild-type growth and all segregants were
sterile, as would be expected if the synthetic lethal mutation and
kex2 were allelic. Moreover, both the kex2
(Kanr) and synthetic lethal allele (KanS)
strains that were ppr2 were extremely sick on YPD medium,
an observation consistent with the very low viability observed in liquid culture with the kex2 ppr2 strain. These
results indicate an allelism or very tight linkage between the mutation
in 44B and KEX2 and further suggest that mutant 44B contains
a loss-of-function mutation in KEX2.
The synthetic lethal mutant 56B was rescued by STT4,
encoding a phosphoinositol-4-kinase. A deletion of STT4 is
lethal in the CH1305 strain background, as disruptions of
stt4 could not be recovered in haploids under several
conditions tested (data not shown). Thus, mutant 56B could not have
suffered a total loss of function in STT4. However, mutant
56B does show phenotypes expected for conditional alleles of
stt4, including temperature sensitivity and rescue by
sorbitol (80) (Table 4). Synthetic lethal mutant 56B
was mated to an STT4 ppr2 strain, and the resulting diploid was disrupted for STT4. Five diploids heterozygous
for the stt4 mutation were identified. Each was
sporulated, and tetrads were analyzed for each of the five. In this
analysis, the STT4 disruption could occur in the diploid at
either the wild-type or the mutant 56B locus (assuming allelism) on the
complementary chromosomes. Any stt4 haploids themselves
would be inviable. Of the five sporulation plates, three showed the 2:2
segregation for viability expected if the disruption had occurred in
the STT4 locus and had replaced mutation 56B, leaving the
wild-type copy of STT4. The other two
stt4 heterozygotes had significantly reduced overall
spore germination. In one case, only 4 of 40 spore positions were
viable, and only 6 of 40 were viable in the second case. Each of
these viable strains had a severe growth defect and appeared red
(nonsectoring). The low viability is not surprising, as previous work
with mutant 56B showed extremely poor spore viability associated with
the synthetic lethal allele. These combined patterns of segregation suggest that mutant 56B represents a partial function allele of STT4.
Characterization of the interaction with SNF2.
SWI2/SNF2 encodes a component of the Swi-Snf
chromatin-remodeling complex and is the founding member of a gene
family associated with chromosome structure changes (42).
SWI2/SNF2 is not an essential gene (1), but the
null mutant is characterized by a number of phenotypes, all shared by
the mutant allele isolated in this screen (Fig.
1). For simplicity, SWI2/SNF2
will be referred to as SNF2. The snf2 strain
cannot grow on medium containing raffinose, sucrose, and galactose
under anaerobic conditions, and this mutant is also an inositol
auxotroph. The raffinose sensitivity of the synthetic lethal mutant was
complemented by cloned SNF2 (Fig. 1), strongly indicating
that the mutation lay in SNF2.
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/PPR2 diploid, CMKy35. The resulting
snf2/SNF2 ppr2/PPR2 diploid, CMKy38, was sporulated,
and 12 tetrads were dissected. In each of the tetrads, two spores gave
rise to healthy colonies while the other two spores were either
inviable or gave rise to poorly growing colonies of variable size. As
the viability of the snf2 strains was problematic in this
strain background, plating modifications were necessary to improve
germination and strain recovery following germination (see Materials
and Methods). Subsequently, over 175 tetrads were analyzed from the
CMKy38 diploid strain to permit a thorough analysis of the genotypes of
every spore position and the number of cells resulting from each
germination event. The results clearly demonstrate that an
snf2 mutation is synthetically lethal in combination with
a ppr2 mutation (Fig. 2).
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strain itself. The
low frequency with which they were identified (105) and
the robust growth suggested that these were suppressor mutants. Interestingly, these putative suppressors obviate swi/snf
mutant phenotypes, such as reduced growth on raffinose- and
inositol-free medium, but do not appear to suppress the
6-azauracil sensitivity of ppr2 strains.
The entire 5.1-kb allele of SNF2 was rescued from the
synthetic lethal mutant. Sequence analysis identified a nonsense
mutation at nucleotide position 3385 that would be expected to produce a protein truncated in the ATPase domain. The genetic connection between PPR2 and SNF2 was examined in detail to
explore the role of the Swi-Snf complex in transcript elongation and
the possible role of TFIIS on a nucleosomal template. However, complex
interactions with auxotrophic markers were examined first.
Early work with SWI/SNF had suggested that leucine
auxotrophy would contribute to the phenotypic severity detected in
swi/snf mutant strains (66). In that report,
swi2/snf2 leu2 double mutant strains were found to require
300 µg of leucine per liter, 10 times the standard concentration (30 µg/ml). To allow comparisons of the interactions between
swi2/snf2 and ppr2 directly, all characterizations were done in medium containing 300 µg of leucine per liter. Additionally, the observed effects were essentially identical in leu2 and LEU2+ backgrounds.
The TRP1+ genotype also contributed to the
severity of phenotypes observed with snf2 strains. This
effect is further described below, but the synthetic interactions
between snf2 and trp1 initially complicated
phenotypic interpretations with ppr2. To avoid this, the
interactions between ppr2 and snf2 were
examined in TRP1+ strains. In CH1305,
snf2 strains are much healthier in the presence of
TRP1 on both rich medium (YPD) and minimal medium
supplemented with tryptophan.
Indeed, suppressors of the double-deletion mutant only appeared
when the strain contained TRP1+. In a
trp1 background, the terminal cell count of the
double-deletion mutant was between 20 and 100 cells after 1 week of
incubation. In the presence of TRP1, the double-deletion
mutant forms a small colony of approximately 100 to 1,000 cells
after 1 week of incubation (Table 5). The
explanation of this effect is not known, but it also extends to
TRP5 (data not shown). As a result of these observations, the interactions between SNF2 and PPR2 were
examined in a TRP+ background.
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Phenotypes of snf2 ppr2 cells.
The haploid
snf2 ppr2 double disruption was inviable, but the
phenotypes of the SNF2/snf2 ppr2/ppr2 heterozygous
diploid CMKy46 were informative. This diploid strain had several
unusual phenotypes, including a high frequency of lysis on rich (YPD) medium and complete lysis in medium of higher osmotic strength, such as
sporulation medium. When the cells are examined microscopically, approximately 45% of the cells are characterized by highly elongated bud-like structures (Fig. 3).
Intriguingly, highly elongated buds are also observed with some alleles
of sth1, an essential SNF2-related component of
the RSC chromatin remodeling complex (19). DAPI staining of
the budded diploid CMKy46 demonstrated that the elongated bud structure
contained a single nucleus. This nucleus was not located
exclusively at the mother bud neck. A localization at the mother bud
neck would have suggested a delay in nuclear division. The addition of
1 M sorbitol suppressed the hyperelongated bud phenotype and also
reduced the doubling time of the diploid. Curiously, this diploid
sporulates after 2 days of growth on YPD medium. Rich YPD medium
contains both nitrogen and glucose, each of which normally represses
the sporulation pathway. Thus, the SNF2/snf2 ppr2/ppr2 diploid is overcoming both of these repressive
sporulation signals, indicating a major deregulation within the cell.
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Strain background affects the genetic interaction between
PPR2 and SNF2.
The growth defect of the CH1305
snf2 strain was more severe than that reported for either
an S288C snf2::HIS3 strain (1) or an S288C
snf2::LEU2 strain (9). Furthermore, the
ppr2 and snf2 alleles show no genetic
interaction in S288C (G. Hartzog and F. Winston, personal
communication). The disruptions of SNF2 that had been made
in S288C differed in both the size and content of the disruption
compared to those made in CH1305. The snf2 disruption in
S288C had replaced 500 bp of SNF2 with HIS3
(1). The disruption in CH1305 replaced the entire open
reading frame of SNF2 with the kanr
gene. Thus, the difference between the PPR2 and
SNF2 genetic interaction in S288C and CH1305 could have been
due to the nature of the disruptions. To test this, the 500-bp region
of SNF2 originally replaced in S288C by HIS3
was replaced with the kanr gene in CH1305. This
CH1305-derived disruption strain (snf2Hind) had the same
growth defects as the CH1305 strain with the complete kanr replacement of SNF2 (data not
shown). This disruption was also synthetically lethal with
ppr2. These results suggest that the strain background,
and not the size or location of the disruption, is the basis for the
difference in both the growth defects and the genetic interaction
between the ppr2 and snf2 alleles in S288C and CH1305.
disruptions were constructed in three
additional genetic backgrounds: Z321, a laboratory strain utilized by
R. Young and N. Woychik (78); W303, a laboratory strain from
R. Rothstein (70); and YPH499, a laboratory strain
originally derived from S288C (64). The complete genotypes
of the strains are listed in Table 1. In each case, a diploid strain
was used to make the heterozygous
SNF2/snf2::kanr disruption. For Z321, the
diploid was PPR2/PPR2. In both W303 and YPH499, a
heterozygous PPR2/ppr2::URA3 diploid was used. All
three snf2 haploid strains derived from these diploids
grew very poorly, although there was subtle variation among the three backgrounds in the severity of the growth defect. For YPH499, the
strain originally derived from S288C, the snf2 strains
were the least healthy of all strains tested. Incubation for 1 week was
required to observe any visible growth. In Z321 and W303, the
snf2 strain had a severe growth defect, but each was much more robust than the YPH499 snf2 strain.
Strains W303 and YPH499/500 were used to examine the genetic
interaction between ppr2 and snf2 across
strain backgrounds. The tetrads derived from both W303 and
YPH499/500 SNF2/snf2 PPR2/ppr2 heterozygous diploids
showed a clear synthetic lethal interaction between
ppr2 and snf2 (Fig.
4; results summarized in Table 5). The
severity of the growth defect caused by an snf2 mutation alone affected the terminal cell count of either double deletion mutant. That is, the growth defect of a snf2 strain was
similar in W303 and CH1305, and the terminal cell counts of the
snf2 ppr2 cells in each background were similar. The
double-deletion mutants of W303 grew to approximately 500 to 1,000 cells after 1 week, and suppressors could be recovered, as was observed
with CH1305. In contrast, the YPH499/500 background gave
snf2 strains that grew extremely slowly, and the terminal
cell count of the snf2 ppr2 segregants was only two to
five cells.
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phenotypes can be suppressed by deletion
of one of the two gene pairs encoding H2A and
H2B (HTA1 and HTB1) (33).
Thus, it was of interest to test whether a similar disruption could suppress the synthetic lethality between snf2 and
ppr2 in CH1305. However, in contrast to S288C,
HTA1-HTB1 is apparently essential in segregants derived from
either a heterozygous snf2/SNF2 ppr2/PPR2
HTA1-HTB1/(hta1-htb1) diploid (CMKy73) or the synthetic lethal
snf2 mutant itself, both in the CH1305 background. The essential nature of the HTA1-HTB1 allele has also been
observed in the W303 genetic background (P. Kaufman, personal communication).
To determine if the difference between the S288C snf2
strain and the CH1305 snf2 strain was caused by one gene,
the two snf2 strains were mated. However, the homozygous
snf2/snf2 diploid was unable to sporulate. This
inability of homozygous snf2 diploids to sporulate has
been observed previously (66). This strain difference is
being further pursued to understand if the differences can be revealing
about transcriptional control affected by chromosome structural changes.
Synthetic lethality with other components of the
SWI/SNF complex.
To determine if the synthetic lethal
effects could be extended to additional SWI/SNF genes,
SWI1 and SNF5 were tested for genetic
interactions with PPR2. These genes were selected as two well-characterized components of the Swi-Snf complex, each required for
its activity. A total of 68 tetrads were examined following sporulation
of a heterozygous swi1/SWI1 ppr2/PPR2 diploid, CMKy60, and synthetic lethality was clearly observed (Fig.
5). The results were very similar to
those obtained with an snf2 mutation. The TRP1
locus affected both the growth of swi1 strains and
the terminal cell count of the swi1 ppr2 strains.
However, the terminal cell count of swi1 ppr2 strains
was higher than observed for the snf2 ppr2 strains.
The swi1 ppr2 strains formed a small colony of approximately
500 to 1,000 cells. Apparent suppressors of the synthetic lethality
again accumulated with a frequency estimated to be 105.
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/SNF5 ppr2/PPR2 diploid, CMKy64. Synthetic
lethality was also observed between null alleles of SNF5 and
PPR2 (Fig. 5). Again, TRP1 affected the
growth of the snf5 strain and the terminal cell count of
the snf5 ppr2 strain. With TRP1, the snf5 ppr2 strain grew to 100 to 1,000 cells.
Suppressors appeared at approximately the same frequency as for
the snf2 ppr2 and swi1 ppr2 strains.
The synthetic lethality for all three tested components of the Swi-Snf
complex was complemented by expression of PPR2 in the double-disruption strain. The carboxy-terminal half of the TFIIS protein was sufficient for functional complementation; this portion of
the protein is sufficient to complement both the
6-azauracil sensitivity of ppr2 strains and
the in vitro activities of TFIIS for cleavage and readthrough by RNA
polymerase II (12, 13, 53; N. Shimasaki and C. M. Kane, submitted for publication).
Lack of common phenotypes.
To further investigate the
synthetic lethal interaction between null alleles of PPR2
and SWI/SNF, ppr2 and swi/snf
strains were tested for common phenotypes that might indicate a gene or set of genes regulated by both TFIIS and the Swi-Snf complex. As
mentioned, swi/snf mutants have several distinct phenotypes, including poor anaerobic growth on raffinose, sucrose, and galactose and inositol auxotrophy (30, 46, 47, 58, 59). In contrast, the ppr2 strain grew identically to wild-type cells on
raffinose, sucrose, and galactose in anaerobic conditions and was not
an inositol auxotroph. Cells disrupted for PPR2 are
sensitive to both 6-azauracil and mycophenolic acid (20,
35), and this sensitivity was detectably greater for
ppr2 strains than for swi1,
snf5, or snf2 strains (data not shown).
| |
DISCUSSION |
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|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
A major implication from these results is that the Swi-Snf complex
may be needed to promote efficient transcription elongation. Previous
work has focused primarily on the effects of the Swi-Snf complex on
preinitiation events, although a connection with DNA replication
(21) and possibly transcript elongation (3, 6, 67) has also been suggested. The synthetic lethal approach was undertaken to help understand the function of TFIIS in vivo, and the
genes identified have suggested potential functional interactions between TFIIS and chromatin-remodeling machinery. Synthetic lethality was observed between a ppr2 mutation and
swi1, snf2, and snf5 mutations. These results strongly suggest that the combined loss of the
function of the Swi-Snf complex and TFIIS forms the basis of the
synthetic lethality. The basic unit of chromatin consists of DNA
wrapped around nucleosomes, which is then compacted into higher-order
structures. Protein complexes which are capable of altering the
structure of chromatin have been identified in S. cerevisiae, Drosophila melanogaster, and metazoans
(reviewed in references 7 and
42). The different chromatin-remodeling complexes
have distinct biochemical activities but share a few basic features.
Each complex contains a subunit with homology to DNA-dependent
ATPases (the SNF2 family), and all tested complexes show ATP-dependent chromatin remodeling of nucleosomal templates. The
two identified chromatin-remodeling complexes in yeast cells are the
Swi-Snf complex (reviewed in references 60 and
77) and the RSC complex (10). The Swi-Snf
complex also associates with at least one RNA polymerase II holoenzyme
complex (75).
It is easy to rationalize overlapping functions between TFIIS and the
Swi-Snf complex. Certainly, the genetic interaction between
swi/snf and ppr2 alleles demonstrates only
that the Swi-Snf complex and TFIIS share some essential function,
whether through completely independent transcriptional control
mechanisms on an essential gene (or genes) or through overlapping
transcription functions on at least some genes. Recently, the need for
a functional Swi-Snf complex throughout transcription has been reported
(3), and it is possible that this requirement occurs during
the elongation process. The involvement of the Swi-Snf complex in
elongation also was suggested previously from the results of in vitro
experiments (6). Indeed, the NPH-I protein of vaccinia
virus, shown to affect elongation, is also an SNF2
homolog (16). In support of a link between elongation and
chromatin remodeling, swi/snf strains are modestly sensitive
to 6-azauracil and mycophenolic acid, both of which reduce
cellular nucleotide pools and presumably slow RNA polymerases
(62). Indeed, the overall transcription levels in the cell
are lower in the presence of 6-azauracil (48). Under
reduced nucleotide concentrations, the polymerase might stall and
arrest more often, and in the absence of the Swi-Snf complex and TFIIS,
it might not efficiently recover from arrest.
The terminal phenotypes of swi/snf ppr2 cells include
loss of osmoregulation and possibly cell cycle control. These
phenotypes suggest a major loss of gene regulation in the cell as
opposed to the specific disruption of a pathway independently regulated by the Swi-Snf complex and TFIIS. Transcription of chromatin templates by RNA polymerase II likely relies on a combination of
chromosome-remodeling and elongation-stimulatory factors for efficient
movement along the template. Eliminating TFIIS or the Swi-Snf complex
individually might impede the polymerase, but neither is essential. The
phenotypes of swi/snf mutants are more severe than those of
ppr2 mutants, and this result emphasizes a role for the
Swi-Snf complex beyond that carried out by TFIIS. Thus, transcript
elongation is likely to be the overlapping function, and additional
roles for the Swi-Snf complex in transcription (7) and
replication (21) have been documented. In addition, the
interaction with PPR2 could result from a combination of
transcription effects, during initiation for the Swi-Snf complex and
elongation for TFIIS.
Subtle differences were observed in the genetic interactions between
ppr2 and each of swi1, snf2,
and snf5 alleles. The combination of snf5
and ppr2 mutations was the most deleterious to the cell.
The double-deletion mutants arrested at an average of 50 cells. The
interaction with an snf2 mutation was slightly less
severe, with the snf2 ppr2 strains arresting at an
average of 200 cells. The combination of swi1 and
ppr2 was the least deleterious, as double mutant strains
continued to grow to an average of 500 cells. One simple explanation
for the differences may be that different null SWI/SNF
mutations disrupt the Swi-Snf complex to different degrees (C. Peterson, personal communication). There may be residual Swi-Snf
complex activity in swi1 strains, not present in
snf5 strains, that allows the swi1 ppr2
cells to survive longer. As ppr2 strains share none of
the swi/snf mutant phenotypes, it does not appear that
PPR2 has a selective role in the regulation of the
identified genes whose expression is impaired in swi/snf
mutants. In addition, preliminary results using microarray analysis do
not highlight genes known to be affected in swi/snf mutants
(C. Seidel and C. M. Kane, unpublished results).
The unusual phenotypes of an SNF2+/snf2
ppr2/ppr2 diploid appear very relevant to the reduced
viability of snf2 ppr2 cells. The results suggest that
genes important in osmoregulation and cell wall maintenance are
especially sensitive to the interaction between SWI/SNF and
PPR2. In this regard, it is noteworthy that swi/snf
ppr2 double-disruption spores can germinate and divide for
several generations, but when these cells are observed under higher
magnification, the morphology of the cells is extremely aberrant,
including highly elongated buds. A high degree of lysis is also
evident. These phenotypes were not observed for swi1, snf2, or snf5 strains in a PPR2
background. Likewise, the ppr2 strain itself does not
exhibit these phenotypes. Taken together, the results from both the
diploid and haploid double-disruption mutants indicate that defects in
osmoregulation and cell wall maintenance may be the primary cause of
the lethality in swi/snf ppr2 cells.
Curiously, mutations in STT4 also cause osmosensitivity and
cell wall defects that can be rescued by osmotic stabilizers. This gene
can complement one of the other recessive mutations that is
synthetically lethal with ppr2 (Table 4). Some
conditional mutants in the essential RSC chromatin-remodeling complex
also show highly elongated buds (11, 19). The mechanism that
results in such buds is not known in either the rsc or the
swi/snf ppr2 mutants. Additionally, temperature-sensitive
alleles of genes encoding members of the RSC complex arrest at the
G2/M boundary, another phenotype shared with conditional
alleles of stt4 (11, 80). Mutations in the
CLN genes, essential regulators of the cell cycle, also led
to elongated buds (49). Thus, the phenotypes of the
snf2/SNF2 ppr2/ppr2 diploid might result from
either cell cycle defects or the loss of osmoregulation and cell wall integrity, but these two hypotheses need not invoke alternate mechanisms. The cell cycle phenotypes might also be related to the
sporulation observed on rich medium of the SNF2/snf2
ppr2/ppr2 diploid. Mutations in many genes lead to
derepressed sporulation, and these include cell cycle mutants. Since
TFIIS is believed to be a general regulator of transcription
elongation, it is certainly reasonable that the sporulation phenotype
of the snf2/SNF2 ppr2/ppr2 diploid might result
from a loss of regulation of several genes important in the cell cycle.
The synthetic lethality between ppr2 and stt4
is worth further comment. A synthetic lethal mutant which contains a
mutant allele of STT4 shares many swi/snf mutant
phenotypes, but it also grows better in the presence of
6-azauracil than without the drug. Perhaps a reduction in
transcription of TFIIS-regulated genes coupled with mutations in this
phosphoinositol 4-kinase cripple the cell, although a more direct
connection to chromatin remodeling is possible (81). In
S. cerevisiae, Stt4 is a likely candidate to create the
precursor to phosphatidylinositol 4,5-biphosphate (17, 34)
and in human cells, phosphatidylinositol 4,5-biphosphate appears to
target the human BAF chromatin remodeling complex to chromatin in vitro
(81). The BAF complex contains BAF53, a human homolog of
Arp7 and Arp9, the two actin-related subunits of the Swi-Snf complex in
S. cerevisiae (8, 61). Mutations in either of
these genes in yeast cells also result in swi/snf mutant phenotypes.
As a final note from this analysis, the identification of
KEX2 in this screen (Table 4) marks the second time it has
shown genetic interactions with the RNA polymerase II transcription machinery (51). The genetic interaction between the
kex2 and ppr2 alleles resulted in severe
sickness in the double-deletion mutant, characterized by a
dramatic reduction in viability. Kex2 is a prohormone protease
located in the Golgi. Null mutations in KEX2 also suppress
mutations in the largest subunit of RNA polymerase II, RPB1
(51). While the nature of this suppression is unknown, Kex2
may process a protein that directly or indirectly modifies the
biochemical properties of RNA polymerase II or the levels of its
nucleotide substrates. The absence of this protein caused by the loss
of Kex2 may create an RNA polymerase II dependent on the activity of
TFIIS. All these possible connections are provocative, but it is
essential to note that although the results might suggest a mechanistic
linkage, mutations in many types of genes confer overlapping phenotypes
(28). Further study is needed to test the interactions among
the genes identified in this screen.
Several complementation groups from this synthetic lethal screen remain to be characterized, but as yet there is no evidence for a functional homolog that shares a mechanistically overlapping essential function with TFIIS. An additional component of the Swi-Snf complex, Tfg3, also has strong genetic interactions with TFIIS, and its genetic relationship to TFIIS is very different from that between TFIIS and other members of the Swi-Snf complex (J. Davie and C. Kane, unpublished data). The results presented here also predict that loss of TFIIS function might be compensated for by activities in addition to the Swi-Snf complex that promote efficient elongation. With a growing number of described proteins that impact elongation by RNA polymerase II, additional interactions with chromatin-remodeling components are likely to be found.
| |
ACKNOWLEDGMENTS |
|---|
We thank Danesh Moazed, Alexander Johnson, Grant Hartzog, Fred Winston, and Marian Carlson for kind gifts of reagents. We thank Danesh Moazed, Grant Hartzog, Fred Winston, Craig Peterson, Jeremy Thorner, Chau Huynh, Jasper Rine, and Paul Kaufman for helpful discussions and sharing unpublished observations.
This work was supported by a grant from the National Institutes of Health awarded to C.M.K. (GM54012). J.K.D. was supported by a Howard Hughes Medical Institute predoctoral fellowship.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202. Phone: (510) 642-4118. Fax: (510) 643-9290. E-mail: kanecm{at}uclink4.berkeley.edu.
Present address: University of Texas M.D. Anderson Cancer
Center, Biochemistry/Molecular Biology, Houston, TX 77030.
| |
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Materials and Methods
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Discussion
References
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