Previous Article | Next Article 
Molecular and Cellular Biology, October 1998, p. 6110-6120, Vol. 18, No. 10
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Functional Characterization of the N Terminus
of Sir3p
Monica
Gotta,
Francesca
Palladino,
and
Susan M.
Gasser*
Swiss Institute for Experimental Cancer
Research, CH-1066 Epalinges/Lausanne, Switzerland
Received 19 March 1998/Returned for modification 6 May
1998/Accepted 9 July 1998
 |
ABSTRACT |
Silent information regulator 3 is an essential component of the
Saccharomyces cerevisiae silencing complex that functions at telomeres and the silent mating-type loci, HMR and
HML. We show that expression of the N- and
C-terminal-encoding halves of SIR3 in trans
partially complements the mating defect of the sir3 null
allele, suggesting that the two domains have distinct functions. We
present here a functional characterization of these domains. The
N-terminal domain (Sir3N) increases both the frequency and extent of
telomere-proximal silencing when expressed ectopically in
SIR+ yeast strains, although we are unable to
detect interaction between this domain and any known components of the
silencing machinery. In contrast to its effect at telomeres, Sir3N
overexpression derepresses transcription of reporter genes inserted in
the ribosomal DNA (rDNA) array. Immunolocalization of Sir3N-GFP and
Sir2p suggests that Sir3N directly antagonizes nucleolar Sir2p,
releasing an rDNA-bound population of Sir2p so that it can enhance
repression at telomeres. Overexpression of the C-terminal domain of
either Sir3p or Sir4p has a dominant-negative effect on telomeric
silencing. In strains overexpressing the C-terminal domain of
Sir4p, elevated expression of either full-length Sir3p or Sir3N
restores repression and the punctate pattern of Sir3p and Rap1p
immunostaining. The similarity of Sir3N and Sir3p
overexpression phenotypes suggests that Sir3N acts as an allosteric
effector of Sir3p, either enhancing its interactions
with other silencing components or liberating the full-length protein
from nonfunctional complexes.
| |
INTRODUCTION |
Chromatin structure plays an
important role in the regulation of gene expression in the eukaryotic
cell. Cytological studies have long suggested that different domains of
the genome are packaged into two structurally different types of
chromatin, heterochromatin and euchromatin (48). In contrast
to euchromatin, heterochromatin is rich in repetitive sequences
and remains constitutively condensed throughout the cell cycle. In
higher eukaryotic cells, these regions are generally poor in coding
sequences and are replicated late in S phase, while most
transcriptionally active genes are located in early replicating,
euchromatic regions of the genome. Important for the genetic
characterization of heterochromatin was the observation that it
influences the transcription of genes transposed nearby. The
resulting variegated repression of the euchromatic gene is known
as position effect variegation (reviewed in reference
20).
Despite the absence of a cytologically visible heterochromatin, the
yeast Saccharomyces cerevisiae has distinct chromosomal regions which, like heterochromatin, confer a heritable state of
transcriptional repression on otherwise functional promoters. Repression at the silent mating-type loci, HML and
HMR (hereafter collectively called HM loci), is
stable, while repression of RNA PolII genes integrated near the
telomeric TG1-3 repeat is variegated (called
telomeric position effect [TPE]), much like the stochastic patterns
of repression observed near centromeric heterochromatin in flies.
In yeast, both these domains are less accessible to nucleolytic and
methylase modification, and they contain a histone, H4, which is
underacetylated on lysines 5 and 16 (reviewed in reference
32). In telomeric regions, the repressed chromatin
state spreads along the chromosome, limited by the dosage of essential
components, again reminiscent of the spread of centric heterochromatin
in flies (16, 33). More recently, variegated expression has
also been noted for reporter genes inserted into the tandemly repeated
ribosomal DNA (rDNA) locus of yeast (4, 40).
A number of proteins are required for both telomeric and mating-type
locus repression. These include repressor activator protein 1 (Rap1p),
the silent information regulators Sir2 to -4, and the N termini of
histones H3 and H4 (1, 17, 21, 23, 34, 44, 45). Of these,
only Rap1p binds telomeric DNA directly, while Sir3p and Sir4p are both
able to form homo- and heteromultimeric complexes (27, 29)
that interact with the N termini of histones H3 and H4 (15).
Combined immunofluorescence and in situ-hybridization experiments have
shown that telomeres are clustered and that Rap1p, Sir3p, and Sir4p
colocalize with telomeric foci in wild-type cells (9).
Immunoprecipitation and cross-linking data confirm that Sir3p, Sir4p,
histones, and Rap1p can be coimmunoprecipitated with subtelomeric DNA
in wild-type cell extracts (16, 43). Sir2p is also part of
this complex and can be cross-linked to telomeric chromatin through its
interaction with Sir4p (43).
In addition to its telomeric localization, Sir2p was shown to be
constitutively bound to the rDNA in a manner independent of Sir3p and
Sir4p (10). This is consistent with the observation that the
variegated repression of a PolII gene inserted in the rDNA repeats, as
well as repression of recombination between rDNA repeats, requires
SIR2 but not SIR3 or SIR4 (11,
40). The presence of Sir2p in the nucleolus suggests a direct
effect on rDNA chromatin, perhaps through modulation of nucleosomal
organization within the RNA PolI or PolIII promoter regions (4, 8,
40). In aging yeast cells, or in strains carrying mutant forms of
Sir4p, Sir3p also relocalizes from telomeres to the nucleolar
compartment (10, 22). Although the function of Sir3p in the
nucleolus is unclear, Sir proteins do affect nuclear events other than
HM and telomeric silencing: mitotic recombination increases
fourfold in sir3-deficient strains, mitotic chromosome loss
increases fivefold in sir4-deficient strains
(31), and deletion of either SIR2, SIR3, or SIR4 increases sensitivity of a
rad52 strain to ionizing radiation (47).
Sir3p plays a unique and central role in chromatin-mediated repression.
Although Sir3p and Sir4p are present in approximately equimolar amounts
(7), only Sir3p is limiting for the propagation of telomeric
silencing (33). In SIR+ cells, TPE
represses genes up to 4 kb from the telomere (core heterochromatin),
while in cells overexpressing SIR3, telomeric repression
extends roughly 20 kb from the telomeric TG1-3 repeat,
coinciding with the spread of Sir3p along the repressed chromatin
(16, 33, 43). The propagation of Sir3p is presumably mediated by interaction of its C-terminal domain with histone tails
(15).
The N-terminal 214 amino acids (aa) of Sir3p have over 50% identity
with the N terminus of Orc1p, the largest subunit of the origin
recognition complex, and gene fusion experiments indicate that the N
terminus of Orc1p can functionally substitute for that of Sir3p
(2). Moreover, several point mutations in the N-terminal domain of Sir3p suppress silencing-deficient mutants in Rap1p and the N
termini of histones H3 and H4, although these domains do not interact
directly (15, 18, 24). Here we report that the ectopic
expression of an N-terminal region (aa 1 to 503, hereafter called
Sir3N) enhances TPE, while that of the C-terminal domain (aa 568 to 978 [Sir3C]) derepresses silencing. Expression of this Sir3N fragment in
the presence of full-length Sir3p also extends telomere-proximal
repression. In a strain overexpressing the C terminus of Sir4p,
elevated expression of Sir3N, like that of full-length Sir3p, is able
to restore both silencing and the punctate staining patterns of Sir3p
and Rap1p.
In contrast to its effect at telomeres, overexpression of Sir3N
derepresses a URA3 reporter gene inserted within the rDNA repeat. Localization of a Sir3N-GFP fusion protein indicates that it
accumulates in the nucleolus in a Sir2p-dependent manner. Intriguingly, Sir3N overexpression leads to enhanced Sir2p staining at telomeres, coincident with the improvement in telomeric silencing, although we
detect no direct interactions between Sir3N and Sir2p, nor between
Sir3N and Sir4C. The hypothesis most consistent with the available data
is that Sir3N counteracts the Sir4C-induced derepression and extends
TPE by acting as an allosteric effector of full-length Sir3p.
| |
MATERIALS AND METHODS |
Plasmid construction.
Standard molecular biology techniques
were used, following published protocols (37). pADH-SIR4 was
described previously (7). pADH-SIR4C, used in this study,
was constructed by replacing the LEU2 gene of pADH-SIR4C
(7) with a HIS3 gene. pADH-SIR3N was constructed
by subcloning a 1.5-kb BamHI-HindIII fragment of SIR3 into the HindIII site of vector pAAH5
after filling in overhangs. The 5' BamHI site of
SIR3 was engineered as described previously (31).
pADH-SIR3 was described earlier as p2µ-ASir3 (26).
pADH-SIR3C was constructed by subcloning a
HindIII-HindIII 2.7-kb fragment into the
HindIII site of vector pAAH5. pMG17 was constructed by
subcloning a 3-kb BglII-BamHI fragment of
SIR3 into the BamHI site of vector p423ADH
(30). The pSIR3N-GFP1 and pSIR3N-GFP2 plasmids were
constructed by cloning a 1.5-kb BamHI-HindIII
fragment of SIR3 as before and a
HindIII-EcoRI fragment containing GFP (pGFP;
Clontech) into p414ADH and p424ADH, respectively (30). For
two-hybrid assays DNA binding domains were fused both before and after
the Sir3N open reading frame. N-terminal fusions lost the phenotypes
associated with SIR3N expression, suggesting that they fold improperly.
Therefore, for all SIR3 constructs used in this paper the N
terminus was kept in its native state.
Yeast media and strains.
All yeast strains are described in
Table 1. UCC18, YHR434, YHR440, and
YHR441 are isogenic, and UCC18 was described previously (1).
UCC518, UCC520, and UCC522 are isogenic (33), as are UCC3107, UCC3203, and UCC3207 (10, 42). Standard media were used for the growth of S. cerevisiae (12); all
cultures were grown at 30°C. Yeast transformation was performed by
the lithium acetate procedure (38), and other manipulations
were as described previously (35).
Repression assays.
The expression of the telomere-proximal
URA3 was monitored by determining the fraction of cells
capable of growth on 5 fluoro-orotic acid (5-FOA)-containing medium,
which allows the growth of ura3
cells but not
URA3+ cells (3, 12). The cells were
grown for 3 to 5 days on selective medium at 30°C. Isolated colonies
were resuspended in water, and 10-fold serial dilutions were spotted
onto synthetic selective medium and onto the same medium containing
0.1% 5-FOA (12). 5-FOA resistance was determined as the
average ratio of colonies formed on 5-FOA medium to colonies formed on
selective medium. The number of colonies for each spot was determined
after 3 to 4 days of growth at 30°C. Several independent
transformants were tested, and the mean was calculated either directly
(usually with eight independent colonies; performed in triplicate) or
as described for standard fluctuation tests (discarding high and low
extremes and taking the mean). We observed no significant differences
in the values thus obtained.
Individual colonies carrying the
ade2-1 mutation and the
wild-type
ADE2 gene adjacent to the left telomere of
chromosome VII
or the right arm of chromosome V (
12,
23,
33)
were streaked
onto medium containing 10 mg of adenine/liter. Within
single yeast
colonies, the appearance of red and white sectors
indicates metastable
repression of the telomeric
ADE2.
Colonies were grown for 3 to
5 days at 30°C and then stored for 1 to
2 weeks at 4°C for pigment
accumulation.
Repression of a
URA3 reporter with a mutated promoter
inserted at the rDNA
[
RDN1::(m
URA3-HIS3)] was measured as
follows: colonies
from the JS231 strain transformed with either the
vector or the
plasmid overexpressing Sir3N, Sir3C, or full-length Sir3p
were
grown for 3 to 5 days at 30°C were resuspended in water. Tenfold
serial dilutions were spotted onto selective media lacking leucine
(to
ensure maintenance of the plasmid) and lacking both leucine
and uracil
to measure repression of the rDNA
URA3 gene. Derepression
of
URA3 results in bigger colonies after 3 days at 30°C.
Quantitative mating.
Quantitative mating was performed
essentially as described previously (41). Strains UCC3107,
the isogenic sir3::TRP1 (GA822), and
the 
tester strain PT2 were grown to a density of 5.0 × 106 to 1.5 × 107 cells per ml in
selective medium lacking leucine and histidine to ensure maintenance of
plasmids. The cells (2.0 × 106) were mixed with
107 cells of the tester strain, collected on a
0.8-µm-pore-size, 25-mm-diameter nitrocellulose filter disk (type AA;
Millipore), and allowed to mate for 6 h at 30°C. The cells were
resuspended in water and sonicated for 5 to 10 s to disperse
clumps. Tenfold serial dilutions were plated on minimal medium to
measure the titer of a/ diploids and on minimal medium
complemented with adenine, tryptophan, and uracil, which allows the
growth of both a/ diploids and the a haploid.
Mating efficiency is expressed as the titer of a/ cells
divided by the titer of a/ cells plus a cells.
Immunofluorescence and antibodies.
Immunofluorescence assays
were performed as described previously (9), using
affinity-purified rabbit antibodies. The anti-Sir3p antibody was raised
against the C-terminal 537 aa of Sir3p. The affinity-purified rabbit
anti-GFP antibody was a kind gift of K. Sawin (Imperial Cancer Research
Fund, London, England) and was used at a 1:800 dilution. Secondary
antibodies coupled to the fluorochrome 5-([4,6-dichlorotriazin-2-yl]
(amino)-fluorescein (DTAF), and CY3 and GFP fluorescence, were
visualized on a Zeiss Axiovert 100 microscope (Zeiss laser scanning
microscope 410) with a 63× Plan-Apochromat objective (1.4 oil) as
previously described (9). Under standard imaging conditions
no signal from one fluorochrome could be detected on the other filter
set. Standardized conditions for the image capture and subtraction of a
background value (about 15% of the maximum signal) were carried out
uniformly on all images.
| |
RESULTS |
Sir3N restores TPE in strains overexpressing Sir4C.
Overexpression of full-length Sir4p or its C-terminal domain (aa 743 to
1358 [Sir4C]) relieves silencing at HM and telomeric loci
(7, 27). This may result either from disruption of the Sir2p-Sir3p-Sir4p complex or from the titration of an unknown, yet
essential, silencing component. Coincident with the loss of silencing,
the punctate staining patterns of Rap1p and Sir3p are disrupted upon
Sir4C overexpression and Sir proteins are found diffused throughout the
nucleus (7). We reasoned that if overexpression of Sir4C
competes for the assembly of the multicomponent complex required for
silencing, then overexpression of the limiting component might restore
TPE.
To identify this limiting component, we screened for multicopy
suppressors of Sir4C overexpression in a
ade2-1 strain
carrying
a telomeric copy of
ADE2 on the left arm of
chromosome VII. In
this strain, with or without the control pADH
vector, silencing
of
ADE2 produces red sectors within white
colonies (Fig.
1) (
12).
Cells
overexpressing the Sir4C terminus, on the other hand, form
white
nonsectoring colonies, due to
ADE2 expression (i.e., loss
of
TPE [Fig.
1]).
View larger version (110K):
[in this window]
[in a new window]
|
FIG. 1.
Overexpression of Sir3N restores sectoring in
Sir4C-overexpressing cells. The strain AJL275-2AVIIL, which carries
ADE2 adjacent to the VIIL telomere, was
transformed with pADH-SIR4C (also called pFP340) and pAAH5 (a);
pADH-SIR4C and pADH-SIR3N (b); pADH-SIR4C and pADH-SIR3 (p2µ-ASIR3
[26]) (c); control vectors (pAAH5 and p2HG) (d);
pADH-SIR3N and p2HG (e); and pADH-SIR3 (p2µ-ASIR3
[26]) and p2HG (f). In all cases two plasmids were
present and isolated colonies were streaked onto medium lacking
histidine and leucine to ensure maintenance of the plasmids. Adenine
concentrations are limiting. The colonies were allowed to grow for 5 days at 30°C. Following incubation, the plates were stored at 4°C
to enhance the pigmentation of the cells.
|
|
A YEp13-based high-copy-number genomic library was introduced into the
Sir4C-overexpressing strain, and transformants were
screened for red
sectoring, indicative of
ADE2 repression. Screening
of
5.0 × 10
3 transformants identified 30 sectored
transformants, each carrying
a different plasmid. Only one
plasmid, however, reproducibly restored
the sectored
phenotype following plasmid isolation and retransformation.
Southern blot analysis and sequencing revealed that this
clone
contained the first 1.8 kb of
SIR3, encoding the
N-terminal 503
aa of the protein (Sir3N [Fig.
2]), expressed under the control
of its
own promoter. No other
SIR genes or known silencing factors
were recovered in the screen. To see whether an increased dosage
of
this domain improves its ability to restore sectoring, we subcloned
this fragment from another
SIR3 vector so that it could be
expressed
under the control of the
ADH promoter. As shown in
Fig.
1, the
resulting plasmid, pADH-SIR3N, is able to restore sectoring
to
levels higher than wild type in a Sir4C-overexpressing strain.
Immunofluorescence with anti-Sir4C antibodies indicates no
destabilization
or down-regulation of the Sir4C fragment (see
below).
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 2.
Functional domains of Sir3p. A schematic representation
of full-length Sir3p and the functional domains revealed by genetic,
two-hybrid, and biochemical studies is shown. Notes: 1, reference
15; 2, two-hybrid data indicate that the Sir4p
binding domain is 3' of aa 494 (6), and unpublished
pull-down data indicate that there is only one site of interaction, not
two as previously suggested (12a, 43), 3, reference
29 (as indicated by the shaded box, the domain
necessary and sufficient for Rap1p interaction has been narrowed down
to aa 455 to 481 of Sir3p, and the Sir3p homodimerization domain has
been defined from aa 762 to the end of the protein
[38a]); 4, reference 2; 5, this study. The two
mutations isolated as suppressors of histone H4 mutants are labeled
SIR3R1 and SIR3R3 (18). See the text
for more details.
|
|
The simplest explanation for the ability of Sir3N to suppress the
phenotype of Sir4C overexpression is that the two domains
interact
directly. However, extensive two-hybrid analysis with
aa 1 to 503 of
Sir3p, either as bait or as prey, failed to demonstrate
an interaction
with Sir4C (data not shown). We know that the Sir3N
fusion used as bait
in these assays is folded correctly, since
it produces the same
phenotypes as nonfused Sir3N (see below),
and the Sir4C construct has
been shown to interact with itself,
Rap1p, and Sir3p (data not shown).
Similarly, two-hybrid data
of Moretti et al. (
29) show that
Sir4C binds a domain of Sir3p
from aa 307 to the end of the protein.
More recently the site
of Sir4p binding was mapped by glutathione
S-transferase interaction
and two-hybrid assays to a central
core of Sir3p (aa 481 to 734
[
38a] and aa 503 to 763 [
12a]) (Fig.
2). Finally, attempts to
coimmunoprecipitate Sir3N with Sir4C overexpressed in yeast were
negative (data not shown). Thus, in vitro binding assays,
two-hybrid
data, and coimmunoprecipitation results all suggest that
Sir3N
suppresses the Sir4C overexpression phenotype through a mechanism
other than direct protein-protein interaction.
Overexpression of the Sir3N- and C-terminal domains have
opposite effects on telomeric silencing.
One mechanism by
which the Sir3N fragment could suppress the loss of silencing due
to Sir4C overexpression is by stabilizing repressed chromatin
structure through interactions with other silencing factors.
Consistent with this, it has been reported that point mutations in the
N terminus of SIR3 (SIR3R1 and SIR3R3) are able to suppress the loss of HML silencing that results
from mutation of the N terminus of histone H4 (18). One of
these SIR3 point mutations also suppresses the loss of
telomeric and HML silencing conferred by mutations in the
Rap1p C-terminal domain (24). This is surprising, since the
binding sites for the histone N termini and for Rap1p, as well as the
Sir3p homodimerization domain, map to regions C-terminal of aa 503 by
two-hybrid and in vitro binding assays (15, 29, 38a). Using
two-hybrid and coimmunoprecipitation methods, we could
confirm the published interactions between Sir3C and Rap1p, Sir4C, and
histones, while Sir3N showed no binding to any of these factors or to
Sir2p (data not shown).
In view of this paradox, we decided to characterize the inherent
functions of the N- and C-terminal domains of Sir3p by overexpressing
these domains in a
ura3 strain carrying a
URA3-marked telomere.
In this strain, transcriptional
silencing of the telomere-proximal
reporter can be measured
quantitatively by the ability of cells
to grow on 5-FOA (
12)
(see Materials and Methods). We confirmed
that overexpression of
full-length Sir3p increases the efficiency
of telomeric silencing by 2 orders of magnitude (Table
2), as
shown
by Renauld et al. (
33). Intriguingly, overexpression of
Sir3N improves telomeric silencing with almost the same efficiency
as
full-length Sir3p (Table
2). This is not a bypass of the regular
silencing mechanism, as the Sir3N-enhanced repression requires
Sir2p,
Sir4p, the histone N termini, and, importantly, full-length
Sir3p
(Table
3). Because the Sir3N domain shows
a high degree
of homology with the N-terminal domain of Orc1p
(
2), which
was shown to bind Sir1p (
46), we
tested whether the Sir3N enhancement
of telomere-proximal
silencing requires
SIR1. However, the Sir3N
effect
is identical in
SIR1+ and
sir1 strains, ruling out the possibility that
Sir3N stabilizes telomeric
silencing by recruiting Sir1p to telomeres
(Table
3).
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Effects of overexpression of the N- and C-terminal
domains of Sir3p on telomeric position effect and requirements for
enhanced repression: fraction of 5-FOAR colonies upon
introduction of overexpression plasmidsa
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Effects of overexpression of the N- and C-terminal
domains of Sir3p on telomeric position effect and requirements for
enhanced repression: fraction of FOAR colonies or
ADE2 repression upon overexpression of Sir3N
or Sir3pa
|
|
Do other domains of Sir3p have the same effect as Sir3N? To test this,
we overexpressed the C-terminal half of Sir3p (Sir3C
[Fig.
2]). In
contrast to the effect of Sir3N, elevated levels
of Sir3C lead to
derepression of the telomere-proximal
URA3 (Table
2). This
derepression phenotype was confirmed by monitoring
ADE2 expression at the VR telomere (Fig.
3)
and in a variety of strain
backgrounds (Table
2). Sir3C-mediated
derepression, although
weaker than that provoked by the overexpression
of Sir4C, probably
reflects the presence of essential homo- and
heterodimerization
motifs, as well as the Rap1 binding sites, in these
two C-terminal
domains (
29,
43). Sir3C may also interfere
with propagation
of the Sir complex along nucleosomes by competing for
the N termini
of histones H3 and H4 (
15).
View larger version (67K):
[in this window]
[in a new window]
|
FIG. 3.
Overexpression Sir3N counteracts the effect of
overexpression of Sir3C at telomeres. The strain UCC3107, which carries
ADE2 adjacent to the VR telomere, was
transformed with plasmids as indicated as well as a second control
plasmid. The control plasmids are the backbone vectors without
SIR gene inserts, namely, pAAH5 and p423ADH (see Materials
and Methods). Plasmids with SIR3 gene inserts are pADH-SIR3N
and pADH-SIR3C (pMG17). In each case, two independent transformants
were streaked onto medium lacking histidine and leucine to ensure
maintenance of the plasmids and with limiting adenine concentrations.
The cells were allowed to grow for 3 to 5 days at 30°C, and the
plates were stored at 4°C to enhance the pigmentation of the cells.
|
|
The effects of Sir3N and Sir3C overexpression on the repression of the
telomere-proximal
URA3 gene were confirmed by using
a
telomere-proximal
ADE2 gene, which gives a red-sectored
phenotype
in
SIR+ cells. Sir3N overexpression
results in completely red colonies,
indicating full repression of the
telomeric marker, while Sir3C-expressing
cells are white (Fig.
1e and
3). Using this assay, we determined
that a shorter N-terminal
fragment, which stops at aa 440 and
lacks the Rap1p-interacting domain
(Fig.
2), also improves TPE.
The combined overexpression of Sir3N and
Sir3C is discussed below.
Expression in trans of the N- and C-terminal
domains of Sir3p restores HML repression in a strain
with SIR3 deleted.
Do the N and C termini of Sir3p
perform different and independent functions within the silencing
complex? To test this, the N- and the C-terminal domains of Sir3p were
overexpressed from separate vectors in a strain carrying a
complete deletion of SIR3 (sir3::TRP1), and both TPE and
mating-type silencing were scored. Although no red sectoring of the
white colonies was detected with an ADE2-marked telomere in
the UCC3107 background (data not shown), we did observe a 100-fold
increase in mating efficiency when both Sir3N and Sir3C were expressed
from separate plasmids in this sir3::TRP1 strain (Table
4). The expression of either domain individually does not restore mating in the absence of Sir3p (Table 4).
Restriction mapping of plasmids recovered from these yeast cells rules
out the possibility that the two plasmids might have recombined to
create an intact SIR3 gene (data not shown).
We cannot conclude that there is no complementation of TPE whatsoever
when the two Sir3p domains are expressed in
trans in
a
sir3 deletion strain, yet, as we never see red sectors in
the
sir3::
TRP1 strain used (GA822), any
complementation must be below
the limit of detection with the
TelV
R::
ADE2 reporter. Importantly,
in
a
SIR3+ background we do observe that Sir3N
expression counteracts the
disruptive effect of Sir3C overexpression at
telomeres. This is
visualized as a high frequency of red colonies, due
to
ADE2 repression,
in contrast to the white colonies of
strains overexpressing Sir3C
(Fig.
3). The ability of Sir3N to
counteract the derepression
mediated by Sir3C may reflect a direct
neutralization of Sir3C
by Sir3N or simply a net improvement of TPE,
due to the strong
increase in silencing mediated by Sir3N. This might
be able to
balance an independent disruptive effect of Sir3C. Finally,
the
ability of Sir3p domains to substitute more efficiently at
HML than at telomeres in the absence of Sir3p may be due
to the redundancy
of silencer organization (reviewed in reference
32). We propose
that the additional nucleation sites
provided by ORC and Abf1p
at silencers may be critical to allow the
separate Sir3p domains
to function in
trans.
Silenced chromatin extends inward from the telomere upon Sir3N
overexpression.
A unique feature of Sir3p is its ability to
propagate a repressed chromatin state inwards from the core of silent
chromatin adjacent to the TG1-3 repeat. Indeed, upon
overexpression of SIR3, silent chromatin and Sir3p itself
spreads as far as 20 kbp from the telomere (16, 33).
Although extended silencing requires SIR2 and
SIR4, these proteins do not propagate stoichiometrically with Sir3p over the extended silencing domain (43). Since
overexpression of Sir3N, like that of Sir3p, improves repression of a
telomere-proximal gene, we next tested whether the N-terminal domain is
sufficient to promote the spread of TPE.
In a set of isogenic
ppr1-deficient strains carrying
URA3 at various distances from the right end of chromosome V
(Ppr1p is
a
trans-activator that enhances
URA3
expression [
33,
36]),
we monitored the efficiency of growth on 5-FOA (FOA
R [Fig.
4]).
Overexpression of Sir3N improves silencing of the
most proximal
URA3 gene (promoter at 2 kb; 5.9 × 10
1 ± 1.7 × 10
1 FOA
R compared to 5.6 × 10
2 ± 0.8 × 10
2 FOA
R
of cells transformed with the vector alone). At 4 kb from the
chromosomal end,
URA3 repression is increased about 20-fold
(5.9
× 10
3 ± 4.2 × 10
3
compared to 3.0 × 10
4 ± 3.76 × 10
4), and at 6 kb it is increased 10-fold (1.4 × 10
4 ± 1.8 × 10
4 compared to 1.6 × 10
5 ± 0.9 × 10
5), over the rate
in control cells (Fig.
4). Although full-length
Sir3p increases
silencing more efficiently at the most internal
site (4.6 × 10
3 ± 3.6 × 10
3 FOA
R at
6 kb), the ability of Sir3N to promote propagation of silent
chromatin
is highly reproducible and statistically significant
(Fig.
4). Other
Sir proteins, such as Sir2p and Sir4p, do not
extend silencing,
although at low levels Sir2p can improve the
efficiency of repression
within the 4 kb of core heterochromatin
(
6).
View larger version (48K):
[in this window]
[in a new window]
|
FIG. 4.
Sir3N overexpression promotes TPE spreading. Strains
UCC518, UCC520, and UCC522 were transformed with pADH (pAAH5),
pADH-SIR3N, and pADH-SIR3 (p2µ-ASIR3 [26]). Colonies
were grown for 3 to 5 days at 30°C on medium lacking leucine, to
ensure maintenance of the plasmids, and resuspended in H2O,
and 10-fold serial dilutions were plated onto medium lacking leucine
( leu) and medium lacking leucine and containing 1 mg of 5-FOA/liter
(5-FOA leu), as described in Materials and Methods. Two independent
transformants for each case are shown. On the right is a schematic
representation of the URA3-marked telomere of strain UCC518
(top; URA3 at 2 kb from the telomere), UCC520 (middle;
URA3 at 4 kb), and UCC522 (bottom; URA3 at 6 kb).
The frequency of resistance to 5-FOA was calculated from eight
independent transformants, each scored in three independent assays. The
means and standard deviations are given in the text.
|
|
Sir3N overexpression derepresses rDNA silencing.
Recently it
has been shown that the variegated expression of PolII genes inserted
in the rDNA requires SIR2, but not SIR3 and
SIR4 (4, 40). Consistently, in logarithmically
growing wild-type cells, Sir2p, but not Sir3p and Sir4p, is found bound to the rDNA repeat within the nucleolus (10). Intriguingly, Sir3p becomes localized to the nucleolus in sir4 mutants and
in aging cells (10, 22). In view of this, we next tested
whether Sir3N affects the efficiency of silencing of a URA3
gene inserted at the rDNA (40), as assayed by the colony
growth rate on medium lacking uracil. Wild-type cells form small,
slow-growing colonies in the absence of uracil (Fig.
5, compare leu with leu ura for
pADH), while the same cells overexpressing Sir3N grow significantly faster (Fig. 5, pADH-SIR3N). This indicates derepression of the URA3 promoter in the rDNA, similar to the derepression
observed when SIR2 is deleted (6, 40).
Interestingly, overexpression of either full-length Sir3p or Sir3C does
not lead to derepression of the URA3 reporter. We conclude
that Sir3N is able to partially relieve transcriptional repression
within the rDNA locus, achieving the opposite of its effect at
telomeres.
View larger version (55K):
[in this window]
[in a new window]
|
FIG. 5.
Sir3N derepresses rDNA silencing. Strain JS231 carrying
the URA3 gene inserted at the rDNA was transformed with
plasmids pADH (pAAH5), pADH-SIR3N, pADH-SIR3C, and pADH-SIR3
(p2µ-ASIR3). Colonies were grown for 3 to 5 days at 30°C on medium
lacking leucine, to ensure maintenance of the plasmids, and resuspended
in H2O, and 10-fold serial dilutions were plated onto
medium lacking leucine (leu) and medium lacking leucine and uracil
(leu ura), as described in Materials and Methods. Eight independent
transformants for each plasmid were tested, of which two are shown.
|
|
Sir3N localizes to the nucleolus in a Sir2p-dependent manner.
Does Sir3N act directly at both the rDNA and telomere-proximal sites,
or are these effects indirect? To help address this question, we
created a Sir3N C-terminal fusion with the green fluorescent protein,
allowing us to follow the distribution of Sir3N in vivo. The fusion
protein confers the same silencing phenotypes as Sir3N alone (data not
shown). When carried on a 2µ plasmid (pSIR3N-GFP2), the fusion
protein produces an intense but diffuse nuclear green fluorescence
visible by direct fluorescence microscopy (Fig.
6A, view a). In many cells we can
identify a portion of the nucleus that is significantly brighter than
the rest of the nucleoplasm, reminiscent of the nucleolar staining
observed for Sir2p (10). The fixed spheroplasts carrying
pSIR3N-GFP2 were then stained with anti-Nop1 antibodies, producing red
fluorescence (Fig. 6A, view b) which was superimposed on the green
GFP fluorescence. The coincidence of the signals (Fig. 6A, view c)
indicates that Sir3N-GFP is indeed enriched in the nucleolar
compartment, although it is not excluded from the rest of the
nucleoplasm.
View larger version (135K):
[in this window]
[in a new window]
|
FIG. 6.
(A) SIR3N-GFP localizes to the nucleolus. The haploid
strain UCC3107 was transformed with either a 2µ-based plasmid (a, b,
and c) or a centromeric plasmid (d and f) expressing Sir3N fused to the
green fluorescent protein under the control of the ADH promoter
(pSIR3N-GFP2 and pSIR3N-GFP1, respectively). The direct fluorescence of
Sir3N-GFP (green) (a), anti-Nop1p staining on the same cells visualized
with a Cy3-conjugated secondary antibody (red) (b), and the merge of
the two stainings (c) are shown. The blue area represents the nucleus.
Colocalization of Sir3N-GFP and Nop1p is white. Cells transformed with
pSIR3N-GFP1 and stained with anti-GFP antibodies (the kind gift of
K. E. Sawin, Imperial Cancer Research Fund, London, England)
visualized by a DTAF-conjugated secondary antibody (green) (d); the DNA
staining of the same cells with POPO-3, which preferentially stains the
nucleolar domain (red) (e); and anti-Sir2p immunofluorescence on a
fixed wild-type diploid strain (GA229) that had been washed in 1%
Triton-0.02% sodium dodecyl sulfate as described previously
(10) (f) are also shown. Sir2p staining is visualized by a
DTAF-conjugated secondary antibody (green), and the DNA is
counterstained with ethidium bromide. Immunofluorescence assays were
performed with affinity-purified antibodies as described in Materials
and Methods. The arrows indicate an apparent looped body, while the
arrowhead indicates the same loop extended. (B) SIR2 but not
SIR4 is necessary for the enrichment of Sir3N-GFP in the
nucleolus. The haploid strains UCC3107 (SIR+) (a
and a'), UCC3203 (sir2::HIS3) (b and
b'), and UCC3207 (sir4::HIS3) (c and
c') were transformed with plasmid pSIR3N-GFP1. The phase-contrast image
(a to c) and the direct fluorescence (a', b', and c') of Sir3N-GFP are
shown. Results identical to those shown in c and c' were obtained for a
sir3::HIS3 strain.
|
|
To see if Sir3N, like Sir2p, is associated with the rDNA
(
10), we used an anti-GFP antibody to detect the fusion
protein
expressed from a centromere plasmid (pSIR3N-GFP1). We observe
diffuse green fluorescence in the nucleoplasm, but also along
an
apparently looped structure within the strongly POPO-3-stained
nucleolus (Fig.
6A, views d and e). This loop was previously shown
to
be the rDNA of chromosome XII (
13), and it stains brightly
with anti-Sir2p antibodies (Fig.
6A, view f). When spheroplasts
are
gently lysed in detergents the rDNA loop extends, yet still
maintains
anti-Sir2p staining, consistent with cross-linking data
that show that
Sir2p precipitates with the rDNA repeats (Fig.
6A, view f). The fact
that Sir3N-GFP produces a similar fluorescence
both in living cells and
in fixed spheroplasts suggests that Sir3N
is also associated with rDNA.
We have previously shown that Sir3p accumulates in the nucleolus in a
sir4 deletion strain, and that it requires both
SIR2 and
UTH4 gene products for this localization
(
10). To see if
Sir2p and Uth4p are required for Sir3N
localization, we scored
for the localization of the Sir3N-GFP fusion
expressed from a
centromere plasmid in strains that lack either
SIR2,
SIR3,
SIR4,
or
UTH4.
The direct fluorescence of Sir3N-GFP is shown in Fig.
6B, below the
phase images of the corresponding living yeast cells.
Whereas the
fluorescence is localized in a nucleolar subdomain
in
SIR+ strains, the signal is dispersed throughout
the nucleoplasm in
a
sir2::
HIS3 strain
(Fig.
6B, views b and b'). On the other hand,
in strains carrying gene
disruptions for
SIR4 (Fig.
6B, views
c and c'),
SIR3, or
UTH4 (data not shown), Sir3N-GFP is
still
enriched in the nucleolus. Thus, the only known silencing factor
required for the nucleolar accumulation of Sir3N is Sir2p. These
results make it likely that derepression of the
URA3
reporter
in the rDNA repeat reflects a direct action of Sir3N.
What happens to Sir2p when Sir3N accumulates in the nucleolus? We have
recently observed that increasing the gene dosage of
SIR2 by
a single copy leads to increased telomeric silencing (reference
6; see Discussion below). This led to the hypothesis
that Sir2p
might be released from the rDNA upon Sir3N overexpression
and
therefore be free to increase silencing at telomeres. To test
this
possibility, we performed immunofluorescence assays with
anti-Sir2p
antibodies on yeast cells transformed with the vector
alone or with the
plasmid overexpressing Sir3N. Although the majority
of the detectable
Sir2p is found in the nucleolus in control cells,
weak foci of staining
can be detected at the nuclear periphery,
colocalizing with telomeres
(
10,
43). When cells expressing
Sir3N are stained with
anti-Sir2p, we observe a higher fluorescent
signal in the nucleoplasm
and more intense telomeric spots than
are observed in the pADH control
(Fig.
7). By performing Western
blotting
on extracts from cells carrying either the pADH vector
or pADH-SIR3N we
can rule out the possibility that Sir2p levels
increase in the
Sir3N-overexpressing cells (data not shown). This
and our
immunofluorescence data are consistent with the idea that
Sir3N
overexpression causes a partial redistribution of Sir2p
from the rDNA
to telomeres, which could explain how Sir3N overexpression
can have
opposite effects on these two sites of repression.
View larger version (82K):
[in this window]
[in a new window]
|
FIG. 7.
Sir2p is enriched at telomeric foci upon overexpression
of Sir3N. The wild-type haploid strain UCC3107, transformed with
plasmids pADH (pAAH5) and pADH-SIR3N, was stained with anti-Sir2p,
detected by a DTAF-conjugated secondary antibody (green). The DNA was
counterstained with POPO-3 (red).
|
|
Overexpression of Sir3p and Sir4p restores telomeric silencing and
telomeric foci.
The redistribution of Sir2p from the rDNA to
telomeres cannot completely account for the effect of Sir3N at
telomeres, since Sir2p overexpression does not extend silencing beyond
4 kb, which both Sir3p and Sir3N can do (Fig. 4) (6).
Indeed, the many parallels between the effects of overexpressing either
Sir3p or Sir3N suggested to us that perhaps Sir3N was able to
upregulate or activate the nuclear pool of full-length Sir3p, resulting
in phenotypes similar to those obtained when SIR3 is
overexpressed. We could rule out the possibility that Sir3N increases
the amount of Sir3p in the nucleus by performing Western blotting on
cells carrying either the pADH vector or pADH-SIR3N. Within the twofold range of accuracy afforded by chemiluminescence, we conclude that there
is no variation in total cellular Sir3p when Sir3N is overexpressed (data not shown).
If Sir3N were to "activate" or allow recruitment of an inactive
population of Sir3p, we would predict that the overexpression
of
full-length Sir3p would restore TPE in a strain overexpressing
Sir4C,
like Sir3N does. As shown in Figure
1c, this is indeed
the case.
Moreover, whereas raising the dosage of full-length
Sir4p derepresses a
telomere-proximal
ADE2 gene, repression can
be restored, and
even enhanced above background levels, by balancing
the higher levels
of Sir4p with elevated levels of Sir3p (Fig.
8). This is visualized as an enhancement
in the frequency of dark-red
colonies when both pGPD-SIR3 and pADH-SIR4
are introduced into
a strain carrying an
ADE2-marked
telomere (Fig.
8). These results
demonstrate that the disruptive effect
of Sir4C or Sir4p overexpression
can be compensated for by increasing
the amounts of Sir3p available
for the assembly of silent chromatin.
View larger version (68K):
[in this window]
[in a new window]
|
FIG. 8.
Overexpression of full-length Sir3p restores silencing
in a strain that overexpress full-length Sir4p. The strain UCC3107,
which carries ADE2 adjacent to the VR telomere,
was transformed with plasmids as indicated as well as a second control
plasmid. The control plasmids are the backbone vector without
SIR gene inserts, namely, pAAH5 and p2HG (see Materials and
Methods). The plasmids with SIR gene inserts are pADH-SIR4
and pGPD-SIR3. Colonies (two independent transformants in each case)
were streaked onto medium lacking histidine and leucine to ensure
maintenance of the plasmids and allowed to grow for 3 to 5 days at
30°C. Following incubation, the plates were stored at 4°C for 1 week to enhance the pigmentation of the cells.
|
|
Loss of silencing correlates tightly with the delocalization of Rap1p
and Sir proteins from telomeric foci (
7,
9,
10,
31). We have
previously shown that in strains overexpressing
either Sir4C or
full-length Sir4p telomeric silencing is relieved
and Rap1p and Sir3p
are delocalized from telomeric foci (
7,
26). Here we show
that the simultaneous overexpression of both
Sir3N and Sir4C (Fig.
9f), like the simultaneous and balanced
overexpression of full-length Sir3p and Sir4p, restores the punctate
staining of full-length Sir3p (Fig.
9c). This correlates with
the
restoration of repression at telomeres. Similarly, Rap1p and
Sir4p foci
are restored under conditions of balanced overexpression
(6). Western
blot analysis confirms that the levels of Sir4p and
Sir4C remain high
when Sir3p or Sir3N are overexpressed, ruling
out a trivial effect of
Sir3N or Sir3p on
SIR4 expression or protein
stability
(reference
26 and data not shown). Thus, we extend
the tight correlation between the restoration of TPE and balanced
levels of Sir3p and Sir4p. In view of this and the fact that all
phenotypes reported here for Sir3N both mimic and depend on full-length
Sir3p, we propose that Sir3N acts by enhancing the pool of functional
Sir3p.
View larger version (63K):
[in this window]
[in a new window]
|
FIG. 9.
Overexpression of Sir3N restores the focal staining
pattern of Sir3p. Yeast cells were stained with anti-Sir3p antibodies
detected by a DTAF-conjugated secondary antibody (white signal). All
signals are within the yeast nuclei, as indicated in the insets, where
the anti-Sir3p signals are superimposed on a DNA stain to reveal the
nuclear shape (see Materials and Methods). Strain EG37 (26)
transformed with the vectors pAAH5 and pRS316 (a), pC-ASir4
(26) and pAAH5 (b), and pC-ASir4 and p2µ-ASir3 (c) and
strain AJL275-2AVIIL transformed with the vectors pAAH5 and
p2HG (d), pADH-SIR4C (pFP340) and pAAH5 (e), and pADH-SIR4C (pFP340)
and pADH-SIR3N (f) are shown.
|
|
| |
DISCUSSION |
Here we show that expression of the N-terminal 503 aa of Sir3p in
an otherwise wild-type strain counteracts the derepression provoked by
Sir4C overexpression. Moreover, expression of Sir3N alone improves and
extends telomere-proximal repression. Both these effects require the
presence of full-length Sir3p and can also be achieved by
overexpression of full-length Sir3p. As with Sir3p, the Sir3N-dependent
improvement of silencing requires SIR2 and SIR4
and the histone N-termini, although not SIR1. Thus,
repression by Sir3N is not achieved by bypassing the normal pathway of
telomere-proximal repression, nor does it function by targeting Sir1p
to telomeric sites.
Surprisingly, we were unable to detect any interaction between Sir3N
and the well-characterized components of the repression machinery; that
is, Sir3N cannot dimerize, nor can we detect Sir3N interaction with
either Sir2p, Sir4p, Rap1p, or the N termini of histones H3 and H4 by
two-hybrid and/or coimmunoprecipitation assays (references 6,
12a, 15, and 38a and data not shown). In
contrast, interaction between the C-terminal domain of Sir3p and the
last four components has been well characterized (Fig. 2) (6, 15,
16, 29, 43). Indeed, the presence of multiple binding sites in
the Sir3 C-terminal domain for components of repressed chromatin is
consistent with its dominant-negative effect on TPE. The fact that the
overexpression of Sir3N and that of Sir3C have opposite effects on
silencing is consistent with the notion that they interact with
different subsets of proteins.
There is conflicting data in the literature as to whether Sir2p
interacts directly with Sir3p (28, 43). However, it is clear
that Sir2p does not coimmunoprecipitate with Sir3p in strains with
SIR4 deleted (43), and in our hands no Sir2p
interactions could be detected with either the Sir3N or Sir3C termini
in two-hybrid assays. It is possible that this is a regulated
interaction or one requiring another factor.
Alternative models for Sir3N-dependent enhancement of
repression.
The Sir3N fragment alone accumulates in the nucleolus
and appears to provoke the release or relocalization of a fraction of Sir2p from the nucleolus to telomeric sites (see below). This effect
cannot, however, account for the extension of silencing observed upon
Sir3N overexpression, since increased Sir2p expression only improves
repression up to 4 kb from the telomeric repeat (6). One
simple explanation for the Sir3N-induced phenotypes would be that Sir3N
expression results in elevated levels of Sir3p, and perhaps also of
Sir2p, in the cell. However, quantitative Western blots show no
significant variation in either of these components when Sir3N is
overexpressed. A second possibility is that Sir3N itself can promote
the propagation of silent chromatin. Recently, Strahl-Bolsinger and
colleagues (16, 43) have shown that transcriptionally inert
regions of the yeast genome can have two distinct forms. One of these,
present as the "core" heterochromatin at telomeres in wild-type
cells, contains the three Sir proteins and Rap1p as structural
components of the repressed chromatin. The second type, which is
induced by overexpression of Sir3p, can only be generated as an
extension from a preexisting domain of the core heterochromatin but
appears to require only the propagation of a Sir3p-histone complex
(43). The N-terminal 503 aa of Sir3p may be able to promote
propagation of a repressed chromatin structure from a preassembled
core. However, this would suggest that the propagation can occur
without direct interaction with histone N termini, since Sir3N is
lacking the domain that binds histone tails. Moreover, it would require
that Sir3N interact with other components of telomeric chromatin, which
was not observed.
In view of the similarity of the effects at telomeres provoked by
overexpression of Sir3N and Sir3p, we favor a third model,
in which
Sir3N increases the pool of full-length Sir3p available
to the
repression machinery. This might be achieved by exerting
an allosteric
effect on Sir3p, which would improve its ability
to interact with other
components of the silencing machinery.
In its simplest form, this
mechanism would imply a physical interaction
between Sir3N and Sir3C;
however, this could not be detected by
a two-hybrid assay.
Alternatively, the pool of active full-length
Sir3p could be increased
by releasing or activating a subpopulation
of Sir3p that is normally
sequestered in a silencing-incompetent
form. This may reflect
interaction with an unidentified third
component which binds
Sir3p through its N terminus, or it could
reflect activation of
Sir3p by posttranslational modification
or a conformational change.
Results currently cannot distinguish
between these possibilities.
Genetic evidence supports the model in which the N terminus of Sir3p
activates its C-terminal domain, namely, the N-terminal
mutations
SIR3R3 and
SIR3R1, which suppress mutations in
the Rap1
C terminus and in histone N termini (
18,
24). In
both cases,
the mutations suppressed by
SIR3R3 are expected
to weaken interaction
between either Rap1p or the histone N termini and
the Sir3 C terminus
(
7,
15,
25). Thus, one explanation for
the suppression
data is that these Sir3 N-terminal mutations activate
full-length
Sir3p by improving the ability of Sir3C to bind other
silencing
factors, such as Rap1p and the histone tails. Consistently,
Park
et al. have shown that a central region of Sir3p inhibits the
silencing initiation function of the C-terminal 144 aa, which,
however,
can be overcome by Sir3N (31a).
Sir3p is a target of regulatory mechanisms.
The ability of
Sir3N and Sir3p overexpression to compensate for the derepression
provoked by Sir4C and Sir4p overexpression underscores how important
the balance between these factors is for proper transcriptional
control. Consistently, Sir3p appears to be the target of multiple
regulatory mechanisms, including one which may involve the sequestering
of the full-length protein in an inactive form. Our model would suggest
that overexpression of Sir3N overcomes this. We do not know whether
this requires a yet-unidentified Sir3p ligand, but if there is a
significant pool of full-length Sir3p that is not participating in a
silencing complex, then it is likely to be localized at telomeric foci, since immunofluorescence does not reveal a significant fraction of
Sir3p elsewhere in the nucleus (Fig. 9).
Recently it was shown that Sir3p is a phosphoprotein and that
hyperphosphorylation by a mitogen-activated protein (MAP) kinase
pathway leads to increased silencing (
42). Interestingly,
although
the phosphorylation sites have not yet been mapped, nearly all
consensus sites for MAP kinases (minimal consensus as PT/S or
T/SP) are
found in the N-terminal half of Sir3p, suggesting another
means by
which this domain might regulate the activity of full-length
Sir3p. As
well as being phosphorylated itself, Sir3N might target
kinases to
other components of the silencing complex to regulate
silencing
efficiency, protein assembly, or turnover.
Competition between domains for a limiting amount of
Sir2p.
Although Sir3N improves telomeric repression, it
antagonizes repression of a URA3 reporter gene
inserted in the rDNA repeats (4, 8, 40). This
repression, as well as the suppression of recombination between rDNA
repeats, is mediated by Sir2p, which is associated with the rDNA
(4, 10, 11, 22, 40). Upon deletion of SIR3 or
SIR4 or overexpression of SIR2, silencing at the
rDNA is increased, suggesting that there is a competition between the
rDNA and telomeres for a limiting amount of Sir2p (40).
Indeed, a moderate increase in Sir2p dosage also leads to increased
repression at telomeres (6). Our data are consistent with
the idea that overexpression of Sir3N causes the release or
displacement of some fraction of the nucleolar Sir2p from the rDNA,
leading to partial derepression of URA3 and enhanced
repression at telomeres. Consistent with this hypothesis, telomeric
foci, as detected by anti-Sir2p antibodies, are more intense when Sir3N is expressed than they are in the control (Fig. 7). This result corroborates increasing evidence that the different loci at which silencing occurs (HM loci, telomeres, and rDNA) compete for
limiting amounts of silencing factors (5, 6, 14, 26).
The interference of Sir3N in the repression pathway of rDNA may reflect
competition between Sir3N and Sir2p for a common third
factor. Since
rDNA repression is poorly characterized on a molecular
level, it is
impossible to say which nucleolar elements might
be influenced by
Sir3N. ORC subunits, Abf1p, Reb1p, and nucleosomes
are all possible
candidates (
2,
19). Moreover, since a significant
pool of
Sir3N is found dispersed in the nucleoplasm, we cannot
rule out the
possibility that this domain acts indirectly to influence
repression.
The fact that Sir3N has opposite effects on TPE and
rDNA repression
supports the hypothesis that the two types of
silencing, although
related, make use of different molecular mechanisms.
The Sir3N terminus is highly enriched in the nucleolar compartment. We
and others have previously found that the relocalization
of Sir3p and
Sir4p to the nucleolus occurs both in old mother
cells and in mutants
that show suppression of precocious-aging
phenotypes (
22,
39). The relocation of Sir3p requires Sir2p
and the product of
UTH4, a gene also implicated in yeast life
span
determination (
22). The data presented here indicate that
the first 503 aa of Sir3p contains information necessary and sufficient
for nucleolar targeting. Since the nucleolar localization of Sir3N,
unlike that of Sir3p, does not require Uth4p, it appears that
Uth4p
overcomes a negative element that impedes the nucleolar
targeting of
full-length Sir3p. Sir3N and Sir3p, on the other
hand, both require
Sir2p for their accumulation in the nucleolus,
indicating that the
presence of Sir2p itself provides or creates
a binding site for Sir3N
and Sir3p.
Whether Sir3N, Sir3p, and/or Sir4p has a unique function in the
nucleolus is unclear. Although deletion of either
SIR3 or
SIR4, like deletion of
SIR2, shortens life span,
only deletion
of
SIR2 affects recombination rates and PolII
expression in the
rDNA (
4,
11,
40). Additional studies are
needed to determine
why Sir2p is necessary for the nucleolar
accumulation of Sir3N
and what Sir3N and Sir3p achieve in the
nucleolus. Although we
are unable to detect Sir2p-Sir3N interaction by
two-hybrid or
coimmunoprecipitation assays (
43), this does
not conclusively
rule out an interaction. Novel approaches are clearly
required
to resolve both this question and the mechanism by which Sir3N
activates the Sir3p holoprotein.
| |
ACKNOWLEDGMENTS |
We acknowledge the expert technical assistance of T. Laroche. We
thank K. Sawin for the anti-GFP antibodies, Ed Hurt for anti-Nop1 antibodies, H. Renauld for strains, and M. Cockell, H. Renauld, D. Shore, A. Lustig, and M. Grunstein for allowing us to
cite unpublished results and for helpful discussions.
M.G. thanks ISREC for a Ph.D. fellowship. F.P. was supported by the
Human Frontiers Science Program. Research in the Gasser laboratory
is supported by the Swiss National Science Foundation, the Human
Frontiers Science Program, and the Swiss League against Cancer.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Swiss Institute
for Experimental Cancer Research, Chemin des Boveresses 155, CH-1066 Epalinges/Lausanne, Switzerland. Phone:
41-21-692-5886. Fax: 41-21-652-6933. E-mail:
sgasser{at}eliot.unil.ch.
Present address: Dept. of Genetics, University of Cambridge,
Cambridge, United Kingdom.
Present address: Dept. of Zoology, University of Fribourg,
Pérolles, Fribourg, Switzerland.
| |
REFERENCES |
| 1.
|
Aparicio, O. M.,
B. L. Billington, and D. E. Gottschling.
1991.
Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae.
Cell
66:1279-1287[Medline].
|
| 2.
|
Bell, S. P.,
J. Mitchell,
J. Leber,
R. Kobayashi, and B. Stillman.
1995.
The multidomain structure of Orc1p reveals similarity to regulators of DNA replication and transcriptional silencing.
Cell
83:563-568[Medline].
|
| 3.
|
Boeke, J. D.,
J. Trueheart,
G. Natsoulis, and G. R. Fink.
1987.
5-Fluoroorotic acid as a selective agent in yeast molecular genetics.
Methods Enzymol.
154:164-175[Medline].
|
| 4.
|
Bryk, M.,
M. Banerjee,
M. Murphy,
K. E. Knudsen,
D. J. Garfinkel, and M. J. Curcio.
1997.
Transcriptional silencing of Ty elements in the RDN1 locus of yeast.
Genes Dev.
11:255-269[Abstract/Free Full Text].
|
| 5.
|
Buck, S. W., and D. Shore.
1995.
Action of a RAP1 carboxy-terminal silencing domain reveals an underlying competition between HMR and telomeres in yeast.
Genes Dev.
9:370-384[Abstract/Free Full Text].
|
| 6.
| Cockell, M., M. Gotta, F. Palladino, S. G. Martin,
and S. M. Gasser. Targeting limiting pools of Sir proteins to
sites of action: a general mechanism for regulated repression. Cold
Spring Harbor Symp. Quant. Biol., in press.
|
| 7.
|
Cockell, M.,
F. Palladino,
T. Laroche,
G. Kyrion,
C. Liu,
A. J. Lustig, and S. M. Gasser.
1995.
The carboxy termini of Sir4 and Rap1 affect Sir3 localization: evidence for a multicomponent complex required for yeast telomeric silencing.
J. Cell Biol.
129:909-924[Abstract/Free Full Text].
|
| 8.
|
Fritze, C. E.,
K. Verschueren,
R. Strich, and R. E. Esposito.
1997.
Direct evidence for SIR2 modulation of chromatin structure in yeast rDNA.
EMBO J.
16:6495-6509[Medline].
|
| 9.
|
Gotta, M.,
T. Laroche,
A. Formenton,
L. Maillet,
H. Scherthan, and S. M. Gasser.
1996.
The clustering of telomeres and colocalization with Rap1, Sir3 and Sir4 proteins in wild-type Saccharomyces cerevisiae.
J. Cell Biol.
134:1349-1363[Abstract/Free Full Text].
|
| 10.
|
Gotta, M.,
S. Strahl-Bolsinger,
H. Renauld,
T. Laroche,
B. K. Kennedy,
M. Grunstein, and S. M. Gasser.
1997.
Localization of Sir2p: the nucleolus as a compartment for silent information regulators.
EMBO J.
16:3243-3255[Medline].
|
| 11.
|
Gottlieb, S., and R. E. Esposito.
1989.
A new role for a yeast transcriptional silencer gene, SIR2, in regulation of recombination in ribosomal DNA.
Cell
56:771-776[Medline].
|
| 12.
|
Gottschling, D. E.,
O. M. Aparicio,
B. L. Billington, and V. A. Zakian.
1990.
Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription.
Cell
63:751-762[Medline].
|
| 12a.
| Grunstein, M. Personal communication.
|
| 13.
|
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].
|
| 14.
|
Hardy, C. F.,
L. Sussel, and D. Shore.
1992.
A RAP1-interacting protein involved in transcriptional silencing and telomere length regulation.
Genes Dev.
6:801-814[Abstract/Free Full Text].
|
| 15.
|
Hecht, A.,
T. Laroche,
S. Strahl-Bolsinger,
S. M. Gasser, and M. Grunstein.
1995.
Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins: a molecular model for the formation of heterochromatin in yeast.
Cell
80:583-592[Medline].
|
| 16.
|
Hecht, A.,
S. Strahl-Bolsinger, and M. Grunstein.
1996.
Spreading of transcriptional repressor SIR3 from telomeric heterochromatin.
Nature
383:92-96[Medline].
|
| 17.
|
Ivy, J. M.,
A. J. S. Klar, and J. B. Hicks.
1986.
Cloning and characterization of four SIR genes of Saccharomyces cerevisiae.
Mol. Cell. Biol.
6:688-702[Abstract/Free Full Text].
|
| 18.
|
Johnson, L. M.,
P. S. Kayne,
E. S. Kahn, and M. Grunstein.
1990.
Genetic evidence for an interaction between SIR3 and histone H4 in the repression of the silent mating loci in Saccharomyces cerevisiae.
Proc. Natl. Acad. Sci. USA
87:6286-6290[Abstract/Free Full Text].
|
| 19.
|
Kang, J. J.,
T. J. Yokoi, and M. J. Holland.
1995.
Binding sites for abundant nuclear factors modulate RNA polymerase I-dependent enhancer function in Saccharomyces cerevisiae.
J. Biol. Chem.
270:28723-28732[Abstract/Free Full Text].
|
| 20.
|
Karpen, G. H.
1994.
Position-effect variegation and the new biology of heterochromatin.
Curr. Opin. Genet. Dev.
4:281-291[Medline].
|
| 21.
|
Kayne, P. S.,
U. J. Kim,
M. Han,
J. R. Mullen,
F. Yoshizaki, and M. Grunstein.
1988.
Extremely conserved histone H4 N terminus is dispensable for growth but essential for repressing the silent mating loci in yeast.
Cell
55:27-39[Medline].
|
| 22.
|
Kennedy, B. K.,
M. Gotta,
D. A. Sinclair,
K. Mills,
D. S. McNabb,
M. Murthy,
S. M. Pak,
T. Laroche,
S. M. Gasser, and L. Guarente.
1997.
Redistribution of silencing proteins from telomeres to the nucleolus is associated with extension in life span in S. cerevisiae.
Cell
89:381-391[Medline].
|
| 23.
|
Kyrion, G.,
K. Liu,
C. Liu, and A. J. Lustig.
1993.
RAP1 and telomere structure regulate telomere position effects in Saccharomyces cerevisiae.
Genes Dev.
7:1146-1159[Abstract/Free Full Text].
|
| 24.
|
Liu, C., and A. J. Lustig.
1996.
Genetic analysis of Rap1p/Sir3p interactions in telomeric and HML silencing in Saccharomyces cerevisiae.
Genetics
143:81-93[Abstract].
|
| 25.
|
Liu, C.,
X. Mao, and A. J. Lustig.
1994.
Mutational analysis defines a C-terminal tail domain of RAP1 essential for telomeric silencing in Saccharomyces cerevisiae.
Genetics
138:1025-1040[Abstract].
|
| 26.
|
Maillet, L.,
C. Boscheron,
M. Gotta,
S. Marcand,
E. Gilson, and S. M. Gasser.
1996.
Evidence for silencing compartments within the yeast nucleus: a role for telomere proximity and Sir protein concentration in silencer-mediated repression.
Genes Dev.
10:1796-1811[Abstract/Free Full Text].
|
| 27.
|
Marshall, M.,
D. Mahoney,
A. Rose,
J. B. Hicks, and J. R. Broach.
1987.
Functional domains of SIR4, a gene required for position effect regulation in Saccharomyces cerevisiae.
Mol. Cell. Biol.
7:4441-4452[Abstract/Free Full Text].
|
| 28.
|
Moazed, D.,
A. Kistler,
A. Axelrod,
J. Rine, and A. D. Johnson.
1997.
Silent information regulator protein complexes in Saccharomyces cerevisiae: a SIR2/SIR4 complex and evidence for a regulatory domain in SIR4 that inhibits its interaction with SIR3.
Proc. Natl. Acad. Sci. USA
94:2186-2191[Abstract/Free Full Text].
|
| 29.
|
Moretti, P.,
K. Freeman,
L. Coodly, and D. Shore.
1994.
Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1.
Genes Dev.
8:2257-2269[Abstract/Free Full Text].
|
| 30.
|
Mumberg, D.,
R. Muller, and M. Funk.
1995.
Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds.
Gene
156:119-122[Medline].
|
| 31.
|
Palladino, F.,
T. Laroche,
E. Gilson,
A. Axelrod,
L. Pillus, and S. M. Gasser.
1993.
SIR3 and SIR4 proteins are required for the positioning and integrity of yeast telomeres.
Cell
75:543-555[Medline].
|
| 31a.
| Park, Y., J. Hanish, and A. J. Lustig. Sir3p domains
involved in the initiation of telomeric silencing in
Saccharomyces cerevisiae. Genetics, in press.
|
| 32.
|
Pillus, L., and M. Grunstein.
1995.
Chromatin structure and epigenetic regulation in yeast, p. 123-146.
In
S. C. R. Elgin (ed.), Chromatin structure and gene expression. IRL press, Oxford, England.
|
| 33.
|
Renauld, H.,
O. M. Aparicio,
P. D. Zierath,
B. L. Billington,
S. K. Chhablani, and D. E. Gottschling.
1993.
Silent domains are assembled continuously from the telomere and are defined by promoter distance and strength, and by SIR3 dosage.
Genes Dev.
7:1133-1145[Abstract/Free Full Text].
|
| 33a.
| Renauld, H. Unpublished data.
|
| 34.
|
Rine, J., and I. Herskowitz.
1987.
Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae.
Genetics
116:9-22[Abstract/Free Full Text].
|
| 35.
|
Rose, M. D.,
F. Winston, and P. Hieter.
1990.
Methods in yeast genetics.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 36.
|
Roy, A.,
F. Exinger, and R. Losson.
1990.
cis- and trans-acting regulatory elements of the yeast URA3 promoter.
Mol. Cell. Biol.
10:5257-5270[Abstract/Free Full Text].
|
| 37.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 38.
|
Schiestl, R. H., and R. D. Gietz.
1989.
High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier.
Curr. Genet.
16:339-346[Medline].
|
| 38a.
| Shore, D. Personal communication.
|
| 39.
|
Sinclair, D. A.,
K. Mills, and L. Guarente.
1997.
Accelerated aging and nucleolar fragmentation in yeast sgs1 mutants.
Science
277:1313-1316[Abstract/Free Full Text].
|
| 40.
|
Smith, J. S., and J. D. Boeke.
1997.
An unusual form of transcriptional silencing in yeast ribosomal DNA.
Genes Dev.
11:241-254[Abstract/Free Full Text].
|
| 41.
|
Sprague, G. F.
1991.
Assay of yeast mating reaction.
Methods Enzymol.
194:77-93[Medline].
|
| 42.
|
Stone, E. M., and L. Pillus.
1996.
Activation of a MAP kinase cascade leads to Sir3p hyperphosphorylation and strengthens transcriptional silencing.
J. Cell Biol.
135:571-583[Abstract/Free Full Text].
|
| 43.
|
Strahl-Bolsinger, S.,
A. Hecht,
L. Kunheng, and M. Grunstein.
1997.
SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast.
Genes Dev.
11:83-93[Abstract/Free Full Text].
|
| 44.
|
Sussel, L.,
D. Vannier, and D. Shore.
1993.
Epigenetic switching of transcriptional states: cis- and trans-acting factors affecting establishment of silencing at the HMR locus in Saccharomyces cerevisiae.
Mol. Cell. Biol.
13:3919-3928[Abstract/Free Full Text].
|
| 45.
|
Thompson, J. S.,
X. Ling, and M. Grunstein.
1994.
Histone H3 amino terminus is required for telomeric and silent mating locus repression in yeast.
Nature
369:245-247[Medline].
|
| 46.
|
Triolo, T., and R. Sternglanz.
1996.
Role of interactions between the origin recognition complex and SIR1 in transcriptional silencing.
Nature
381:251-253[Medline].
|
| 47.
|
Tsukamoto, Y.,
J. Kato, and H. Ikeda.
1997.
Silencing factors participate in DNA repair and recombination in Saccharomyces cerevisiae.
Nature
388:900-903[Medline].
|
| 48.
|
Weiler, K. S., and B. T. Wakimoto.
1995.
Heterochromatin and gene expression in Drosophila.
Annu. Rev. Genet.
29:577-605[Medline].
|
Molecular and Cellular Biology, October 1998, p. 6110-6120, Vol. 18, No. 10
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Sampath, V., Yuan, P., Wang, I. X., Prugar, E., van Leeuwen, F., Sternglanz, R.
(2009). Mutational Analysis of the Sir3 BAH Domain Reveals Multiple Points of Interaction with Nucleosomes. Mol. Cell. Biol.
29: 2532-2545
[Abstract]
[Full Text]
-
Taddei, A., Van Houwe, G., Nagai, S., Erb, I., van Nimwegen, E., Gasser, S. M.
(2009). The functional importance of telomere clustering: Global changes in gene expression result from SIR factor dispersion. Genome Res
19: 611-625
[Abstract]
[Full Text]
-
Buchberger, J. R., Onishi, M., Li, G., Seebacher, J., Rudner, A. D., Gygi, S. P., Moazed, D.
(2008). Sir3-Nucleosome Interactions in Spreading of Silent Chromatin in Saccharomyces cerevisiae. Mol. Cell. Biol.
28: 6903-6918
[Abstract]
[Full Text]
-
van Welsem, T., Frederiks, F., Verzijlbergen, K. F., Faber, A. W., Nelson, Z. W., Egan, D. A., Gottschling, D. E., van Leeuwen, F.
(2008). Synthetic Lethal Screens Identify Gene Silencing Processes in Yeast and Implicate the Acetylated Amino Terminus of Sir3 in Recognition of the Nucleosome Core. Mol. Cell. Biol.
28: 3861-3872
[Abstract]
[Full Text]
-
Connelly, J. J., Yuan, P., Hsu, H.-C., Li, Z., Xu, R.-M., Sternglanz, R.
(2006). Structure and Function of the Saccharomyces cerevisiae Sir3 BAH Domain. Mol. Cell. Biol.
26: 3256-3265
[Abstract]
[Full Text]
-
Michel, A. H., Kornmann, B., Dubrana, K., Shore, D.
(2005). Spontaneous rDNA copy number variation modulates Sir2 levels and epigenetic gene silencing. Genes Dev.
19: 1199-1210
[Abstract]
[Full Text]
-
Wang, X., Connelly, J. J., Wang, C.-L., Sternglanz, R.
(2004). Importance of the Sir3 N Terminus and Its Acetylation for Yeast Transcriptional Silencing. Genetics
168: 547-551
[Abstract]
[Full Text]
-
Huang, Y.
(2002). Transcriptional silencing in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Nucleic Acids Res
30: 1465-1482
[Abstract]
[Full Text]
-
Xie, W., Gai, X., Zhu, Y., Zappulla, D. C., Sternglanz, R., Voytas, D. F.
(2001). Targeting of the Yeast Ty5 Retrotransposon to Silent Chromatin Is Mediated by Interactions between Integrase and Sir4p. Mol. Cell. Biol.
21: 6606-6614
[Abstract]
[Full Text]
-
Stone, E. M., Reifsnyder, C., McVey, M., Gazo, B., Pillus, L.
(2000). Two Classes of sir3 Mutants Enhance the sir1 Mutant Mating Defect and Abolish Telomeric Silencing in Saccharomyces cerevisiae. Genetics
155: 509-522
[Abstract]
[Full Text]
-
Enomoto, S., Johnston, S. D., Berman, J.
(2000). Identification of a Novel Allele of SIR3 Defective in the Maintenance, but Not the Establishment, of Silencing in Saccharomyces cerevisiae. Genetics
155: 523-538
[Abstract]
[Full Text]
-
Cockell, M. M., Perrod, S., Gasser, S. M.
(2000). Analysis of Sir2p Domains Required for rDNA and Telomeric Silencing in Saccharomyces cerevisiae. Genetics
154: 1069-1083
[Abstract]
[Full Text]
-
COCKELL, M., GOTTA, M., PALLADINO, F., MARTIN, S.G., GASSER, S.M.
(1998). Targeting Sir Proteins to Sites of Action: A General Mechanism for Regulated Repression. Cold Spring Harb Symp Quant Biol
63: 401-412
[Abstract]
Top
Abstract
Introduction
