Previous Article | Next Article 
Molecular and Cellular Biology, March 1999, p. 2351-2365, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A New Member of the Sin3 Family of Corepressors Is Essential
for Cell Viability and Required for Retroelement Propagation
in Fission Yeast
Van Dinh
Dang,1
Michael J.
Benedik,1,
Karl
Ekwall,2,
Jeannie
Choi,1
Robin C.
Allshire,2 and
Henry
L.
Levin1,*
Laboratory of Eukaryotic Gene Regulation,
National Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, Maryland 20892,1
and MRC Human Genetics Unit, Western General Hospital,
Edinburgh EH4 2XU, Scotland2
Received 14 August 1998/Returned for modification 23 October
1998/Accepted 25 November 1998
 |
ABSTRACT |
Tf1 is a long terminal repeat (LTR)-containing retrotransposon that
propagates within the fission yeast Schizosaccharomyces pombe. LTR-retrotransposons possess significant similarity to retroviruses and therefore serve as retrovirus models. To determine what features of the host cell are important for the proliferation of
this class of retroelements, we screened for mutations in host genes
that reduced the transposition activity of Tf1. We report here the
isolation and characterization of pst1+, a gene
required for Tf1 transposition. The predicted amino acid sequence of Pst1p possessed high sequence homology with the Sin3 family
of proteins, known for their interaction with histone deacetylases. However, unlike the SIN3 gene of Saccharomyces
cerevisiae, pst1+ is essential
for cell viability. Immunofluorescence microscopy indicated that Pst1p
was localized in the nucleus. Consistent with the critical role
previously reported for Sin3 proteins in the histone acetylation
process, we found that the growth of the strain with the
pst1-1 allele was supersensitive to the specific histone
deacetylase inhibitor trichostatin A. However, our analysis of strains
with the pst1-1 mutation was unable to detect any changes in the acetylation of specific lysines of histones H3 and H4 as measured in bulk chromatin. Interestingly, the pst1-1
mutant strain produced wild-type levels of Tf1-encoded proteins and
cDNA, indicating that the defect in transposition occurred after
reverse transcription. The results of immunofluorescence
microscopy showed that the nuclear localization of the Tf1 capsid
protein was disrupted in the strain with the pst1-1
mutation, indicating an important role of pst1+
in modulating the nuclear import of Tf1 virus-like particles.
| |
INTRODUCTION |
Retroviruses and
retrovirus-like transposons belong to a widely
distributed family of eukaryotic elements called long terminal repeat
(LTR)-containing retroelements. Although these LTR-containing elements
have been found in hosts as diverse as yeast and mammals, striking
structural and functional conservation has been observed in the
mechanisms responsible for their life cycles. The similarities include the proteolytic processing of precursor proteins, the assembly
of particles, the reverse transcription of cDNA, and the integration of
the cDNA into the host genome (10). Although retroviruses
and retrotransposons possess a requirement for many components of
host physiology, only a few host genes that control retroviral
proliferation have been identified. One reason for this general lack of
information is the difficulty of studying host organisms with high
genetic complexity. One approach to understanding the propagation of
retroelements is to study yeast retrotransposons, a family of elements
that possess an extensive similarity to retroviruses and thus serve as
a retrovirus model system (10, 63).
We study Tf1, an LTR-containing retrotransposon isolated from the
fission yeast Schizosaccharomyces pombe. This retroelement has coding sequences for Gag, protease (PR), reverse transcriptase (RT), and integrase (IN) proteins (40, 42). In vivo assays for transposition demonstrate that Tf1 is highly active and
generates transposition frequencies varying from 2 to 20%
(41). The results of sucrose gradient sedimentation revealed
that Tf1 Gag, IN, mRNA, and reverse transcription products all assemble
into virus-like particles (VLPs) that contain a 26-fold molar excess of
Gag relative to IN (4, 38, 41).
In eukaryotic cells, genomic DNA is assembled with chromosomal
proteins, particularly histones, to form a complex structure called
chromatin. The level of chromatin condensation plays a key role in many
cellular processes, including the regulation of transcription (68,
79), the replication of DNA (61, 72), the segregation
of chromosomes (2, 19, 20, 25), and the integration of
retroviral cDNA into host targets (56-59). In the last
few years, factors capable of altering the structure of chromatin have
been identified (35). One important class of modification is
the acetylation of lysine residues in the core histones (13, 68,
80). Genetic and biochemical studies have revealed a correlation between histone acetylation and the transcriptional activity of promoters associated with acetylated histones (31, 44, 62, 79). Hyperacetylation of core histones destabilizes nucleosomes and correlates with gene activation, while hypoacetylation stabilizes nucleosomes and correlates with gene repression. Recently, factors containing histone acetylase and deacetylase activities have been identified (68, 79). Among these factors, the family of Sin3 proteins have been shown to play the role of scaffolding elements required for the formation and function of histone deacetylase complexes (54, 79). Indeed, each Sin3 protein contains four paired amphipathic helix (PAH) domains, which have been proposed to
mediate protein-protein interactions (77). Sin3 proteins have been shown to use distinct domains to form histone
deacetylase complexes and to interact with specific DNA-binding
proteins (8, 79). Thus, Sin3p has been proposed to play
an important role in recruiting the histone deacetylase activity
to specific regions of chromosomes, causing targeted changes in
chromatin structure and expression (8, 54, 79).
To identify genes of S. pombe that are required for Tf1
activity, we mutagenized cultures and screened for strains that were unable to support Tf1 transposition. In this paper, we report the
isolation of the pst1+ (for pombe SIN
three) gene in S. pombe that is a homologue of the
SIN3 gene of Saccharomyces cerevisiae. The
pst1+ gene is shown here to be important for Tf1
transposition and the ability of Tf1 cDNA to homologously recombine
with Tf1 sequences contained in plasmids. Immunoblotting results
indicated that the pst1-1 mutation did not cause changes in
acetylation levels of key lysines in histones H3 or H4 when measured in
bulk histone preparations. Nevertheless, the histone deacetylase
inhibitor trichostatin A (TSA) was found to reduce the activity
of the Tf1 element. Interestingly, strains with the pst1-1
allele exhibited pseudohyphal growth in the presence of TSA. Further
analysis revealed that the pst1-1 mutation caused a block in
the nuclear import of Tf1 Gag, and we propose that the mislocalization
of Gag is responsible for the loss of Tf1 transposition.
| |
MATERIALS AND METHODS |
Media.
The S. pombe media were prepared as
described previously (39, 46). Agar plates containing
sporulation medium with malt extract (ME; Bio 101, La Jolla, Calif.)
were prepared according to the manufacturer's instructions. Phloxin B
was used at 5 µg/ml to differentiate diploid cells from their haploid
progeny. TSA (WAKO BioProducts) was dissolved in dimethyl sulfoxide and
used at concentrations of 0.1 to 5 µg/ml. Ten micromolar vitamin B1 (thiamine) was added to minimal medium to repress the nmt1
promoter. 5-Fluoroorotic acid (5-FOA) (U.S. Biologicals, Swampscott,
Mass.) was used at 1 mg/ml in Edinburgh minimal medium (EMM). Yeast
extract plus supplements (YES) 5-FOA-G418 plates were made from YES
medium containing 1 mg of 5-FOA/ml and 500 µg of Geneticin (Gibco)/ml (corrected for purity).
Plasmid construction.
Many plasmids used for this study were
constructed by PCR cloning techniques. For this purpose, the plasmids
were created in duplicate from independent PCRs and the properties of
each plasmid were studied in parallel. The oligonucleotides and
plasmids used in this study are listed in Table
1.
The wild-type allele of the
pst1 gene was isolated by
transforming the
pst1-1 mutant (YHL5018) with a genomic
library (kindly
provided by P. Young) and selecting for strains that
showed wild-type
levels of transposition and cDNA recombination.
The library plasmid
pHL1380 contained a 9-kb genomic insert with the
entire coding
sequence of the
pst1+ gene. To
create an integrative version of the
pst1+ gene,
an
Eco47III-
NheI (5.6-kb) fragment was isolated
from pHL1380
and cloned into the integrative vector pHL481 at the
SmaI and
XbaI sites. The resulting plasmid was
named pHL1383 and contained
the entire open reading frame (ORF) of
pst1+ and 393 nucleotides of the promoter.
Vector pHL481 was created
by removing from pJK148 the 742-nucleotide
ClaI fragment (
34).
The
NotI-
ClaI fragment with
pst1+ was isolated from pHL1383 and cloned into
Bluescript KS(+) (Stratagene)
at the
NotI and
ClaI sites to generate pHL1385-17. The insert
with
pst1+ from this plasmid was then transferred
into the
S. pombe plasmid
pSP1 (
16) at the unique
NotI and
XhoI sites to generate plasmid
pHL1386-1.
To create a frameshift in the coding sequence of
pst1+, the plasmid pHL1385-17 was digested
with
EcoNI, treated with Klenow
enzyme (Boehringer), and
recircularized with T4 ligase (United
States Biochemical, Cleveland,
Ohio). The presence of the newly
created frameshift in the resulting
plasmid pHL1412-2 was confirmed
by sequencing. The 5.6-kb
pst1 frameshift-containing insert of
pHL1412-2 was
isolated and cloned into the replicative vector
pSP1 to generate
pHL1413-22.
The
pst1::his3 allele was constructed as
follows. The putative translation start site of
pst1+ (GGATGG) was first replaced by a unique
BamHI site (GGATCC) by
using the fusion PCR technique
described previously (
38). Primers
HL395, HL396, HL397, and
HL398 were used to generate from the
template plasmid pHL1380 a 2.7-kb
fusion PCR product that contained
997 nucleotides of the
pst1+ promoter, a unique
BamHI site
at the ATG codon of
pst1+, and 1.7 kb of its
coding sequence. This PCR product was digested
with
SphI and
XhoI and cloned into two plasmids, pHL1385-17 and
pHL1386-1,
at the
SphI and
XhoI sites to generate pHL1459-2
and
pHL1460-2, respectively. A wild-type version of pHL1460-2, i.e.,
without the artificial
BamHI site, was created and named
pHL1458-3.
The entire
pst1+ coding sequence of
pHL1459-2 was then removed by digesting it
with
BamHI-
StyI and replaced by a 2-kb
his3+ marker that was generated from the plasmid
pAF1 (
52) by PCR
with the primers HL440 and HL441. The
resulting plasmid was named
pHL1581-1.
The HA-tagged allele of
pst1+ included a triple
copy of the HA epitope that was inserted at the C terminus of the
pst1+ coding sequence in the plasmid pHL1458-3.
For this purpose, the
oligonucleotides HL335 and HL467 were used to
generate a 1.1-kb
PCR product that contained 1 kb of the
pst1+ C terminus and a triple-HA epitope. HL335
annealed upstream of
the unique
EcoRI site located within
the
pst1+ coding sequence. HL467 annealed just
upstream of the stop codon
of
pst1+, and its
nucleotide sequence contained a triple-HA epitope and
the last three
amino acids of
pst1+, including a
StyI site. This
EcoRI-
StyI fragment
with the HA
sequence was used to replace the C-terminal
EcoRI-
StyI fragment
of
pst1+ carried by pHL1458-3. The resulting
plasmid was named pHL1626-2.
The junction of
pst1+ and the HA epitope was sequenced, and the
expression of the HA-tagged
pst1+ was verified
by immunoblot analysis with a monoclonal anti-HA
antibody (Babco) (data
not
shown).
The FLAG-tagged Gag version of Tf1 was constructed by inserting a FLAG
epitope (Eastman Kodak, New Haven, Conn.) at the C
terminus of Tf1 Gag
as described previously (
6).
To determine the location of the mutation in the
pst1-1
allele, we cloned the chromosomal copy of the
pst1-1 allele
by PCR
and determined its nucleotide sequence. Genomic DNA of a yeast
strain with the
pst1-1 mutation (YHL5356) was extracted and
used
as a PCR template. The oligonucleotides used in this experiment
are listed in Table
1. The sequence of the wild-type allele of
pst1+ carried in the plasmid pHL1385-17 was also
determined.
Yeast strain construction.
The yeast strains used in this
study are listed in Table 2. To integrate
the wild-type allele of pst1+ into its
chromosomal locus, the integrative plasmid pHL1383 was linearized with
SphI and used to transform the pst1-1 mutant
YHL5356. Stable Leu+ transformants were selected, and DNA
blot analysis was performed to verify the integration of a single copy
of the plasmid pHL1383 at the original pst1+
locus (data not shown). The resulting integrant was YHL6211. The two
strains YHL5761 and YHL5762 were from spores resulting from the mating
of two haploid strains, YHL1101 and YHL5356. Strain YHL6403-2 was the
diploid resulting from the mating of two haploid strains, YHL6381 and
YHL6382 (gifts from R. Ohi and K. Gould). To construct the heterozygous
diploid strain YHL6604, the 3.5-kb insert of the plasmid pHL1581-1 was
isolated and used to transform the YHL6403-2 strain. His+
prototroph transformants were selected, and the replacement of one of
the two wild-type pst1+ alleles by the
pst1::his3 allele was checked by DNA blot
analysis (data not shown). Plasmids were introduced into S. pombe strains by lithium acetate transformation (46).
Transposition and homologous recombination assays.
Tf1
transposition frequencies were determined as described previously
(39). Briefly, Tf1 transposition was monitored by placing a
neo-marked Tf1 element under the control of an inducible nmt1 promoter. The neo gene allowed cells to grow
in the presence of 500 µg of G418/ml. S. pombe
strains that contained a Tf1-neo plasmid were grown as
patches on EMM-Ura dropout agar plates in the absence of thiamine to
induce transcription of the nmt1-Tf1-neo fusion.
After 4 days of 32°C incubation, these plates were then replica
printed to medium containing 5-FOA to eliminate the
URA3-Tf1-neo plasmid (11). Finally,
5-FOAr patches were printed to medium containing both 5-FOA
and G418 and incubated at 32°C for 2 days to detect strains that
became resistant to G418 as the result of insertions of the
neo-marked Tf1 element into the genome. Wild-type Tf1
produced confluent G418r patches, whereas mutations in
element-encoded components or host factors reduced the growth on the
G418 plates. Quantitative measurements of transposition frequencies
were performed as follows. Strains were grown as patches of cells on
EMM-Ura dropout agar for 4 days at 32°C. These cells were then
resuspended at an optical density at 600 nm (OD600) of 1.0 and diluted approximately 100-fold. Approximately 0.1 ml of the cells
was then spread onto 5-FOA plates, and the resulting colonies
(approximately 7,000/plate) were replica printed to YES plates
containing FOA and 500 µg of G418/ml (41). The transposition frequency was the percentage of the FOA-resistant colonies that were also G418 resistant.
The presence of Tf1 cDNA in the nucleus was examined by cDNA
recombination assays, which were conducted according to the method
of
Atwood et al. (
3). This protocol is similar to the
transposition
assay in that strains with the
neoAI-marked
Tf1 plasmid were first
grown as patches on agar plates that contained
EMM (plus 10 µM
thiamine and dropout powder) and then replica printed
to similar
EMM plates that lacked thiamine. After 4 days of 32°C
incubation,
the plates were replica printed directly to YES medium that
contained
500 µg of G418/ml. Recombination between cDNA and
cellular transposon
sequences was scored on the G418 plates after
48 h of growth at
32°C.
Nucleic acid preparation and analysis.
cDNA preparation
(4) and total RNA extraction (17) were performed
as described previously. The protocols used for DNA and RNA blot
analyses were adopted from previously published works (4,
17). To measure the expression level of the neoAI gene carried on the transposition assay plasmid pHL449-1, the RNA blot was
hybridized with the oligonucleotide HL2 used as a strand-specific probe, which was 5' radiolabelled with polynucleotide kinase
(Boehringer). An EcoRI DNA fragment specific for Tf1 Gag
(42) was used as a probe to detect Tf1 mRNA.
Protein extraction and immunoblot detection of Tf1 Gag and
IN.
Total proteins were extracted from cells grown under inducing
conditions (absence of thiamine) by a previously published protocol (4). Protein pellets were collected, and an equal volume of 2× sample buffer (4) was added. The mixture was boiled for 3 min, and 25 µg of total protein from each sample was loaded on
sodium dodecyl sulfate (SDS)-10% polyacrylamide gels for immunoblot analysis. Standard electrotransfer techniques were used (70) with Immobilon-P membranes (Millipore). The detection method used was
the ECL system as described by the manufacturer (Amersham), except that
the secondary antibody, horseradish peroxidase-conjugated donkey
anti-rabbit immunoglobulin, was used at 1:10,000 dilution. The primary
polyclonal antisera used for each filter were from production bleeds
660 (anti-Gag) and 657 (anti-IN) (42).
Indirect immunofluorescence. (i) Localization of the FLAG-tagged
Gag.
The anti-FLAG M2 monoclonal antibody (Eastman Kodak), and
fluorescein isothiocyanate (FITC)-Oregon green 488-goat anti-mouse immunoglobulin G (IgG) antibody (Molecular Probes) were used for immunofluorescence experiments. To visualize nuclear DNA, cells were
stained with 1 µg of DAPI (4',6'-diamino-2-phenylindole)/ml. Detection of FLAG-tagged Gag in intact cells was achieved by incubating mutant and wild-type cells containing the FLAG-tagged Gag under inducing conditions. Cells containing the Tf1 plasmid without FLAG,
pHL449-1, were used as negative controls. Approximately 5 × 107 stationary-phase cells (OD600, 10 to 11)
were harvested and fixed with 3.7% formaldehyde (F1268; Sigma) at
32°C for 40 min. The cells were next incubated in 0.1 M phosphate
buffer, 1.2 M sorbitol, 0.01% 
-mercaptoethanol, and 0.1 mg of 100T
zymolase/ml at 32°C for 40 min. The resulting spheroplasts were
washed five times in 1 ml of 0.1 M phosphate buffer-1.2 M sorbitol and
then resuspended in 0.2 ml of the same buffer and transferred onto
polylysine-coated slides. The cells were then immunostained with
primary antibody (anti-FLAG M2; 1:1,000 dilution) and secondary
antibody (FITC-Oregon green-anti-mouse IgG; 1:500 dilution),
respectively. The cells were then mounted on glass slides with mounting
solution (1 mg of p-phenylenediamine [P-1519; Sigma]/ml-1
µg DAPI/ml in 50% glycerol). The cells were examined by using a
Zeiss Axioscope equipped with UV and FITC optics. Images were collected
and imported into Adobe Photoshop version 4.0.1 for figure presentation.
(ii) Nuclear localization of HA-tagged Pst1p.
The yeast
strain YHL912 was transformed with plasmids containing either the
HA-tagged pst1+ (pHL1626-2) or the untagged
pst1+ (pHL1458-3). Cells of the resulting
transformants were grown to an OD600 of 0.3, and
approximately 5 × 107 cells were harvested by
centrifugation. Immunostaining of cells was performed by the same
procedure as described above for FLAG-Gag localization. Primary and
secondary antibodies were monoclonal anti-HA(Ab) (MMS-101P;
Babco) (1:1,000 dilution) and FITC-Oregon green
488-anti-mouse IgG (Molecular Probes) (1:1,000 dilution), respectively.
Histone preparations and analysis.
Cells grown in rich YES
medium were harvested at log phase (OD600 of 1).
S. pombe histones were isolated from whole-cell
extracts as described previously (20) with minor
modifications. Histone samples (2 µg) were run on SDS-15%
polyacrylamide gels and then electroblotted to nitrocellulose filters.
Immunoblot analysis was performed to determine the acetylation levels
of histone H4, using antisera raised against specific acetylated lysine
residues in histone H4 (7, 71). Global acetylation levels of
histones were also determined with rabbit polyclonal IgG antibodies
against acetylated histones H3 and H4 (Upstate Biotechnology). Specific acetylation levels of the individual lysines AcK5-H4, AcK8-H4, AcK12-H4, and AcK16-H4 were determined with specific antibodies obtained from B. Turner (R41-K5, R232-K8, R101-K12, and R252-K16, respectively). Calf histones were used as molecular weight markers (Boehringer). Highly acetylated human histones, which were used as a
positive control, were a gift from A. Wolffe. Rabbit IgG was detected
by ECL (Amersham) with horseradish peroxidase-conjugated anti-rabbit
IgG according to the manufacturer's instructions.
| |
RESULTS |
Genetic screen to identify yeast strains deficient for Tf1
transposition.
To identify genes in S. pombe that
contribute to the propagation of the Tf1 element, we used ethyl
methanesulfonate to mutagenize a strain that contained an active copy
of Tf1 in a transposition assay plasmid (38). Mutagenized
cells were screened for strains that showed reduced levels of
transposition. Tf1 activity in individual colonies was monitored by
using an assay that detected the resistance to G418 caused by the
insertion of neo-marked Tf1 elements into the genome of
S. pombe cells (41). The assay plasmid
(pHL449-1) carried a copy of Tf1 (Tf1-neoAI) that was placed
under the control of the inducible nmt1 promoter. The Tf1
element in the assay plasmid was marked with a bacterial neo
gene that was inserted in the orientation opposite to Tf1 transcription
(Fig. 1B). An artificial intron was
inserted into the neo gene to block the translation of the
neo mRNA. The orientation of the intron was opposite to that of neo transcription so that the intron could only be
spliced out of the Tf1 transcript, and thus the neo gene
could provide G418 resistance only after the Tf1 transcript was reverse
transcribed. Cells harboring the plasmid with Tf1-neoAI were
induced for transposition by activating the expression of the
nmt1 promoter. Before the induced colonies were tested for
resistance to G418, the plasmid with Tf1-neoAI was subjected
to counterselection by replica printing cells to medium that contained
5-FOA. Thus, only cells that received a transposition event (conferring
a G418r phenotype) and subsequently lost the assay plasmid
(conferring a 5-FOAr phenotype) papillated on medium that
contained G418 and 5-FOA. Figure 1B presents the results of a
transposition assay and shows that a patch of cells that
initially contained wild-type Tf1-neoAI produced
confluent growth on a G418-5-FOA plate. A frameshift mutation in the N
terminus of IN was shown to block IN expression and reduce the
frequency of G418r by 34-fold (3). In addition,
a frameshift in PR blocks the expression of RT and IN and produced no
resistance to G418 (Fig. 1B).
View larger version (46K):
[in this window]
[in a new window]
|
FIG. 1.
Genetic screens used to identify strains with decreased
Tf1 transposition and cDNA recombination. (A) The transposition
assay plasmid, pHL449-1, contained the URA3 gene of
S. cerevisiae and the Tf1 element fused to an inducible
nmt1 promoter. The BxtXI restriction sites
referred to in the text are shown. The shaded portion of the element
represents the single ORF that encodes the Gag, PR, RT, and IN
proteins. The triangles depict the LTRs. The Tf1 element was
genetically marked with a bacterial neo gene, and the arrow
indicates the orientation of neo transcription. An
artificial intron (AI) was inserted into the neo sequence,
and the 5' and 3' splice sites are shown. (B) Transposition was induced
to high levels by growing cells on medium that activated the
nmt1 promoter (the absence of thiamine) and was detected by
the G418-resistant phenotype acquired when the neo-marked
Tf1 element transposed. The extended arrows labeled with neo
represent the Tf1 mRNA before and after the intron is spliced out.
After splicing and reverse transcription, the neo gene
carried on Tf1 cDNA became functional and was used to detect either
the integration of the cDNA into genomic sequence (left panel) or
its homologous recombination with other sources of transposon sequences
(right panel). The manipulations required for the transposition and
recombination assays are described in parentheses. The wild-type strain
(YHL1282) and a strain with the pst1-1 mutation (YHL5406)
were subjected to the transposition and cDNA recombination assays.
These two strains contained the wild-type version of the
nmt1-Tf1-neoAI assay plasmid (pHL449-1). The two
control strains were the wild-type strain containing the
nmt1-Tf1-neoAI assay plasmid with a frameshift
(fs) created either in IN (YHL1554) or in PR (YHL1836) of Tf1.
|
|
Mutagenized strains that appeared to have significantly less
transposition activity were then screened for trivial causes
of reduced
growth on the G418-5-FOA plates. The original strain
we mutagenized
contained an integrated copy of
LacZ under the
control of
the
nmt1 promoter so that each candidate suspected
of
possessing defects in transposition could be tested for reduced
promoter function. We also retransformed each candidate with a
fresh
copy of the Tf1-
neoAI plasmid to identify which strains
were
defective for transposition simply due to mutations in the
assay
plasmid. In addition, we tested strains with a version of
Tf1 that
contained the
arg3+ gene as a transposition
marker. In this way we could exclude
candidates that showed low growth
on the G418-5-FOA plates due
to alterations specific to the
metabolism of G418. A set of several
strains with reduced transposition
was identified after we screened
a total 2,500 independent colonies.
This report describes the
analysis of a mutation in a gene that we
named
pst1+.
The strain with the
pst1-1 mutation was backcrossed with a
wild-type strain three times, and 20 tetrads were dissected. Of
the 16 tetrads that produced four viable spores, all exhibited
a 2:2
segregation of the transposition defect, indicating that
the reduced
Tf1 activity was due to a mutation in a single gene.
Figure
1B shows
the transposition defect of a
pst1-1 mutant strain
that was
a product of the three backcrosses. We also found that
in the context
of diploid strains, this mutation was recessive
for the reduced level
of transposition (data not shown). After
the three backcrosses,
additional tests were conducted to determine
whether the mutation in
the
pst1-1 allele lowered the transcription
of Tf1 in the
context of the assay plasmid. Total RNA was extracted
from strains that
were grown under inducing conditions (absence
of thiamine) and was
subjected to RNA blot analysis. The results
presented in Fig.
2 showed that the
pst1-1
mutation allowed normal
levels of Tf1 transcripts to be expressed from
the Tf1-
neoAI plasmid.
There was also the possibility that
the
pst1-1 mutation reduced
the activity of the
neo promoter so that even if transposition
occurred
normally, the insertion events would go undetected. The
same RNA blot
shown in Fig.
2 was hybridized with a
neo-specific
probe,
and the mutation in the
pst1-1 allele was found to have
no
effect on
neo mRNA levels. Once we established that Tf1
mRNA
levels were normal in these strains, we considered the
possibility
that the
pst1-1 mutation could have indirectly
reduced the level
of one or all of the Tf1 proteins. Figure
3A is an immunoblot
of proteins extracted
from cultures harvested in both stationary
and exponential phases. The
levels of Gag and IN proteins that
accumulated in cells with the
pst1-1 mutation were indistinguishable
from those of
wild-type cells. Because IN is the last protein
encoded by the single
ORF of Tf1 (
41), the normal levels of
IN indicated that all
Tf1 proteins were translated with wild-type
efficiency. No IN is
detected in stationary-phase cells because
of a degradation mechanism
that creates a significant molar excess
of Gag relative to IN and RT
(
4).
View larger version (38K):
[in this window]
[in a new window]
|
FIG. 2.
RNA blot analysis of Tf1 and neoAI
transcripts. (A) Schematic presentation of the two transcripts of
interest. The largest rectangle represents the Tf1 element, and the
triangles indicate the LTRs. The wavy arrows represent Tf1 and
neo mRNAs and their transcription orientations. The 5'
and 3' splice sites of the artificial intron (AI) inserted within the
neo sequence are shown. The transcription of Tf1 was under
control of the inducible nmt1 promoter, while neo
transcription was driven by its own promoter. (B) Total RNA was
extracted from cells that either did (YHL5406) or did not (YHL1282)
(wild type [WT]) contain the pst1-1 mutation. The rRNAs
(top panel), stained with ethidium bromide, were used to monitor equal
loading of the lanes. Hybridizations were done with
32P-radiolabelled probes specific for either Tf1 mRNA
(middle panel) or neo mRNA (bottom panel).
|
|
View larger version (46K):
[in this window]
[in a new window]
|
FIG. 3.
Effects of the pst1-1 mutation on the
accumulation of Tf1 proteins and cDNA. (A) Levels of Tf1 Gag and IN
accumulated in strains that were wild type (WT) (YHL1282) or contained
the pst1-1 mutation (YHL5406). The immunoblots of
S. pombe extracts were made from cells harvested either
at log phase or at stationary phase. The filters were probed
simultaneously with anti-Gag and anti-IN antisera. The positions of
molecular mass standards are shown. The positions of Gag and IN are
indicated by arrows. (B) DNA blot used to measure the levels of Tf1
cDNA produced in the strain with the pst1-1 mutation
(YHL5406). The three control strains were the wild-type strain
containing versions of Tf1 that either were wild-type (YHL1282) or
contained a frameshift (fs) mutation in IN (YHL1554) or a frameshift
mutation in PR (YHL1836). DNA was extracted from S. pombe strains induced for Tf1 expression. The DNA was digested
with BstXI and probed with neo-specific sequence.
The 2.1-kb band was generated by Tf1 cDNA, while the 9.5-kb band
was produced by vector sequence and was used to monitor equal
loading.
|
|
pst1-1 mutation caused a defect in a late step of the
transposition pathway.
To measure the magnitude of the
transposition defect caused by the pst1-1 mutation, we
subjected strains to a previously developed quantitative assay
(3). Patches of cells induced for Tf1 expression were
resuspended in liquid and spread onto agar medium that contained 5-FOA.
Approximately 5,000 to 10,000 5-FOA-resistant colonies per plate were
then replica printed to plates with G418 and 5-FOA to determine what
fraction of the cells induced for transposition received an insertion
of Tf1-neo. The results of the quantitative transposition
assay presented in Table 3 show that the
strain with the pst1-1 mutation produced 10-fold fewer
transposition events than did the wild-type strain.
The mutagenized strains were also screened with an assay that measured
homologous recombination between copies of Tf1 cDNA
and Tf1 plasmid
sequences (
3). Wild-type levels of this recombination
indicate that normal levels of Tf1 cDNA are produced and that
this
cDNA is transported to the nucleus. The homologous recombination
assay was performed with the same Tf1-
neoAI plasmid shown in
Fig.
1. As described above, the
neo marker of the
Tf1-
neoAI plasmid
was initially unable to provide resistance
to G418 because the
intron in
neo could only be spliced from
the Tf1 mRNA. The reverse
transcripts of the spliced Tf1 mRNA,
when present in the nucleus,
homologously recombine with the
Tf1-
neoAI plasmid to generate
a G418-resistant version of
the assay plasmid. The difference
between the transposition and
recombination assays was that cells
tested for recombination were
printed from the induction medium
directly to plates with G418 without
first selecting against the
presence of the plasmid on plates with
5-FOA. The recombination
assay shown in Fig.
1B shows that a wild-type
copy of Tf1-
neoAI produced confluent growth on agar medium
that contained G418.
The confluent growth produced by a version of
Tf1-
neoAI that lacked
IN and the lack of G418 resistance due
to a frameshift just upstream
of RT were demonstrations used to
indicate that the homologous
recombination assay detects products of
reverse transcription
even in the absence of IN activity
(
3). Figure
1B also shows
that the
pst1-1
mutation caused a reduction in the homologous
recombination of Tf1
cDNA compared to the wild-type strain and
the strain with the
frameshift in IN. The magnitude of the recombination
defect was
determined by subjecting the strains to the quantitative
version of the
homologous recombination assay (
3). Patches
of yeast cells
induced for Tf1 expression were resuspended in
liquid and spread
directly onto plates that contained G418. The
number of colonies that
grew on G418 was divided by the total
number of viable cells plated to
determine the fraction of cells
that generated resistance to G418.
Table
3 shows that the strain
with the
pst1-1 mutation
produced eightfold-lower levels of cDNA
recombination than the
wild-type strain. However, the recombination
defect in the
pst1-1 mutant strain represented a threefold decrease
when compared to the levels of recombination produced by a strain
without IN (YHL1554 [Table
3]). This may be considered a more
appropriate comparison, since the lack of IN generates a baseline
level
of resistance to G418 that does not include
transposition.
The results of the recombination assays suggested that either the
levels of Tf1 reverse transcripts were reduced by the
pst1-1 mutation or that normal levels of cDNA were produced but they
accumulated in a compartment that was inaccessible to the
Tf1-
neoAI plasmid. To investigate these possibilities, we
used a previously
published method of DNA blot analysis to measure the
accumulated
levels of Tf1 cDNA in cells that contained the
pst1-1 mutation
(
3,
39). Liquid cultures of cells
induced for Tf1 expression
were extracted for total DNA that was then
digested with
BstXI
and subjected to DNA blot analysis. The
results presented in Fig.
3B show that the wild-type
Tf1-
neoAI produced a measurable amount
of a 2.1-kb fragment
of cDNA detected with a
neo-specific probe.
This
BstXI fragment is derived from the terminal sequence of Tf1
that is the final region to be reverse transcribed and therefore
represents the levels of full-length products. The DNA extracted
from
control strains showed that the frameshift in IN did not
reduce the
intensity of the 2.1-kb band, whereas the frameshift
just upstream of
RT blocked the accumulation of cDNA. In comparison,
the strain with
the
pst1-1 mutation generated normal levels of
the 2.1-kb
fragment of cDNA. A 9.5-kb band resulted from the
BstXI
digestion of the Tf1-
neoAI plasmid, and this species served
as
an internal control for levels of DNA loaded in each
lane.
The fact that the strain with the
pst1-1 mutation showed a
defect in cDNA recombination despite its ability to produce
wild-type
levels of cDNA suggested that the general recombination
machinery
could be defective. To investigate this possibility, the
frequencies
of homologous recombination between two defective alleles
of the
ade6 gene were measured by testing for crossover
events that generated
adenine prototrophy. The plasmid pade6-M375-M26
(
55) that contained
an allele of
ade6 with a
double mutation at the beginning of the
ORF was transformed into two
yeast strains that both contained
a chromosomal allele of
ade6 with a point mutation near the end
of the ORF,
ade6-M210. One of the yeast strains contained the
pst1-1 allele, while the other was
pst1+. Two independent colonies of each strain
were grown for 2 days
in liquid EMM lacking uracil, and mitotic
recombination frequencies
were determined by plating the cells onto EMM
agar lacking adenine.
The number of Ade
+ prototrophs
compared to the number of cells viable on rich medium
(YES) represented
the frequency of recombination between the two
point mutations at the
ade6 locus. As shown in Table
4, the frequency
of mitotic recombination
was not significantly affected by the
pst1-1 mutation.
Taken together, the accumulation of normal amounts of Tf1 cDNA, the
low levels of cDNA recombination, and the wild-type recombination
at the
ade6+ locus indicated that the
transposition defect caused by the
pst1-1 mutation occurred
in a late step in the retrotransposition pathway,
i.e., after reverse
transcription. To gain further information
about the defect caused by
the
pst1-1 allele, we isolated the
wild-type allele of the
pst1+ gene from a library of genomic
DNA.
pst1+ is a homologue of the
SIN3 genes found in mammals and S. cerevisiae.
The reduced levels of transposition and cDNA
recombination caused by the pst1-1 mutation served as the
basis for cloning the wild-type copy of the
pst1+ gene. For this purpose, we transformed a
library of plasmids that contained S. pombe genomic DNA
into a strain with the pst1-1 mutation and screened for
complementation of the transposition defect. The plasmid library
consisted of a multicopy vector containing genomic inserts that
averaged 10 kb in length (kindly provided by P. Young). Out of a total
of 130,000 colonies screened, only one plasmid was isolated for its
ability to complement both the transposition and cDNA recombination
defects caused by the pst1-1 mutation. Initial sequence data
from this insert revealed that its sequence was available from the
S. pombe genome project (accession no. Z54140).
Figure
4A is a diagram of the isolated
fragment with the significant ORFs and the restriction sites used for
subsequent experiments.
The sequence began with a truncated ORF whose
predicted amino
acid sequence had 58.6% identity to that of the
product of the
GFA1 gene of
S. cerevisiae.
The second part of the genomic fragment
contains the entire ORF for a
hypothetical protein with an amino
acid sequence showing 36.6%
identity to the ySin3p protein of
S. cerevisiae
(
77). Subsequent subcloning studies showed that
the 5.6-kb
Eco47III-
NheI fragment, which contained the
homologue
of
SIN3, was responsible for the efficient
complementation of
the defects due to the
pst1-1 mutation
(Fig.
4). We also found
that a frameshift generated at the beginning of
the second exon
of
pst1+ severely reduced the
complementation activity of the plasmid
(Fig.
4). These data clearly
indicated that the second ORF, which
encodes a homologue of ySin3p, was
the source of the complementation.
To test whether this gene was
allelic to
pst1+, we subcloned it into an
integrative vector that contained the
leu1+ gene
of
S. pombe. The resulting plasmid was integrated into
the
genome of a haploid with the
pst1-1 allele at the site
of the
complementing ORF. A single integrated copy of this genomic
fragment
was found to retain its ability to complement the
pst1-1 transposition
defect (Fig.
4B). The resulting strain
was crossed with a wild-type
pst1+ haploid
strain, and 16 tetrads were dissected. All four spores
of these tetrads
possessed wild-type transposition and cDNA recombination
activity
(data not shown). Taken together, these data indicate
that the
homologue of
SIN3 was allelic to
pst1+. We named this gene
pst1+ as an abbreviation for
pombe
SIN-three no. 1.
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 4.
Isolation and characterization of the
pst1+ gene of S. pombe. (A)
Schematic representation of the original genomic fragment that
complemented the transposition defect caused by the pst1-1
mutation. The ORF corresponding to the pst1+
allele is indicated by a large arrow, whereas the truncated ORF with
high homology to the GFA1 gene of S. cerevisiae is also shown, with the location of its ATG codon. The
restriction sites referred to in the text are shown. The
Eco47III-NheI fragment responsible for the
complementation of the pst1-1 transposition defect is
indicated as a shaded region. The locations of the point mutation (stop
codon) and the artificial frameshift in pst1 are also shown.
(B) Transposition assay. Wild-type (WT; YHL4988) and pst1-1
(YHL4994) are the strains in the top row. The three lower rows are the
pst1-1 strain that contained an additional allele of the
pst1 gene. The name and copy number of each additional
allele are indicated in parentheses. The second, third, and fourth rows
are strains YHL6214, YHL6221, and YHL6222, respectively. The
transposition assay was carried out as described in Materials and
Methods.
|
|
To gain further information about the role of
pst1+ in the propagation of Tf1, we cloned the
chromosomal copy of the
pst1-1 allele by PCR and determined
its nucleotide sequence. Two independent
PCR products of each region
were cloned and sequenced (see Materials
and Methods for details). The
sequences of the cloned PCR products
were compared to the wild-type
pst1+ sequence retrieved from the
S. pombe database as well as the
sequence of the wild-type
pst1+ clone isolated from the
S. pombe library. A single G
A substitution
was found in the
sequence of the
pst1-1 allele that converted
codon 1305 from
a tryptophan residue into a stop codon. The result
of this nonsense
mutation was predicted to stop translation of
the
pst1
mRNA, producing a truncated protein that lacked 217 amino
acids
from its C terminus (Fig.
4A and
5A).
Putative splicing signals were predicted in the
pst1+ ORF and reported in the
S. pombe database (accession no.
Z54140).
A conserved splice
branch and acceptor sequence TACTAATTATTTGATTAG
(
43) can be found in the
pst1+
gene at nucleotides 249 to 266, relative to the ATG codon. The
5'
splice site (GTATGT; nucleotides 130 to 135) is also
present.
We demonstrated that this putative intron is spliced in vivo
by
determining the sequence of the
pst1+
mRNA by a reverse-PCR technique (data not shown). The spliced
message is predicted to encode a polypeptide of 1,522 amino acids
(171 kDa) (Fig.
5A). As mentioned above,
sequence comparisons
showed that Pst1 protein (Pst1p) possessed
significant similarity
to ySin3p of
S. cerevisiae and
its mammalian mSin3A and mSin3B
counterparts (
5,
77).
Furthermore, the examination of the
S. pombe sequence
database identified a second Sin3 homologue
that we named
pst2+ (accession no.
Z98559). Figure
5A shows
the predicted amino
acid sequence of Pst1p and indicates the position
of the four
PAH domains. The PAH motif consists of two amphipathic
helices
separated by a loop of about 20 amino acids (
77),
which is similar
to the amphipathic helical structure found in the
tetratricopeptide
repeat motif and the helix-loop-helix motif. It has
been suggested
that the amphipathic helices participate in protein
dimerization
or in the mediation of protein-protein interactions
(
23,
26,
29,
47,
66,
75). The sequences of Pst1p and Pst2p
of
S. pombe were aligned with the sequences of ySin3p,
mSin3A, and mSin3B.
A schematic representation of this alignment was
generated by
the program MACAW version 2.0.5 (Fig.
5B).
Examination of the
alignments revealed that the predicted
amino acid sequence of
S. pombe Pst1p and Pst2p
displayed blocks of similarity to the
S. cerevisiae
and mammalian Sin3 proteins throughout their respective
ORFs (Fig.
5B). The regions of highest similarity between the
Pst and Sin3
proteins were centered around the four PAH domains,
suggesting that
these regions in the
S. pombe proteins possess
important functions. In addition, the histone deacetylase-interaction
domain (HID) located between PAH3 and PAH4 (
37) and the
region
just following PAH4 are highly conserved in all Sin3 and Pst
proteins.
View larger version (41K):
[in this window]
[in a new window]
|
FIG. 5.
Amino acid sequence for Pst1 protein. (A) The deduced
amino acid sequence encoded by the pst1+ gene of
S. pombe is shown. The ORF is interrupted by an intron
in the gene whose position is indicated by the arrowhead. Each pair of
boxes connected by underlining represents a PAH motif; all the pairs
have been labeled PAH1 to PAH4. The solid circle denotes the position
of the pst1-1 point mutation where a tryptophan residue was
replaced by a stop codon. (B) Deduced amino acid sequences encoded by
pst1+ and pst2+ of
S. pombe are schematically shown aligned to that
encoded by SIN3 of S. cerevisiae and
mammalian mSIN3A and mSIN3B, using MACAW algorithms (65).
The length of each protein is given. The solid boxes correspond to the
four conserved PAH domains. The shaded boxes indicate the locations of
HIDs, and the hatched boxes are conserved regions just downstream of
PAH4. The scores of sequence similarity given in parentheses for each
conserved domain represent the percentages of the amino acids in
alignments that are similar within all five proteins. The open boxes
represent nonconserved regions.
|
|
pst1+ is an essential gene.
Previous
studies demonstrated that the budding yeast SIN3 is required
for normal sporulation of diploid cells (50, 67, 73) but is
not required for viability (77). To test whether pst1+ is required for sporulation or cell
viability, we deleted just the ORF of pst1+ from
one allele of a diploid strain and replaced it with the his3+ marker gene. Only 5 of 40 dissected
tetrads produced two viable spores and two dead spores, while the rest
of the tetrads (35) produced one or no viable spores. In
addition, all of the germinating spores were auxotrophic for histidine,
suggesting that the deletion of pst1+ and its
replacement by the his3+ marker may inhibit
spore germination and/or viability. The heterozygous diploid was
subjected to random spore analysis, and none of the 500 germinating
spores was prototrophic for histidine. Although these results suggested
that the pst1+ gene was required for viability,
we tested the possibility that the gene was only required for spore
germination. A plasmid-shuffling technique was used for this purpose.
From the diploid described above, we generated a haploid strain that
contained the his3+ substitution of the
pst1+ gene by introducing a wild-type copy of
pst1+ carried on a LEU2 plasmid. The
haploid was transformed with a second plasmid that contained
pst1+ as well as the S. cerevisiae
URA3 marker. This strain was first grown as a patch and then
streaked on EMM that lacked uracil but contained leucine to
allow for random loss of the LEU2 plasmid. These colonies
were then replica printed to an agar plate that contained uracil and
not leucine and to another agar plate that contained uracil,
leucine, and 5-FOA. The 5-FOA selects against the presence of the
URA3-expressing plasmid (11). The growth phenotypes were scored after a 2-day incubation either at 25, 32, or
37°C. After a second printing to plates that contained 5-FOA, the
colonies that lacked the copy of pst1+ on the
LEU2 plasmid did not grow on the 5-FOA plate. In comparison, the colonies that retained the LEU2 plasmid did form
5-FOA-resistant colonies. These results provided strong evidence that
pst1+ was essential for vegetative growth.
Previous studies performed with Sin3 counterparts of other organisms
strongly suggested a critical role for Sin3 proteins
in histone
deacetylation (
54,
79). We therefore conducted
a series of
experiments to address whether
pst1+ of
S. pombe was also involved in these processes and
whether
alterations in histone deacetylation affected Tf1
transposition.
Cellular localization of HA-tagged Pst1 protein.
The role of
Sin3 proteins in remodeling chromatin structure and regulating
transcription, as shown in previous studies of mammalian and budding
yeast Sin3 proteins, suggested that Sin3p is a nuclear protein. Indeed,
budding yeast Sin3p and mammalian mSin3B proteins are located in the
nucleus (1, 77). We found that Pst1p was also localized in
the nucleus, by using indirect immunofluorescence microscopy. For this
purpose, we created a yeast strain with a plasmid that contained
pst1+ with a triple-HA epitope inserted at the
C-terminal end. In this strain, HA-tagged pst1+
was expressed from its own promoter, and the presence of the HA epitope
in the sequence of pst1+ was found not to alter
its ability to complement the transposition and cDNA recombination
defects (data not shown). S. pombe cells were grown to
early log phase (OD600, 0.3), fixed with formaldehyde, prepared for immunofluorescence staining with an anti-HA monoclonal antibody (HA.11; Babco), and subjected to immunofluorescence
microscopy. The results presented in Fig.
6 indicate that the FITC signals corresponding to HA-tagged Pst1p were distributed throughout the nucleus, as visualized by DAPI staining of chromosomal DNA (Fig. 6B).
The control strain contained a plasmid with the
pst1+ allele that lacked the HA tag. This strain
produced no FITC signal (Fig. 6A), indicating that the signals
generated by the experimental strain were specific for HA-tagged Pst1p.
Nuclear localization of Pst1p was also observed when it was expressed
from a single integrated copy of the HA-tagged
pst1+ allele (data not shown). The fact that
S. pombe Pst1p is a nuclear protein is consistent with
its assumed role in the deacetylation of histones.
View larger version (63K):
[in this window]
[in a new window]
|
FIG. 6.
Cellular localization of Pst1p. Yeast cells with a
plasmid copy of pst1+ that either did not (A) or
did (B) contain an HA epitope were grown in YES medium to an
OD600 of 0.3. The cells were harvested and subjected to
immunofluorescence microscopy. The two right panels show the FITC
signals obtained with the anti-HA antibody, and the two left panels
show the locations of nuclei as indicated by DAPI counterstaining.
|
|
A strain with the pst1-1 mutation was highly sensitive
to the histone deacetylase inhibitor TSA.
To investigate the
possibility that Pst1p plays a role in the deacetylation of histones,
we tested the ability of a strain with the pst1-1
mutation to grow in the presence of the histone deacetylase inhibitor
TSA. As shown in Fig. 7A, a strain
with the pst1+ allele was able to grow in the
presence of 8 nM TSA whereas an isogenic strain with the
pst1-1 allele grew much more slowly, such that no colonies
were visible after 3 days of incubation at 32°C. We also measured the
growth rates of the same strains in liquid cultures. Figure 7B shows
the growth curves of the wild-type strain and the strain with the
pst1-1 mutation in the presence of increasing TSA
concentrations ranging from 0 to 16 nM. In the absence of TSA, the
growth of the two strains was indistinguishable. When added at
concentrations as high as 16 nM, TSA only caused small changes in the
growth of the wild-type strain whereas the growth of the strain with
the pst1-1 allele was strongly inhibited, and no growth was
observed when TSA was used at 16 nM. The supersensitivity of the strain
with the pst1-1 mutation to TSA was consistent with the
proposed involvement of S. pombe Pst1p in the histone
deacetylation process. Remarkably, when grown in liquid medium with 8 nM TSA, the strain with the pst1-1 mutation exhibited a
morphological change by shifting from the unicellular growth mode to a
filamentous growth mode based on the formation of pseudohyphal cells
(Fig. 7A). Extensive filamentous growth led to the formation of
mycelium-like structures that displayed pseudohyphae with
branches and lateral buds. In comparison, exposure to TSA did not cause
any apparent morphological change to the strain with the
pst1+ allele (Fig. 7A).
View larger version (59K):
[in this window]
[in a new window]
|
FIG. 7.
Effect of the pst1-1 mutation on cell growth
in the presence of TSA. (A) Supersensitivity of the strain with the
pst1-1 mutation to TSA. Cells of strains that contained
either pst1+ (YHL1282) or pst1-1
(YHL5406) allele were streaked on EMM-Ura. Growth in the presence (+)
(middle panel) or absence ( ) of 8 nM TSA (left panel) was scored
after 3 days at 32°C. The pseudohyphal growth mode of the cells with
the pst1-1 mutation when grown in liquid medium supplemented
with 8 nM TSA is also shown (bottom right panel), while the wild-type
cells maintained the yeast form (top right panel). (B) Growth rates of
strains with pst1+ and pst1-1 allele
in increasing concentrations of TSA. Cell growth in EMM-Ura was
monitored by following OD600 as a function of time. The
concentrations of TSA used for this experiment are also presented.
|
|
TSA inhibited Tf1 transposition.
If Pst1p of S. pombe is similar to the Sin3 proteins of other organisms it would
work together with histone deacetylases to change the acetylation
pattern of chromatin. If the mutation in the pst1-1 strain
inhibited Tf1 transposition as the result of reduced deacetylase
function then exposure to TSA could cause the reduction of Tf1
transposition by inhibiting the histone deacetylase activity associated
with Pst1p. To test this prediction, we performed quantitative
transposition assays on strains grown in the presence and absence
of TSA. In these experiments, TSA was present at 8 nM in all steps
of the transposition assay. As shown in Fig.
8A, TSA treatment during transposition
significantly inhibited Tf1 activity in the strain with the
pst1+ allele (wild type). The reduction in
transposition caused by TSA treatment was similar in magnitude to the
low transposition levels caused by the mutation in the
pst1-1 strain. This finding suggests that the
pst1-1 mutation may affect Tf1 transposition through the
histone deacetylation pathway.
View larger version (41K):
[in this window]
[in a new window]
|
FIG. 8.
Effects of either the pst1-1 mutation or
exposure to TSA on Tf1 transposition and histone acetylation of bulk
chromatin. (A) Transposition activity of Tf1 was measured in wild-type
(wt; YHL1282) and pst1-1 mutant (YHL5406) strains, using the
quantitative transposition assay. If present, the concentration of TSA
was 8 nM. The transposition level of the wild-type strain in the
absence of TSA was defined as 100%. (B) Acetylation levels of bulk
chromatin in wild-type (YHL1282) and pst1-1 mutant (YHL5406)
strains. Whole-cell extracts of histones were isolated from cells grown
in EMM-Ura medium and harvested at an OD600 of 1. Histone
samples of two independent cultures of each strain, either wild-type
(lanes 1 and 2) or the strain with the pst1-1 mutation
(lanes 3 and 4), were separated on SDS-polyacrylamide gels,
electroblotted onto nitrocellulose membranes, and probed with
appropriate antibodies. Lane 5, histone extract of the wild-type strain
grown in the presence of 8 nM TSA. Lane 6, highly acetylated human
histones that were used as molecular weight markers. Coomassie staining
was used to monitor equal loading (not shown). The blots were performed
individually and assembled for the figure.
|
|
If the
pst1-1 mutation reduced transposition due to changes
in chromatin acetylation then large changes in the levels of
acetylation
may have occurred. To investigate this possibility, we
measured
the acetylation of specific lysines in bulk preparations of
histones
H3 and H4. In these experiments, cells with and without the
pst1-1 mutation were harvested from cultures grown under the
same conditions
that were used for the quantitative transposition
assay. Histones
were extracted from whole cells by established methods
(
20),
electrophoretically separated on
SDS-polyacrylamide gels, transferred
to nitrocellulose membranes, and
probed with appropriate antibodies.
Since histone H4 has been known to
be mainly acetylated at the
specific lysine residues K5, K8, K12, and
K16 (
13,
68,
80),
we used antibodies that were raised
against histone H4 with acetylation
specifically at these lysines
(
7,
71). We also used antibodies
against histone H3
acetylated at multiple lysines (Upstate Biotechnology).
Data presented
in Fig.
8B show that neither the
pst1-1 mutation
nor the
treatment with 8 nM TSA significantly affected the global
acetylation
levels of lysines 5, 8, 12, and 16 of histone H4 or
the bulk level of
acetylation of lysines in histone H3. This suggests
that the
pst1-1 mutation did not cause a global change in the
acetylation of histones. In contrast, it was shown previously
that an
extended exposure of
S. pombe cells to very high
concentrations
of TSA, such as 80 nM, resulted in an overall increase
in acetylation
at lysine 5 of histone 4 (
20).
Localization of the Tf1 complex is disrupted in the
pst1-1 strain.
One interesting characteristic of cells
with the pst1-1 mutation was the low levels of homologous
recombination that occurred between Tf1 cDNA and cellular sequences
of Tf1 (Fig. 1B). The fact that cells with the pst1-1 allele
produced normal levels of cDNA led us to test the possibility that
cDNA recombination was low because of a defect in nuclear import.
To investigate the possibility that the
pst1-1 mutation
reduced the nuclear localization of Tf1 material, we studied the
localization
of Tf1 Gag. This protein was chosen because it is present
in high
molar ratios in a large macromolecular complex with Tf1
cDNA and
IN (
41). We studied the localization of Gag in
stationary-phase
cells as expressed from the Tf1-
neoAI
plasmid. For this purpose,
a FLAG epitope was inserted near the C
terminus of Gag. The expression
level of Gag was unaffected by the FLAG
epitope, as determined
by immunoblot analysis with Gag(Ab) and
FLAG(Ab), and the resulting
transposon, Tf1(FLAG)-
neoAI,
possessed wild-type levels of transposition
and homologous
recombination activity (data not shown). Wild-type
cells that
were induced for the expression of
Tf1(FLAG)-
neoAI were subjected to immunofluorescence
staining with the M2 anti-FLAG
monoclonal antibody and the Oregon green
488 goat anti-mouse IgG
conjugate. We found that the majority of
wild-type cells produced
a single primary focus of Gag signal within
the nucleus, as demonstrated
by the colocalization of the anti-FLAG
antibody and nuclear staining
by DAPI (Fig.
9A). For this purpose, the fluorescence
image produced
by the anti-FLAG antibody was merged with an inverted
black-and-white
image of the nucleus generated by DAPI counterstaining.
We found
that no FLAG signal was observed from cells that were not
induced
for Tf1(FLAG)-
neoAI expression (Fig.
9A), indicating
that the
anti-FLAG signals were specific for Tf1. A totally different
scenario
was observed in the strain with the
pst1-1
mutation. FLAG-Gag
appeared to form multiple aggregates spread
quasirandomly throughout
the cytoplasm (Fig.
9B). Indeed, very few
nuclei appeared to contain
Gag, suggesting that the nuclear
localization of FLAG signals
was disrupted in this strain. Figure
10 presents an analysis of
97 nuclei
from cells with the
pst1-1 mutation and 82 nuclei from
wild-type cells. We categorized the cells into three classes reflecting
the position of the Gag signal within the cells: (i) a single
predominant signal that overlapped with and was thought to be
located within the nucleus, (ii) multiple Gag signals observed
predominantly in the cytoplasm but some Gag also overlapping with
the
DAPI staining, and (iii) single or multiple signals clearly
positioned
outside the nucleus. The data presented in Fig.
10 indicate
that the
pst1-1 mutation greatly reduced the nuclear
localization
of Gag.
View larger version (46K):
[in this window]
[in a new window]
|
FIG. 9.
Immunofluorescence analysis of Tf1 FLAG-Gag. (A)
Immunofluorescence of Tf1 Gag in wild-type cells. The wild-type strain
YHL5896 contained a FLAG-Gag version of the Tf1-neoAI
plasmid (pHL1277). Cultures were grown under inducing conditions in
EMM-Ura. In the top panels, the green FITC signals are specific for the
FLAG-Gag protein and the blue signals indicate the locations of nuclei
counterstained with DAPI. In the control experiments (bottom panels),
the plasmid pHL449-1 was used in place of pHL1277 in the same wild-type
strain. The upper right panel is a merge of the FLAG-Gag signals
produced by YHL5895 with an inverted black-and-white image of its DAPI
stain. The merge was generated with Adobe Photoshop version 4.0 with
the screen function set at 65% opacity. (B) Same experiment shown in
panel A, except the yeast strains contained the pst1-1
mutation (YHL5356). The top panel shows a strain with the FLAG-Gag
version of the Tf1-neoAI plasmid (pHL1277), and the bottom
panel shows a strain with the control plasmid pHL449-1.
|
|
View larger version (27K):
[in this window]
[in a new window]
|
FIG. 10.
Distribution of Gag in strains with
pst1+ and pst1-1 alleles.
Immunofluorescence-stained cells with the wild-type and
pst1-1 alleles from the experiments described in the legend
to Fig. 9 were analyzed for the localization of Gag relative to the
locations of nuclei. The localization data are from approximately 100 cells. The cells were categorized into three classes. (i) Cytoplasm:
all of the Gag signals were exclusively within the cytoplasm. (ii)
Nucleus plus cytoplasm: the cells had significant amounts of
Gag in both the cytoplasm and the nucleus. (iii) Nucleus: all the Gag
in the cells was contained in the nucleus.
|
|
| |
DISCUSSION |
The application of a genetic assay for retrotransposition has
allowed us to generate strains of S. pombe with host
mutations that greatly reduce the function of Tf1. The yeast strain
with the pst1-1 allele was found to have a mutation that
lowered the frequency of transposition 10-fold and lowered homologous
recombination of cDNA 8-fold. Despite this lack of transposition
activity, we found that normal levels of element-encoded proteins and
cDNA were produced, indicating that a defect occurred in a late
step of transposition, i.e., after reverse transcription.
Chromatin remodeling factor Pst1p is required for
retrotransposition.
The pst1+ gene was
found to contain coding sequence for a protein of 1,522 amino
acids that possessed four PAH motifs and high sequence similarity to
the S. cerevisiae ySin3 protein as well as its
counterparts in mammals (5, 77). In budding yeast, the
single ySin3 protein was initially inferred to be a corepressor based
on the observation that a LexA-Sin3 fusion can repress
transcription when brought to a heterologous promoter (78).
Indeed, ySin3p together with histone deacetylase
Rpd3p negatively regulates a diverse set of yeast genes
(74).
Recently, ySin3p, Rpd3p, and specific DNA-binding proteins have been
found to form a large multiprotein complex (
32,
33)
that
represses transcription by targeting histone deacetylase
activity to specific regions of chromatin (
31,
32,
62).
Two
Sin3 proteins, mSin3A and mSin3B, have been isolated from
mice
(
5). Both mSin3A and mSin3B have been shown to function
as
corepressors that interact with proteins belonging to the Mad
family of
DNA-binding proteins. The resulting complex antagonizes
the activation
and transformation functions of the oncoprotein
Myc (
5,
64).
In addition, mammalian Sin3 proteins have recently
been shown to form
large multiprotein complexes that include mammalian
histone
deacetylases (mRPD3, HDAC1, and HDAC2); DNA-binding repressors,
such as Mad (
27,
37), YY1 (
81), and MeCP2
(
30,
49);
and transcriptional repressors for nuclear
receptors, such as
SMRT (
48) and N-CoR (
1,
28).
The formation of complexes
containing Sin3 proteins and histone
deacetylases has also been
proposed to be the mechanism used by
mammalian cells to target
histone deacetylase activity, thereby
repressing transcription
(
24,
54,
68,
79).
Interestingly, BLAST-based alignments revealed that
S. pombe also has two distinct members of the Sin3 gene family,
pst1+ and a second gene we suggested be called
pst2+. The presence of a second gene encoding a
Sin3-like protein in
S. pombe is in contrast to the
single
SIN3 gene of
S. cerevisiae.
In this
respect,
S. pombe is similar to mammalian cells. We
found
that both Pst1p and Pst2p displayed blocks of similarity to
S. cerevisiae and mammalian Sin3 proteins throughout
their respective
ORFs. In particular, high levels of conservation exist
in the
regions around the four PAH domains and the HID located between
PAH3 and PAH4 (
37). This suggests that Pst1p and Pst2p are
functionally
related proteins. However, Pst1p and Pst2p possess
significant
sequence divergences. The most pronounced differences
between
Pst1p and Pst2p are at the N and C termini of the two proteins.
In addition, Pst2p has a shorter amino-terminal region than Pst1p
and
has deletions relative to Pst1p in the regions between PAH1
and PAH2.
Interestingly, similar types of sequence divergences
and truncations in
the mammalian mSin3B relative to mSin3A have
also been reported
(
5). The obvious and interesting question
is whether the two
genes
pst1+ and
pst2+ in
S. pombe play distinct roles in specific cellular
functions
and, if so, whether their roles are conserved from
S. pombe to
mammals. We demonstrated that the deletion
of the chromosomal
copy of
pst1+ in a haploid
cell caused cell death, indicating an essential
role for
pst1+ in vegetative growth. This result
suggested that the two Pst
proteins in
S. pombe are not
interchangeable.
pst1+ is an essential gene that contributes
to histone deacetylation.
The fact that the single
SIN3 gene of S. cerevisiae is not required
for viability suggests that the chromatin remodeling due to
Sin3p-mediated histone deacetylation does not play an essential role in
the function of chromatin either at centromeres or at heavily
transcribed regions of the genome. It was therefore surprising that the
pst1+ gene of S. pombe was
essential for growth. This finding raises the distinct possibility that
Sin3 proteins of other organisms could possess vital functions. The
presence of distinct homologues of Sin3 proteins in S. pombe and the similarity of these proteins to their mammalian
counterparts suggest the possibility that Sin3 proteins of mammals may
also be essential for viability.
Our observation that the growth of a strain with the
pst1-1
mutation was severely inhibited by TSA was consistent with the
possibility that TSA targeted the essential function of
pst1+. Since TSA is an inhibitor of histone
deacetylases, this result
indicates that an essential function of
pst1+ is its recruitment of deacetylases and its
contribution to the
deacetylation of histones. In addition, the
supersensitivity of
the
pst1-1 strain to TSA suggests that
the
pst1-1 mutation resulted
in reduced levels of histone
deacetylase
activity.
When TSA was added at sublethal concentrations, the strain with
the
pst1-1 mutation exhibited slow growth and an extensive
filamentous morphology. This finding suggests that
S. pombe required
the corepressor Pst1p to maintain its yeast
morphology. A similar
situation has been reported for the pathogenic
yeast
Candida albicans,
which requires another corepressor,
the general transcription
corepressor TUP1, to maintain its yeast
morphology (
12). When
this repression is lifted under
inducing environmental conditions,
C. albicans shifts its
growth mode from unicellular budding to
growth that is pathogenic,
invasive, and hyphal (
51). In this
respect, the TSA
sensitivity of the
pst1-1 strain could be used
as a screen
to identify mutations in deacetylation mechanisms.
In addition, given
the general lack of information about the mechanisms
controlling
pseudohyphal growth in
S. pombe, the strain with the
pst1-1 mutation could be used to identify mutations in genes
responsible
for initiating pseudohyphal
growth.
Inhibition of Tf1 transposition by TSA.
Exposure of a
wild-type strain of S. pombe to TSA significantly
reduced the transposition frequency of Tf1. Intriguingly, the reduction
in transposition caused by TSA treatment was similar in magnitude to
the lowered transposition levels caused by the pst1-1
mutation. An interesting question was whether the mutation in
pst1-1 affected transposition through histone
deacetylation or through an independent mechanism. The
sensitivity of Tf1 to TSA supported the idea that the low levels of
transposition observed in a pst1-1 strain were due to a
decrease in histone deacetylase activity. However, we cannot ignore the
possibility that the defects in Tf1 function caused by TSA treatment
were the indirect consequences of changes in other cellular functions
(82).
It has recently been shown that in
S. pombe, TSA
treatment increased histone acetylation in centromeric chromatin,
resulting
in perturbations of centromere functions that included loss
of
centromere silencing, delocalization of centromere proteins,
chromosome
lagging in anaphase, and perturbation of chromosome
segregation
(
20). Interestingly, the strain with the
pst1-1 mutation also
exhibited the same types of defects
(
19a), indicating that
pst1+ may be
required to maintain the underacetylated state at centromeres.
To see
whether strains with the
pst1-1 mutation possessed a general
change in histone deacetylation, we measured acetylation
levels
of specific lysines within bulk preparations of histones from
wild-type and
pst1-1 cells. Our results indicated that
neither
the
pst1-1 mutation nor the exposure of cells to 8 nM TSA caused
a measurable change in the acetylation levels of
histones. This
finding suggested that the
pst1-1 mutation
only affected histone
acetylation at specific regions of chromatin.
Because
S. pombe has at least two histone deacetylases
(
53), the interesting
question is which histone deacetylase
activity works with
pst1+ in regulating specific
locations of
chromatin.
Potential specificity of
pst1+ versus
pst2+ is suggested by the location of the
pst1-1 mutation. The mutation in the
pst1-1 allele created a stop codon in the C terminus such that the four
conserved PAH domains were still encoded in the truncated product.
In
addition, sequence analysis of the C termini of Sin3 proteins
did not
identify any conserved motifs. One possibility is that
the deletion of
the C-terminal domain of Pst1p reduced its ability
to bind a specific
DNA-binding protein and this caused local changes
in chromatin
organization that could not be corrected by
Pst2p.
We have reported here that the mutation in the
pst1-1 allele
disrupted the nuclear localization of the Tf1-encoded Gag. Indeed,
we
found that wild-type cells, when allowed to reach stationary
phase,
imported the Tf1 Gag protein into the nucleus, whereas
a severe
mislocalization of this protein was observed in cells
with the
pst1-1 mutation. The
pst1-1 strain exhibited a
20.6-fold
increase in the number of cells with multiple aggregates of
Gag
localized exclusively in the cytoplasm. The localization of Gag
within the nucleus has been observed for some retroviruses. For
example, the matrix protein of human immunodeficiency virus (HIV)
is a
component of the preintegration complex and possesses nuclear
localization signal activity that contributes to the nuclear import
of
HIV protein in nondividing cells (
14,
15,
21,
22).
Although
there is no direct evidence that the nuclear import of
Gag is required
for Tf1 transposition, several observations suggest
the presence of Gag
in the nucleus contributes to the propagation
of Tf1. Previous analysis
of Tf1 proteins in sucrose gradients
indicated that in cells grown to
stationary phase, the bulk of
Gag and IN are coassembled into large
macromolecular complexes
called VLPs that also contain cDNA
(
41). Therefore, the results
of the sucrose gradient
fractionation and the immunolocalization
of Gag indicate that these
components are likely present together
in the nucleus as VLPs. This
colocalization suggests that Gag
and cDNA (and likely RT and IN)
were components of a complex that,
in wild-type cells, was transported
together as a unit. In addition,
our finding that the
pst1-1
mutation reduced the frequency of
cDNA recombination with plasmid
sequences supported the idea that
Gag and cDNA were both excluded
from the nucleus. In a study of
the nuclear localization of Ty1 protein
in
S. cerevisiae, mutations
in the nuclear localization
signal of IN caused substantial defects
in transposition but only
modest reductions in cDNA recombination
(
45). The
greater dependence of Ty1 transposition on IN transport
may reflect
differences in the import mechanisms of IN and cDNA.
This is in
contrast to the equivalent levels of defects in the
transposition and
cDNA recombination activities of Tf1 caused
by the
pst1-1 mutation.
We have recently shown that a mutation in a gene that encodes a
component of the nucleopore complex, Nup124p, also inhibited
the
nuclear import of Tf1 Gag (
6). In contrast to the phenotype
of the strain with the
pst1-1 mutation, in which Gag seemed
to
be distributed randomly throughout the cytoplasm, the mutation
in
the
nup124+ gene caused large aggregates of Gag
to adhere to the exterior
of the nuclear envelope. The different
phenotypes of the
pst1-1 and
nup124-1 mutations
strongly suggests that there are multiple
steps in the nuclear import
mechanism of the capsid protein
Gag.
We found that a mutation in the
pst1 gene significantly
inhibited the transposition activity of Tf1 and this lack of
transposition
correlated with a defect in the nuclear import of Tf1
Gag. One
possible explanation for these phenotypes was that the
modification
in Pst1p resulted in the altered expression of a protein,
thus
far unidentified, that participates in the transport of Tf1 into
the nucleus. This indirect contribution of
pst1 to
transposition
is consistent with the ability of the
SIN3
gene of
S. cerevisiae to regulate the expression of
many different genes (
74). In
addition, the localization of
Pst1p to the nucleus limited the
possible contributions that it could
make to the import of Gag.
However, an alternative hypothesis that may
be just as likely
as the indirect mechanism is that the mutation in the
pst1 gene
perturbed the function of the histone deacetylase
complex and
this in turn caused specific alterations in chromatin
acetylation
that directly reduced the integration efficiency of the Tf1
preintegration
complex (PIC). The influence of chromatin structure on
the integration
of retroelement cDNA has already been reported for
the integration
of some yeast retrotransposons (
18,
36,
83,
84) as well
as HIV (
56-59). Although it is reasonable
to propose that altered
histone acetylation could reduce Tf1
integration, it is more difficult
to explain how a change in chromatin
structure could change the
localization of Gag to cytoplasmic. Since
little is known about
target site selection by Tf1, the possibility
exists that, like
Ty5 (
84), Tf1 could avoid the disruption
of host genes by specifically
selecting regions of heterochromatin as
targets of insertion.
If nuclear pores contribute to the organization
of heterochromatin,
as has been suggested (
69), and if
heterochromatin mainly occupies
the perinuclear areas (
76),
the insertion of cDNA into heterochromatin
targets could be coupled
to the nuclear localization of the PIC,
as recently suggested by Boeke
and Devine (
9). In this way,
a defect in the association of
the Tf1 PIC with target sites that
possess the proper chromatin
structure could result in the accumulation
of Tf1 proteins in the
cytoplasm. An observation consistent with
this model is the appearance
of Gag fluorescence as single subnuclear
signals adjacent to the
nuclear envelope. An alternative model
that is unrelated to the
acetylation of histones is the possibility
that direct acetylation and
deacetylation of Tf1 proteins could
be responsible for their import and
activity. To reach a better
understanding of how Pst1p contributes to
Tf1 transposition, we
will undertake a detailed analysis of target site
selection as
well as an examination of Tf1 nuclear
import.
| |
ACKNOWLEDGMENTS |
We thank S. Forsburg, R. Ohi, K. Gould, and Gerald R. Smith
for providing strains and plasmids. We also thank B. Turner, P. Young,
and A. Wolffe for providing the S. pombe library and antibodies.
K.E. was supported by MFR project grant 11821.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Eukaryotic Gene Regulation, National Institute of Child Health and
Human Development, National Institutes of Health, Bethesda, MD
20892-2780. Phone: (301) 402-4281. Fax: (301) 496-8576. E-mail:
Henry_Levin{at}nih.gov.
Present address: University of Houston, Houston, TX 77204-5934.
Present address: Karolinska Institutet, Department of Biosciences,
Novum, S-141 57 Huddinge, Sweden.
| |
REFERENCES |
| 1.
|
Alland, L.,
R. Muhle,
H. Hou, Jr.,
J. Potes,
L. Chin,
N. Schreiber-Agus, and R. A. DePinho.
1997.
Role for N-CoR and histone deacetylase in Sin3-mediated transcriptional repression.
Nature
387:49-55[Medline].
|
| 2.
|
Allshire, R. C.,
E. R. Nimmo,
K. Ekwall,
J. P. Javerzat, and G. Cranston.
1995.
Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation.
Genes Dev.
9:218-233[Abstract/Free Full Text].
|
| 3.
|
Atwood, A.,
J. Choi, and H. L. Levin.
1998.
The application of a homologous recombination assay revealed amino acid residues in an LTR-retrotransposon that were critical for integration.
J. Virol.
72:1324-1333[Abstract/Free Full Text].
|
| 4.
|
Atwood, A.,
J. Lin, and H. Levin.
1996.
The retrotransposon Tf1 assembles virus-like particles with excess Gag relative to integrase because of a regulated degradation process.
Mol. Cell. Biol.
16:338-346[Abstract].
|
| 5.
|
Ayer, D. E.,
Q. A. Lawrence, and R. N. Eisenman.
1995.
Mad-Max transcriptional repression is mediated by ternary complex formation with mammalian homologs of yeast repressor Sin3.
Cell
80:767-776[Medline].
|
| 6.
| Balasundaram, D., M. Benedik, V.-D. Dang, and
H. Levin. Nup124p is a nuclear pore factor of Schizosaccharomyces
pombe that possesses a specialized function required for the nuclear
import of the retrotransposon Tf1. Submitted for publication.
|
| 7.
|
Belyaev, N. D.,
A. M. Keohane, and B. M. Turner.
1996.
Histone H4 acetylation and replication timing in Chinese hamster chromosomes.
Exp. Cell Res.
225:277-285[Medline].
|
| 8.
|
Bestor, T. H.
1998.
Gene silencing. Methylation meets acetylation.
Nature
393:311-312[Medline].
|
| 9.
|
Boeke, J. D., and S. E. Devine.
1998.
Yeast retrotransposon: finding a nice quiet neighborhood.
Cell
93:1087-1089[Medline].
|
| 10.
|
Boeke, J. D., and J. P. Stoye.
1997.
Retrotransposons, endogenous retroviruses, and the evolution of retroelement.
Cold Spring Harbor Laboratory Press, Plainview, N.Y.
|
| 11.
|
Boeke, J. D.,
J. Trueheart,
G. Natsoulis, and G. R. Fink.
1987.
5-Fluoro-orotic acid as a selective agent in yeast molecular genetics.
Methods Enzymol.
154:164-175[Medline].
|
| 12.
|
Braun, B. R., and A. D. Johnson.
1997.
Control of filament formation in Candida albicans by the transcriptional repressor TUP1.
Science
277:105-109[Abstract/Free Full Text].
|
| 13.
|
Brownell, J. E., and C. D. Allis.
1996.
Special HATs for special occasions: linking histone acetylation to chromatin assembly and gene activation.
Curr. Opin. Genet. Dev.
6:176-184[Medline].
|
| 14.
|
Bukrinsky, M. I.,
S. Haggerty,
M. P. Dempsey,
N. Sharova,
A. Adzhubel,
L. Spitz,
P. Lewis,
D. Goldfarb,
M. Emerman, and M. Stevenson.
1993.
A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells.
Nature
365:666-669[Medline].
|
| 15.
|
Bukrinsky, M. I.,
N. Sharova,
T. L. McDonald,
T. Pushkarskaya,
W. G. Tarpley, and M. Stevenson.
1993.
Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection.
Proc. Natl. Acad. Sci. USA
90:6125-6129[Abstract/Free Full Text].
|
| 16.
|
Cottarel, G.,
D. Beach, and U. Deuschle.
1993.
Two new multi-purpose multicopy Schizosaccharomyces pombe shuttle vectors, pSP1 and pSP2.
Curr. Genet.
23:547-548[Medline].
|
| 17.
|
Dang, V. D.,
M. Valens,
M. Bolotin-Fukuhara, and B. Daignan-Fornier.
1994.
A genetic screen to isolate genes regulated by the yeast CCAAT-box binding-protein Hap2p.
Yeast
10:1273-1283[Medline].
|
| 18.
|
Devine, S. E., and J. D. Boeke.
1996.
Integration of the yeast retrotransposon Ty1 is targeted to regions upstream of genes transcribed by RNA polymerase III.
Genes. Dev.
10:620-633[Abstract/Free Full Text].
|
| 19.
|
Ekwall, K.,
J. P. Javerzat,
A. Lorentz,
H. Schmidt,
G. Cranston, and R. Allshire.
1995.
The chromodomain protein Swi6: a key component at fission yeast centromeres.
Science
269:1429-1431[Abstract/Free Full Text].
|
| 19a.
| Ekwall, K., and R. C. Allshire. Unpublished
data.
|
| 20.
|
Ekwall, K.,
T. Olsson,
B. M. Turner,
G. Cranston, and R. C. Allshire.
1997.
Transient inhibition of histone deacetylation alters the structural and functional imprint at fission yeast centromeres.
Cell
91:1021-1032[Medline].
|
| 21.
|
Gallay, P.,
V. Stitt,
C. Mundy,
M. Oettinger, and D. Trono.
1996.
Role of the karyopherin pathway in human immunodeficiency virus type 1 nuclear import.
J. Virol.
70:1027-1032[Abstract].
|
| 22.
|
Gallay, P.,
S. Swingler,
J. Song,
F. Bushman, and D. Trono.
1995.
HIV nuclear import is governed by the phosphotyrosine-mediated binding of matrix to the core domain of integrase.
Cell
83:569-576[Medline].
|
| 23.
|
Goebl, M., and M. Yanagida.
1991.
The TPR snap helix: a novel protein repeat motif from mitosis to transcription.
Trends Biochem. Sci.
16:173-177[Medline].
|
| 24.
|
Grunstein, M.
1997.
Histone acetylation in chromatin structure and transcription.
Nature
389:349-352[Medline].
|
| 25.
|
Guacci, V.,
E. Hogan, and D. Koshland.
1994.
Chromosome condensation and sister chromatid pairing in budding yeast.
J. Cell Biol.
125:517-530[Abstract/Free Full Text].
|
| 26.
|
Hanes, S. D., and R. Brent.
1989.
DNA specificity of the bicoid activator protein is determined by homeodomain recognition helix residue 9.
Cell
57:1275-1283[Medline].
|
| 27.
|
Hassig, C. A.,
J. K. Tong,
T. C. Fleischer,
T. Owa,
P. G. Grable,
D. E. Ayer, and S. L. Schreiber.
1998.
A role for histone deacetylase activity in HDAC1-mediated transcriptional repression.
Proc. Natl. Acad. Sci. USA
95:3519-3524[Abstract/Free Full Text].
|
| 28.
|
Heinzel, T.,
R. M. Lavinsky,
T. M. Mullen,
M. Soderstrom,
C. D. Laherty,
J. Torchia,
W. M. Yang,
G. Brard,
S. D. Ngo,
J. R. Davie,
E. Seto,
R. N. Eisenman,
D. W. Rose,
C. K. Glass, and M. G. Rosenfeld.
1997.
A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression.
Nature
387:43-48[Medline].
|
| 29.
|
Hirano, T.,
N. Kinoshita,
K. Morikawa, and M. Yanagida.
1990.
Snap helix with knob and hole: essential repeats in S. pombe nuclear protein nuc2+.
Cell
60:319-328[Medline].
|
| 30.
|
Jones, P. L.,
G. J. Veenstra,
P. A. Wade,
D. Vermaak,
S. U. Kass,
N. Landsberger,
J. Strouboulis, and A. P. Wolffe.
1998.
Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription.
Nat. Genet.
19:187-191[Medline].
|
| 31.
|
Kadosh, D., and K. Struhl.
1998.
Histone deacetylase activity of Rpd3 is important for transcriptional repression in vivo.
Genes. Dev.
12:797-805[Abstract/Free Full Text].
|
| 32.
|
Kadosh, D., and K. Struhl.
1997.
Repression by Ume6 involves recruitment of a complex containing Sin3 corepressor and Rpd3 histone deacetylase to target promoters.
Cell
89:365-371[Medline].
|
| 33.
|
Kasten, M. M.,
S. Dorland, and D. J. Stillman.
1997.
A large protein complex containing the yeast Sin3p and Rpd3p transcriptional regulators.
Mol. Cell. Biol.
17:4852-4858[Abstract].
|
| 34.
|
Keeney, J. B., and J. D. Boeke.
1994.
Efficient targeted integration at leu1-32 and ura4-294 in Schizosaccharomyces pombe.
Genetics
136:849-856[Abstract].
|
| 35.
|
Kingston, R. E.,
C. A. Bunker, and A. N. Imbalzano.
1996.
Repression and activation by multiprotein complexes that alter chromatin structure.
Genes. Dev.
10:905-920[Abstract/Free Full Text].
|
| 36.
|
Kirchner, J.,
C. M. Connolly, and S. B. Sandmeyer.
1995.
Requirement of RNA polymerase III transcription factors for in vitro position-specific integration of a retroviruslike element.
Science
267:1488-1491[Abstract/Free Full Text].
|
| 37.
|
Laherty, C. D.,
W. M. Yang,
J. M. Sun,
J. R. Davie,
E. Seto, and R. N. Eisenman.
1997.
Histone deacetylases associated with the mSin3 corepressor mediate mad transcriptional repression.
Cell
89:349-356[Medline].
|
| 38.
|
Levin, H. L.
1995.
A novel mechanism of self-primed reverse transcription defines a new family of retroelements.
Mol. Cell. Biol.
15:3310-3317[Abstract].
|
| 39.
|
Levin, H. L.
1996.
An unusual mechanism of self-primed reverse transcription requires the RNase H domain of reverse transcriptase to cleave an RNA duplex.
Mol. Cell. Biol.
16:5645-5654[Abstract].
|
| 40.
|
Levin, H. L., and J. D. Boeke.
1992.
Demonstration of retrotransposition of the Tf1 element in fission yeast.
EMBO J.
11:1145-1153[Medline].
|
| 41.
|
Levin, H. L.,
D. C. Weaver, and J. D. Boeke.
1993.
Novel gene expression mechanism in a fission yeast retroelement: Tf1 proteins are derived from a single primary translation product.
Embo. J.
12:4885-4895[Medline]. (Erratum, 13:1494, 1994.)
|
| 42.
|
Levin, H. L.,
D. C. Weaver, and J. D. Boeke.
1990.
Two related families of retrotransposons from Schizosaccharomyces pombe.
Mol. Cell. Biol.
10:6791-6798[Abstract/Free Full Text].
|
| 43.
|
Mertins, P., and D. Gallwitz.
1987.
Nuclear pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe strictly requires an intron-contained, conserved sequence element.
Embo J.
6:1757-1763[Medline].
|
| 44.
|
Mizzen, C. A., and C. D. Allis.
1998.
Linking histone acetylation to transcriptional regulation.
Cell. Mol. Life Sci.
54:6-20[Medline].
|
| 45.
|
Moore, S. P.,
L. A. Rinckel, and D. J. Garfinkel.
1998.
A Ty1 integrase nuclear localization signal required for retrotransposition.
Mol. Cell. Biol.
18:1105-1114[Abstract/Free Full Text].
|
| 46.
|
Moreno, S.,
A. Klar, and P. Nurse.
1991.
Molecular genetic analysis of fission yeast Schizosaccharomyces pombe.
Methods Enzymol.
194:795-823[Medline].
|
| 47.
|
Murre, C.,
P. S. McCaw, and D. Baltimore.
1989.
A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins.
Cell
56:777-783[Medline].
|
| 48.
|
Nagy, L.,
H. Y. Kao,
D. Chakravarti,
R. J. Lin,
C. A. Hassig,
D. E. Ayer,
S. L. Schreiber, and R. M. Evans.
1997.
Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase.
Cell
89:373-380[Medline].
|
| 49.
|
Nan, X.,
H. H. Ng,
C. A. Johnson,
C. D. Laherty,
B. M. Turner,
R. N. Eisenman, and A. Bird.
1998.
Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex.
Nature
393:386-389[Medline].
|
| 50.
|
Nasmyth, K.,
D. Stillman, and D. Kipling.
1987.
Both positive and negative regulators of HO transcription are required for mother-cell-specific mating-type switching in yeast.
Cell
48:579-587[Medline].
|
| 51.
|
Odds, F. C.
1994.
Pathogenesis of Candida infections.
J. Am. Acad. Dermatol.
31:S2-S5[Medline].
|
| 52.
|
Ohi, R.,
A. Feoktistova, and K. L. Gould.
1996.
Construction of vectors and a genomic library for use with his3-deficient strains of Schizosaccharomyces pombe.
Gene
174:315-318[Medline].
|
| 53.
|
Olsson, T. G. S.,
K. Ekwall,
R. C. Allshire,
P. Sunnerhagen,
J. F. Partridge, and W. A. Richardson.
1998.
Genetic characterisation of hda1+, a putative fission yeast histone deacetylase gene.
Nucleic Acids Res.
26:3247-3254[Abstract/Free Full Text].
|
| 54.
|
Pazin, M. J., and J. T. Kadonaga.
1997.
What's up and down with histone deacetylation and transcription?
Cell
89:325-328[Medline].
|
| 55.
|
Ponticelli, A. S., and G. R. Smith.
1992.
Chromosomal context dependence of a eukaryotic recombinational hot spot.
Proc. Natl. Acad. Sci. USA
89:227-231[Abstract/Free Full Text].
|
| 56.
|
Pruss, D.,
F. D. Bushman, and A. P. Wolffe.
1994.
Human immunodeficiency virus integrase directs integration to sites of severe DNA distortion within the nucleosome core.
Proc. Natl. Acad. Sci. USA
91:5913-5917[Abstract/Free Full Text].
|
| 57.
|
Pruss, D.,
R. Reeves,
F. D. Bushman, and A. P. Wolffe.
1994.
The influence of DNA and nucleosome structure on integration events directed by HIV integrase.
J. Biol. Chem.
269:25031-25041[Abstract/Free Full Text].
|
| 58.
|
Pryciak, P. M.,
A. Sil, and H. E. Varmus.
1992.
Retroviral integration into minchromosomes in vitro.
EMBO J.
11:291-303[Medline].
|
| 59.
|
Pryciak, P. M., and H. E. Varmus.
1992.
Nucleosomes, DNA binding proteins, and DNA sequence modulate retroviral integration target site selection.
Cell
69:769-780[Medline].
|
| 60.
|
Rattray, A. J., and L. S. Symington.
1994.
Use of a chromosomal inverted repeat to demonstrate that the RAD51 and RAD52 genes of Saccharomyces cerevisiae have different roles in mitotic recombination.
Genetics
138:587-595[Abstract].
|
| 61.
|
Roth, S. Y., and C. D. Allis.
1996.
Histone acetylation and chromatin assembly: a single escort, multiple dances?
Cell
87:5-8[Medline].
|
| 62.
|
Rundlett, S. E.,
A. A. Carmen,
N. Suka,
B. M. Turner, and M. Grunstein.
1998.
Transcriptional repression by UME6 involves deacetylation of lysine 5 of histone H4 by RPD3.
Nature
392:831-835[Medline].
|
| 63.
|
Sandmeyer, S. B.
1992.
Yeast retrotransposons.
Curr. Opin. Genet. Dev.
2:705-711[Medline].
|
| 64.
|
Schreiber-Agus, N.,
L. Chin,
K. Chen,
R. Torres,
G. Rao,
P. Guida,
A. I. Skoultchi, and R. A. DePinho.
1995.
An amino-terminal domain of Mxi1 mediates anti-Myc oncogenic activity and interacts with a homolog of the yeast transcriptional repressor SIN3.
Cell
80:777-786[Medline].
|
| 65.
|
Schuler, G. D.,
S. F. Altschul, and D. J. Lipman.
1991.
A workbench for multiple alignment construction and analysis.
Proteins
9:180-190[Medline].
|
| 66.
|
Sikorski, R. S.,
M. S. Boguski,
M. Goebl, and P. Hieter.
1990.
A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis.
Cell
60:307-317[Medline].
|
| 67.
|
Sternberg, P. W.,
M. J. Stern,
I. Clark, and I. Herskowitz.
1987.
Activation of the yeast HO gene by release from multiple regulators.
Cell
48:567-577[Medline].
|
| 68.
|
Struhl, K.
1998.
Histone acetylation and transcriptional regulatory mechanisms.
Genes Dev.
12:599-606[Free Full Text].
|
| 69.
|
Sukegawa, J., and G. Blobel.
1993.
A nuclear pore complex protein that contains zinc finger motifs, binds DNA, and faces the nucleoplasm.
Cell
72:29-38[Medline].
|
| 70.
|
Towbin, H.,
T. Staehelin, and J. Gordon.
1979.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Natl. Acad. Sci. USA
76:4350-4354[Abstract/Free Full Text].
|
| 71.
|
Turner, B. M.,
A. J. Birley, and J. Lavender.
1992.
Histone H4 isoforms acetylated at specific lysine residues define individual chromosomes and chromatin domains in Drosophila polytene nuclei.
Cell
69:375-384[Medline].
|
| 72.
|
Turner, B. M., and L. P. O'Neill.
1995.
Histone acetylation in chromatin and chromosomes.
Semin. Cell Biol.
6:229-236[Medline].
|
| 73.
|
Vidal, M.,
A. M. Buckley,
F. Hilger, and R. F. Gaber.
1990.
Direct selection for mutants with increased K+ transport in Saccharomyces cerevisiae.
Genetics
125:313-320[Abstract].
|
| 74.
|
Vidal, M.,
R. Strich,
R. E. Esposito, and R. F. Gaber.
1991.
RPD1 (SIN3/UME4) is required for maximal activation and repression of diverse yeast genes.
Mol. Cell. Biol.
11:6306-6316[Abstract/Free Full Text].
|
| 75.
|
Voronova, A., and D. Baltimore.
1990.
Mutations that disrupt DNA binding and dimer formation in the E47 helix-loop-helix protein map to distinct domains.
Proc. Natl. Acad. Sci. USA
87:4722-4726[Abstract/Free Full Text].
|
| 76.
|
Wakimoto, B. T.
1998.
Beyond the nucleosome: epigenetic aspects of position-effect variegation in Drosophila.
Cell
93:321-324[Medline].
|
| 77.
|
Wang, H.,
I. Clark,
P. R. Nicholson,
I. Herskowitz, and D. J. Stillman.
1990.
The Saccharomyces cerevisiae SIN3 gene, a negative regulator of HO, contains four paired amphipathic helix motifs.
Mol. Cell. Biol.
10:5927-5936[Abstract/Free Full Text].
|
| 78.
|
Wang, H., and D. J. Stillman.
1993.
Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein.
Mol. Cell. Biol.
13:1805-1814[Abstract/Free Full Text].
|
| 79.
|
Wolffe, A. P.
1997.
Transcriptional control. Sinful repression.
Nature
387:16-17[Medline].
|
| 80.
|
Wolffe, A. P.,
D. Pruss,
B. M. Turner, and L. P. O'Neill.
1996.
Targeting chromatin disruption: transcription regulators that acetylate histones.
Cell
84:817-819[Medline].
|
| 81.
|
Yang, W. M.,
C. Inouye,
Y. Zeng,
D. Bearss, and E. Seto.
1996.
Transcriptional repression by YY1 is mediated by interaction with a mammalian homolog of the yeast global regulator RPD3.
Proc. Natl. Acad. Sci. USA
93:12845-12850[Abstract/Free Full Text].
|
| 82.
|
Yoshida, M.,
S. Horinouchi, and T. Beppu.
1995.
Trichostatin A and trapoxin: novel chemical probes for the role of histone acetylation in chromatin structure and function.
Bioessays
17:423-430[Medline].
|
| 83.
|
Zou, S.,
N. Ke,
J. M. Kim, and D. F. Voytas.
1996.
The Saccharomyces retrotransposon Ty5 integrates preferentially into regions of silent chromatin at the telomeres and mating loci.
Genes Dev.
10:634-645[Abstract/Free Full Text].
|
| 84.
|
Zou, S., and D. F. Voytas.
1997.
Silent chromatin determines target preference of the Saccharomyces retrotransposon Ty5.
Proc. Natl. Acad. Sci. USA
94:7412-7416[Abstract/Free Full Text].
|
Molecular and Cellular Biology, March 1999, p. 2351-2365, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Cowley, S. M., Iritani, B. M., Mendrysa, S. M., Xu, T., Cheng, P. F., Yada, J., Liggitt, H. D., Eisenman, R. N.
(2005). The mSin3A Chromatin-Modifying Complex Is Essential for Embryogenesis and T-Cell Development. Mol. Cell. Biol.
25: 6990-7004
[Abstract]
[Full Text]
-
Kim, M.-K., Claiborn, K. C., Levin, H. L.
(2005). The Long Terminal Repeat-Containing Retrotransposon Tf1 Possesses Amino Acids in Gag That Regulate Nuclear Localization and Particle Formation. J. Virol.
79: 9540-9555
[Abstract]
[Full Text]
-
Dannenberg, J.-H., David, G., Zhong, S., van der Torre, J., Wong, W. H., DePinho, R. A.
(2005). mSin3A corepressor regulates diverse transcriptional networks governing normal and neoplastic growth and survival. Genes Dev.
19: 1581-1595
[Abstract]
[Full Text]
-
Griffith, J. L., Coleman, L. E., Raymond, A. S., Goodson, S. G., Pittard, W. S., Tsui, C., Devine, S. E.
(2003). Functional Genomics Reveals Relationships Between the Retrovirus-Like Ty1 Element and Its Host Saccharomyces cerevisiae. Genetics
164: 867-879
[Abstract]
[Full Text]
-
Teysset, L., Dang, V.-D., Kim, M. K., Levin, H. L.
(2003). A Long Terminal Repeat-Containing Retrotransposon of Schizosaccharomyces pombe Expresses a Gag-Like Protein That Assembles into Virus-Like Particles Which Mediate Reverse Transcription. J. Virol.
77: 5451-5463
[Abstract]
[Full Text]
-
Huang, Y.
(2002). Transcriptional silencing in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Nucleic Acids Res
30: 1465-1482
[Abstract]
[Full Text]
-
Singleton, T. L., Levin, H. L.
(2002). A Long Terminal Repeat Retrotransposon of Fission Yeast Has Strong Preferences for Specific Sites of Insertion. Eukaryot Cell
1: 44-55
[Abstract]
[Full Text]
-
Gonzalez-Lopez, C. I., Szabo, R., Blanchin-Roland, S., Gaillardin, C.
(2002). Genetic Control of Extracellular Protease Synthesis in the Yeast Yarrowia lipolytica. Genetics
160: 417-427
[Abstract]
[Full Text]
-
Behrens, R., Hayles, J., Nurse, P.
(2000). Fission yeast retrotransposon Tf1 integration is targeted to 5' ends of open reading frames. Nucleic Acids Res
28: 4709-4716
[Abstract]
[Full Text]
-
Dang, V.-D., Levin, H. L.
(2000). Nuclear Import of the Retrotransposon Tf1 Is Governed by a Nuclear Localization Signal That Possesses a Unique Requirement for the FXFG Nuclear Pore Factor Nup124p. Mol. Cell. Biol.
20: 7798-7812
[Abstract]
[Full Text]
-
Balasundaram, D., Benedik, M. J., Morphew, M., Dang, V.-D., Levin, H. L.
(1999). Nup124p Is a Nuclear Pore Factor of Schizosaccharomyces pombe That Is Important for Nuclear Import and Activity of Retrotransposon Tf1. Mol. Cell. Biol.
19: 5768-5784
[Abstract]
[Full Text]
-
Huang, Y., Hamada, M., Maraia, R. J.
(2000). Isolation and Cloning of Four Subunits of a Fission Yeast TFIIIC Complex That Includes an Ortholog of the Human Regulatory Protein TFIIICbeta. J. Biol. Chem.
275: 31480-31487
[Abstract]
[Full Text]
Top
Abstract
Introduction
