Department of Microbiology, College of
Physicians & Surgeons of Columbia University, New York, New York
10032,1 and Department of Molecular
Biology, Sciences II, University of Geneva, 1211 Geneva 4, Switzerland2
Received 14 June 2001/Returned for modification 13 July
2001/Accepted 28 August 2001
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INTRODUCTION |
Related forms of transcriptional
silencing in the budding yeast Saccharomyces cerevisiae
occur at silent mating type loci (HML and HMR)
and immediately adjacent to the TG1-3 repeats at
telomeres (reviewed in references 15, 21, and
36). Both mating type and telomeric silencing (referred to
hereafter as telomere position effect [TPE]) require a common set of
trans-acting factors that include a complex of silent
information regulators (Sir2, Sir3, and Sir4 proteins) and the
amino-terminal tails of the core histones H3 and H4. Although the
precise molecular mechanisms underlying silencing are not known,
genetic and biochemical evidence indicates that it results from the
"spreading" of Sir protein complexes from sites of initiation
(HM silencers or telomeres) to nearby chromatin.
Significantly, both Sir3p and Sir4p can interact directly with the
histone H3 and H4 tails in vitro (23), pointing to a possible mechanism for the formation of a closed, or repressive, chromatin structure. Consistent with the idea that Sir2/3/4 protein complexes bring about repression through direct interactions with nucleosomes, silencing is neither promoter nor polymerase specific but
instead appears to have a general effect either on chromatin accessibility (19, 34, 54) or on the subsequent action of chromatin-bound factors (51a).
A key question, whose answer is still not clearly understood, is how
silencing is initiated only at specific chromosomal sites. None of the
Sir proteins appear to recognize DNA directly, and the initiation of
silencing thus requires a set of DNA-binding factors to recruit the Sir
proteins to their sites of action on the chromosome. At telomeres this
is accomplished in part by Rap1p, whose binding sites are contained
within the TG1-3 repeat tracts that comprise the
telomeric DNA (28, 32, 47). Rap1p also binds to three of
the four mating type gene silencers, where it collaborates with the
origin recognition complex (ORC) and/or Abf1p to initiate silencing
(6, 52). Strikingly, none of these three DNA-binding
factors is specific to silencers. Rap1p and Abf1p binding sites are
found in numerous promoter regions, where they typically act to
stimulate transcription (7). Likewise, ORC binding sites
are found at all known origins of DNA replication, most of which are
not silencers (the only known exception being the HMR-E
silencer). Therefore, a full understanding of the role of these
proteins in the initiation of silencing must also explain why they have
quite different functions in other contexts.
An important clue to the molecular mechanism of Rap1p action at
telomeres and silencers came from the identification of both Sir3p and
Sir4p as Rap1p-interacting proteins in the two-hybrid system
(47). Significantly, Rap1 and Sir3p can interact directly in vitro in the absence of other yeast proteins. These observations, together with other studies (11, 32), suggested that the
role of Rap1p in the initiation of silencing is to recruit Sir proteins to the chromosome by direct protein-protein interactions with Sir3p and
perhaps Sir4p. Strong support for this comes from the observation that
direct targeting of Sir proteins to HMR, by the use of Gal4p
DNA-binding domain (GBD)-Sir hybrids, bypasses
the requirement for the normal silencer binding sites (10,
40). Similarly, GBD-Sir and LexA-Sir
hybrids can restore silencing when targeted to a specific telomere in
cells deleted for the carboxy-terminal Sir interaction domain of Rap1p
(10, 37, 40).
To better understand the mechanisms underlying the recruitment of Sir3p
and Sir4p to silencers and telomeres, we extended our analysis of the
interactions of these two proteins with Rap1p. Using the two-hybrid
system and a glutathione S-transferase (GST) hybrid pulldown
assay, we have identified a Rap1p interaction domain of Sir3p that is
both necessary and sufficient for the interaction with the carboxyl
terminus of Rap1p. This short region of Sir3p, between amino acids 455 and 481, does not mediate Sir3p self-interaction or the Sir3p
interaction with Sir4p, and it does not correspond to a previously
characterized histone interaction domain (23). Deletion of
this domain debilitates Rap1p-dependent targeted silencing but has much
weaker effects on normal HM locus and telomeric silencing.
In addition, the silencing defect caused by these Sir3p mutations is
weaker than that obtained with Rap1p carboxy-terminal deletions, which
themselves cause defects in Sir3p binding (47).
We provide evidence that this difference is due to the ability of Rap1p
to interact independently with Sir4p. Significantly, overexpression of
Sir4p suppresses the HMR silencing defect displayed by Sir3p
mutants unable to bind Rap1p. These data support a model in which the
establishment of silencing at HM loci and at telomeres involves the recruitment of the Sir complex to the chromosome via a set
of cooperative interactions, involving direct binding of both Sir3p and
Sir4p to the carboxyl terminus of Rap1p. Taken together with previous
studies of Sir3p and Sir4p binding to histones (23), these
new insights into Rap1p-Sir interactions also suggest a mechanism by
which Rap1p may participate not only in initiation but also in the
propagation and maintenance of silent chromatin.
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MATERIALS AND METHODS |
Yeast strains and silencing assays.
Growth and manipulation
of yeast strains were done according to standard procedures
(51). The yeast two-hybrid reporter strain CTY10-5D
(MATa ade2-1 trp1-901 leu2-3,112 his3-200 gal4
gal80 URA3::lexAop-lacZ) was used in all studies
involving LexA fusion proteins. This strain, and its derivatives with
HIS3 disruptions of SIR1, SIR2,
SIR3, SIR4, or RIF1, has been
described elsewhere (47). All strains used for
transcriptional silencing assays are isogenic to strain W303-1B
(MAT
ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1
ura3-1) (59).
The two strains used for targeted silencing with
GBD/Rap1p fusions are
Leu
derivatives of two strains (relevant
genotypes:
AE(UASG)3hmr::TRP1 gal4::LEU2 sir3::URA3 and
EB(UASG)3hmr::TRP1
gal4::LEU2 sir3::URA3) described
previously (8). These strains were created as follows. First, the gal4::LEU2 gene was disrupted by
gene replacement using a
leu2::(UASG)::ADE2
construct in which the regulatory sequences of the ADE2 gene
have been replaced with UASG, allowing
Gal4p-dependent expression of the gene. The disruption was obtained by
cotransformation with plasmid pMA210 (38), which expresses
Gal4p, followed by selection on plates of synthetic complete (SC)
medium lacking Ade and His (SC
AdeHis).
Leu transformants were identified by replica
plating and then grown in liquid yeast extract-peptone-dextrose (YEPD)
medium to allow loss of the pMA210 plasmid. The cultures were spread on
YEPD plates, and cells with the desired phenotype
(Leu Ade
His) were selected. These cells were then
sequentially transformed, first with GBD/Rap1p
fusions (8) in the HIS3 integrating vector pRS303 (53) and then with mutant sir3 alleles
cloned into a low-copy-number CEN vector.
The strains used for the analysis of transcriptional silencing at the
HMR locus and at telomeres are derived from a series of
HMR::TRP1 and
HMR::ADE2 strains or from a
URA3-Tel VIIL strain, all of which have been
described previously (57, 58). sir3 disruption
derivatives of these strains were obtained by gene replacement using a
sir3::HIS3 deletion/disruption construct
(HMR::TRP1, HMR::ADE2, and URA3-Tel VIIL
strains) or a sir3::URA3 deletion/disruption (HMR::ADE2) strain. The HIS3
disruption removes all Sir3p coding sequences between amino acids 108 and 945. The URA3 disruption deletes the entire Sir3p open
reading frame (978 amino acids) up to position 972. Both gene
disruptions cause a complete loss of repression of the reporter gene
and can be complemented by a plasmid-borne copy of SIR3. For
the experiments with mutant alleles of SIR3, the cells were
transformed with SIR3 deletions cloned into plasmid pRS415
(LEU2 CEN) (53).
Assays for silencing using the HMR::TRP1
reporter were performed by spotting 10-fold serial dilutions of
cultures grown in the appropriate synthetic selective medium as
described previously (57). Repression was tested in
HMR::ADE2 strains by examining colony color
after 3 days of growth of the transformants at 30°C, followed by
storage of the plates at 4°C for 1 or more days. Determination of the
fraction of 5-fluoroorotic acid-resistant
(5-FOAr) and Ura+ cells in
URA3-Tel VIIL strains was done as follows. Independent colonies were grown in SCLeu liquid medium overnight, diluted to an
appropriate concentration, and then spread on SCLeu, SCLeuUra, and SCLeu+5-FOA plates. Colonies were counted after 3 days at 30°C,
and results from three or more independent cultures were used to
calculate an average value.
Plasmids.
The LexA protein and all LexA fusion proteins were
expressed from plasmid pBTM116 (2µm origin, TRP1
pADH1-lexA) (2). All LexA/Rap1p hybrids and the
LexA/Sir4p(839-1358) and LexA/Sir3p(307-978) constructs have been
described elsewhere (47). LexA/Sir1p, LexA/lamin, and
LexA/Adh1p were gifts from Rolf Sternglanz (State University of New
York at Stony Brook). All the Gal4p activation domain
(GAD) hybrids described here were
expressed from plasmid pACTII (2µm origin, LEU2,
pADH1-GAD). The SIR3 fragment used to
create GAD/Sir3p(307-978) was obtained from a
LexA/Sir3p(307-978) fusion described previously (47). All
the other GAD/Sir3p constructs used here were
obtained by two- or three-way ligation of SIR3 fragments to
the pACTII vector.
The construction of some of these constructs required intermediate
cloning steps in pUC19 or pIC series plasmids. All the carboxy-terminal
deletions of SIR3 in the pACTII vector have the same
amino-terminal junction as GAD/Sir3p(307-978)
and were created by cutting the SIR3 sequence with a
suitable restriction enzyme followed by repair of the end with the
Klenow fragment or T4 DNA polymerase and ligation to a flushed site in
the polylinker of the vector. All amino-terminal
GAD/Sir3p fusions other than
GAD/Sir3p(307-978) were created by using a
restriction sites in the SIR3 open reading frame or by
creating a site at a suitable position by PCR. More detailed
information on these constructs is available upon request.
GAD/Sir4p(839-1358) was obtained by ligating a
BamHI-SalI fragment of SIR4 to the
pACTII vector cut with BamHI and XhoI.
GAD/Sir4p(839-1275) was obtained by removing
SIR4 sequences beyond the EcoRV site. The
internal deletions of SIR3 were created either by
joining the flushed ends of DNA fragments cut at suitable restriction sites or by PCR cloning. All deletions cause the removal of
SIR3 sequences without causing the insertion of amino acids
that are not normally present in the protein. Allele 440-502 was
created by joining an amino-terminal fragment cut with EcoRI
followed by Klenow repair of the end to a carboxy-terminal fragment cut with HindIII and repaired with Klenow. Mutant
482-502 was created in the same manner by joining a
BsrFI end repaired with Klenow to the same blunt
HindIII end as above. All remaining SIR3
deletions were constructed using PCR by creating restriction sites at
new positions and subsequently removing sequences between the novel site and an existing site in SIR3.
Alleles 440-480 and 440-454 were constructed by creating an
EcoRI site at positions 481 and 455, respectively, followed by removal of SIR3 sequences between this novel site and the
EcoRI site at amino acid position 439. Allele 456-479
was constructed by creating a BsrFI site at amino acid
position 455 followed by ligation to the BsrFI site at
position 480. Mutant 333-357 was constructed by creating a novel
EagI site at position 333, and alleles 358-437 and
398-437 were constructed by creating new EcoRI sites at
positions 357 and 397, respectively. Fragments from these deletion
constructs of SIR3 were cloned in the pACTII vector for
two-hybrid studies, in a pT7-Sir3p construct for in vitro studies, and
in the pRS415 and pRS425 plasmids to test for transcriptional
silencing in vivo. The GST/Rap1p and pT7-Sir3p(1-978) constructs
used in this work have been described previously.
pT7-Sir4p(839-1358) and pT7-Sir4p(839-1275) are derived from the
corresponding GAD/Sir4p constructs. Constructs
used for the overexpression of SIR1 and SIR4 were created by cloning a gene fragment in the 2µm
URA3 vector pRS426 and in the CEN URA3
vector pRS316, respectively.
Transcriptional activation assays.
Transcriptional
activation assays using LexA hybrids or LexA and
GAD hybrid combinations were performed as
previously described (47).
In vitro protein-binding assays.
Protein expression and
purification and all in vitro binding studies were performed as
described previously (47).
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RESULTS |
Amino acids 455 to 481 of Sir3p are sufficient to mediate a
two-hybrid interaction with Rap1p but not Sir3p or Sir4p.
We
showed previously that the carboxy-terminal two-thirds of Sir3p (amino
acids 307 to 978) can interact with Rap1p, Sir4p, and itself in
two-hybrid assays (47). Here we have extended this
analysis to ask whether a defined region of Sir3p that is specifically
involved in binding to Rap1p could be identified. Beginning with a
construct expressing GAD/Sir3p(307-978), two sets of GAD/Sir3p hybrids with progressive
carboxy- or amino-terminal deletions of Sir3p were created and tested
for the ability to interact with a LexA/Rap1p(679-827) hybrid. As
shown in the top panel of Fig. 1,
GAD/Sir3p fusions with amino-terminal endpoints at positions 307, 356, 439, and 455 (and continuing to the carboxyl terminus at position 978) do not significantly differ in the
strength of their interaction with LexA/Rap1p(679-827).
However, two shorter fusions,
GAD/Sir3p(481-978) and
GAD/Sir3p(503-978), are completely defective
in this interaction. The failure of these latter two GAD/Sir3p constructs to give a signal in the
two-hybrid assay is not due to lack of expression of a functional
protein, since both fusion proteins interact normally with
LexA/Sir3p(307-978) (data not shown). From these results, we place
the amino-terminal endpoint of the minimal region of Sir3p required for
the two-hybrid interaction with Rap1p between amino acids 455 and
481.
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FIG. 1.
Interaction of a series of GAD/Sir3p
truncations with LexA/Rap1p(679-827) in the two-hybrid system.
GAD/Sir3p fusions with progressive amino- and
carboxy-terminal deletions of SIR3 sequences were
assayed for their interaction with LexA/Rap1p(679-827) using the
two-hybrid reporter strain CTY10-5D. Transcriptional activation,
measured as units of  -galactosidase (B-gal) activity, was normalized
to a value of 10,000 U for LexA/Gal4p(768-881)
[(LexA/GAD)], which was included as a control in all
experiments (not shown in the figure).
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The analysis of a series of carboxy-terminal deletions gives a more
complex picture than the amino-terminal set. Deletion of Sir3p
sequences between amino acids 978 and 910 does not significantly affect
the strength of the two-hybrid interaction with Rap1p, whereas two
larger deletions (truncations at positions 798 and 762) completely
abolish the two-hybrid signal (Fig. 1). Again, loss of the Rap1p
interaction with these two latter constructs is not due to lack of
expression of functional GAD/Sir3p hybrids, since
both of these fusions can interact with LexA/Sir4p(839-1358) as
strongly as GAD/Sir3p(307-978) (data not
shown). Surprisingly, with Sir3p deletions beyond amino acid 762, the two-hybrid signal with LexA/Rap1p is restored, weakly in the
case of GAD/Sir3p(307-734) and
GAD/Sir3p(307-685), but to high levels with
truncations at positions 598, 503, and 481 of Sir3p. The interaction is
abolished again with further deletions to endpoints at amino acids 455 and 439. Taken together, these data suggest that the carboxy-terminal limit of the minimal Sir3p region required for the interaction with
Rap1p may lie between amino acids 481 and 455. Although we do not know
why the carboxy-terminal truncations ending between residues 685 and
798 of Sir3p weaken or abolish interactions with Rap1p in the
two-hybrid assay, we note that this region of Sir3p has been linked to
its binding to core histone N-terminal tail sequences
(23). Perhaps, in the Sir3p truncations in question, histone interaction regions become exposed in such a way as to titrate
out the two-hybrid interaction with Rap1p.
To rule out the possibility that the amino-terminal 307 amino
acids of Sir3p that were not included in this analysis also contain
sequences able to mediate an interaction with Rap1p, we tested a
GAD/Sir3p(1-503) and a
GAD/Sir3p(1-439) fusion. Only the first of
these two constructs is able to give a signal in a two-hybrid assay
with LexA/Rap1p(679-827) (data not shown), suggesting that the
first 439 amino acids of Sir3p do not contain regions that are able to
interact with Rap1p. This result, as well as all data shown in Fig. 1,
were replicated with LexA/Rap1p(635-827) and
LexA/Rap1p(653-827) hybrids.
We have shown previously that LexA/Rap1p carboxy-terminal fusions
interact genetically with the endogenous SIR2,
SIR3, SIR4, and RIF1 genes.
Specifically, mutations in any one of these four genes enhance a
cryptic activation potential of certain LexA/Rap1p hybrids. In
addition, Rif1p and Sir3p appear to compete for binding to the carboxyl
terminus of Rap1p in the two-hybrid assay (47). To ask if
the analysis reported above was influenced by competition between the
GAD/Sir3p fusions and endogenous Sir2, Sir3,
Sir4, or Rif1 protein, we repeated these experiments in a set of
reporter strains containing null mutations in each of the corresponding genes. Only minor quantitative differences between mutant and wild-type
reporters were observed (data not shown), arguing against the
possibility of significant perturbation by any of the endogenous factors tested.
The data described above suggest that amino acids 455 to 481 of Sir3p
may contain all of the sequences required for a two-hybrid interaction
with the carboxyl terminus of Rap1p. To test this hypothesis
directly and to determine whether this small Sir3p fragment also
mediates homodimerization or association with Sir4p, we constructed a
GAD/Sir3p(455-481) fusion and tested it with LexA/Rap1p(679-827), LexA/Sir3p(307-978), and
LexA/Sir4p(839-1358). As shown in Table
1,
GAD/Sir3p(455-481) displays a significant association with LexA/Rap1p(679-827), demonstrating that a
fragment of only 27 amino acids of Sir3p is sufficient to mediate a
two-hybrid interaction with the carboxyl terminus of Rap1p. In
contrast, this small Sir3p fragment does not give a signal with
either LexA/Sir3p(307-978) or LexA/Sir4p(839-1358),
suggesting that amino acids 455 to 481 of Sir3p are not
sufficient to mediate a Sir3p self-interaction or binding to
Sir4p.
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TABLE 1.
Short stretch of 27 amino acids of Sir3p is sufficient to
mediate a two-hybrid interaction with Rap1p but not Sir3p or Sir4p
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Deletion of amino acids 456 to 479 of Sir3p specifically abolishes
the two-hybrid interaction with Rap1p.
To test the hypothesis that
the region between amino acids 455 and 481 in Sir3p is necessary for
the association with Rap1p but not required for either Sir3p
self-interaction or binding to Sir4p, we constructed a series of
GAD/Sir3p(307-978) fusions with small
deletions spanning the region between amino acid positions 333 and 502. As shown in Table 2, the deletion of 24 amino acids of Sir3p between positions 456 and 479 completely abolishes
the interaction with LexA/Rap1p(679-827), as do two larger
deletions encompassing the same region (440-480 and 440-502).
None of these three mutations significantly impair the interaction with LexA/Sir4p(839-1358) or LexA/Sir3p(307-978) hybrids
(Table 2), demonstrating the specificity of their effect on the
interaction with Rap1p.
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TABLE 2.
Deletion of as few as 24 amino acids of Sir3p abolishes
the interaction with Rap1p but not Sir3p or Sir4p in the two-hybrid
system
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To determine whether Sir3p sequences adjacent to the short region
between amino acids 456 and 479 play a role in this interaction, constructs with deletions amino-terminal to position 456 (440-454, 398-437, 358-437, and 333-357) and one deletion
carboxy-terminal of position 479 (482-502) were created. As shown
in Table 2, none of these constructs show any defect in the
interaction with LexA/Rap1p(679-827),
LexA/Sir4p(839-1358), or LexA/Sir3p(307-978). The results
of this analysis did not change when the interactions were tested in
two-hybrid reporter strains carrying mutations in either
SIR2, SIR3, SIR4, or RIF1
(data not shown). In summary, these data demonstrate that amino acids
456 to 479 of Sir3p are necessary for the interaction with the carboxyl
terminus of Rap1p in the two-hybrid system but do not play any
detectable role in binding of Sir3p to itself or Sir4p.
Amino acids 455 to 481 of Sir3p are required for binding to
carboxyl terminus of Rap1p in vitro.
Using a GST pulldown assay,
we showed previously that Sir3p can bind to the carboxyl terminus of
Rap1p (47). The same approach was used to test whether
Sir3p sequences required for Rap1p interaction in the two-hybrid assay
are also necessary for binding in vitro (see Materials and Methods for
details). Three 35S-labeled Sir3p mutant proteins
were analyzed, two of which (456-479 and 440-480) contain
deletions that abolish the Rap1p-Sir3p two-hybrid interaction. The
third mutant (440-454) has a deletion of sequences that do not
appear to play a role in the Rap1p interaction, as judged by the
two-hybrid assay.
As shown in Fig. 2, wild-type Sir3p and
Sir3p(440-454) are both able to bind to the
GST/Rap1p(562-827) fusion. In contrast, Sir3p(456-479) and
Sir3p(440-480) show a strong impairment in binding to
GST/Rap1p. These data confirm the results of the two-hybrid analysis of
the interaction between Rap1p and Sir3p and indicate that amino acids
456 to 479 of Sir3p are required for strong binding to the carboxyl
terminus of Rap1p in vitro. Previous studies have shown that this
region of Sir3p does not appear to be required for in vitro binding to
the amino termini of histones H3 and H4 (23). Taken
together, the results described above identify amino acids 455 to 481 of Sir3p as a Rap1p interaction domain of this protein.
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FIG. 2.
Sir3p mutants with a deletion of amino acids 456 to 479 are defective in binding to GST/Rap1p(562-827) in vitro. See
Materials and Methods for details of the binding assay. Right, in
vitro-produced labeled wild-type (WT) and mutant proteins (from a
separate gel) before addition to GST/Rap1p(562-827) or GST-agarose
beads. Left, material bound to the GST/Rap1p beads in each lane
[Sir3p(1-978), Sir3p(456-479), Sir3p(440-480), and
Sir3p(440-454)] has the same mobility as the primary
high-molecular-weight translation product in the right panel (note that
the order of lanes in the two gels is not the same). Lane M, size
standards.
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Targeted silencing by GBD/Rap1p hybrids requires Rap1p
interaction domain of Sir3p.
If the Rap1p interaction domain of
Sir3p identified above plays any role in repression of transcription in
vivo, its deletion should cause an impairment of silencing. In
addition, mutations of Sir3p that do not affect the interaction with
Rap1p, Sir4p, or Sir3p should not cause a silencing defect unless other
functions required for silencing were affected. To test these ideas, we first used a targeted silencing assay (8) in which
GBD/Rap1p hybrids are tethered to mutated
HMR-E silencer elements containing Gal4p binding sites
(UASG). In this assay, the restoration of silencing is critically dependent upon a small carboxy-terminal domain
of Rap1p(667-827) with which Sir3p interacts.
Two reporter strains with different mutant HMR-E silencers
were used in these experiments. In the first strain, the
HMR-E silencer is deleted for the ORC binding site (A) and
the Rap1p-binding site (E), and the two elements are replaced by
three UASG sites [AE(UASG)3].
In the second strain, the Rap1p-binding site and the
Abf1p-binding site (B) are removed and replaced by the same UASG trimer
[EB(UASG)3].
In both strains, the MATa1 gene at
HMR is replaced by TRP1, and silencing is
measured by the ability of the strains to grow on medium lacking
tryptophan (57). To target the Rap1p carboxyl terminus to
these altered silencers, a construct expressing a
GBD/Rap1p(653-827) fusion protein is integrated at the HIS3 locus. To test for the
SIR3 dependence of targeted silencing, the chromosomal copy
of SIR3 is disrupted in these strains and either the
wild-type or a series of mutant SIR3 alleles (the same
mutations tested in the two-hybrid analysis introduced into the
full-length SIR3 gene) are expressed from a low-copy-number
centromere-containing (CEN) vector.
The top panel of Fig. 3 shows results
obtained with the
AE(UASG)3::TRP1
strain. As expected, the TRP1 gene is efficiently repressed
(as judged by an approximately 100-fold decrease in the ability to form
colonies on medium lacking tryptophan) in the presence of wild-type
SIR3 on a plasmid (top row) and is completely derepressed
(full growth on Trp medium) in cells transformed with the control
vector alone (bottom row). Significantly, four SIR3 alleles
with a deletion of the Rap1p interaction domain (456-479, 440-480, 440-502, and 356-502) display an almost complete
loss of repression (Fig. 3, rows 2 to 5). In contrast, Sir3p deletions that did not cause a defect in the two-hybrid interaction with Rap1p
(440-454, 398-437, 358-437, and 333-357) support
silencing in this assay as well as wild-type Sir3p (Fig. 3, rows 6 to 9).
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FIG. 3.
Targeted silencing of an
hmr::TRP1 reporter by a
GBD/Rap1p(653-827) hybrid with different
HMR-E silencer deletions and a series of
SIR3 alleles. Silencing is measured by comparing the
ability of cells to grow in the absence (SCTrp) and presence
(SC) of tryptophan. Each row consists of spots representing
5-µl aliquots from a set of 10-fold serial dilutions of an overnight
liquid culture. Photographs were taken after 2 to 3 days of growth at
30°C.
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The results obtained with the same set of SIR3 alleles in
the
EB(UASG)3::TRP1
strain (bottom panel of Fig. 3) are qualitatively similar. In this
strain, however, targeted silencing in the SIR3 wild-type
background appears much stronger, and the derepressing effect of the
Rap1p interaction domain deletions (rows 2 to 5) is correspondingly
weaker than in the
AE(UASG)3
silencer strain. Nonetheless, we again observe little or no silencing
defect for the SIR3 alleles with deletions outside of the
Rap1p interaction domain (rows 6 to 10). This difference between the
two silencers suggests that the ORC plays a more important role than
Abf1p in recruitment of the silencing complex to the chromosome in
cooperation with Rap1p (60) (see Discussion). In both
strains these results were replicated using
GBD/Rap1p hybrids expressing either a longer or a
shorter fragment of the carboxyl terminus of Rap1p (data not shown),
suggesting that the different effect of the SIR3 mutations in the two strains is not the result of a peculiar feature of the
GBD/Rap1p(653-827) hybrid.
The direct targeting of either Sir3p or Sir4p to the HMR
locus in cells lacking a functional HMR-E silencer is also
sufficient to restore repression (40). In order to
determine whether the defect displayed by the SIR3
alleles tested above was specific to Rap1p-dependent silencing, we
analyzed GBD-Sir3p(1-978)- and GBD-Sir4p(1-1358)-dependent repression in
the presence of the same SIR3 mutants tested above. None of
the SIR3 alleles displays a noticeable silencing defect with
either GBD/Sir3p or
GBD/Sir4p (data not shown). These data
demonstrate that the defect caused by the SIR3 mutations is
bypassed when either Sir3p or Sir4p is targeted to the silencer by a
Rap1p-independent mechanism. Therefore, these small SIR3
deletions do not cause a general impairment of repression and are thus
more likely to specifically affect a step in which Sir3p is recruited
by Rap1p to the silencer.
Deletion of Rap1p interaction domain of Sir3p impairs silencing at
HMR locus when the silencer ORC binding site is
deleted.
We next tested the effect of SIR3 mutations on
silencing initiated by wild-type silencers at HMR as well as
two mutated but fully functional silencers in which the redundant ORC
(A element) or Abf1p (B element) binding sites at HMR-E were
deleted (5, 26, 57). As shown in the top and bottom panels
of Fig. 4, both wild-type and mutant
alleles of SIR3 are able to restore full silencing in both
HMR::TRP1 and
hmrB::TRP1 cells. Only control cells
lacking a functional copy of SIR3 are fully derepressed (last row of each panel in Fig. 4). In contrast, Sir3p mutants with a
deletion of the Rap1p interaction domain (456-479, 440-480, 440-502, and 356-502) display a partial silencing defect in hmrA::TRP1 cells (middle panels of Fig.
4). In these mutants, the ability to grow on medium lacking tryptophan
is increased about 103-fold compared with cells
expressing wild-type Sir3p (first rows of the panels), but is reduced
102- to 103-fold
compared with cells transformed with the CEN plasmid control (last rows of the panels). Significantly, deletion of Sir3p sequences amino-terminal of the Rap1 interaction domain (440-454,
398-437, 358-437, and 333-357) does not cause any
impairment of repression in HMRA::TRP1
cells.
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FIG. 4.
Transcriptional silencing at the HMR
locus with different HMR-E silencer deletions and a
series of SIR3 alleles. The assays were performed as
described for Fig. 3.
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The fact that Sir3p mutants defective in the Rap1p interaction have a
partial silencing defect in hmrA::TRP1
cells but no impairment in HMR::TRP1 or
hmrB::TRP1 cells differs from results obtained with Rap1p mutants defective in Sir3p interaction, in which
there is a complete silencing defect in both
hmrA::TRP1 and
hmrB::TRP1 cells but no loss of repression
in HMR::TRP1 cells (47). To test
the possibility that these Sir3p mutants have a weak silencing defect
that our assay was not sensitive enough to detect, we tested one of
them (456-479) in strains in which the ADE2 reporter is
integrated at HMR in cells containing wild-type or mutant
HMR-E silencer (A and B)
(58). The ADE2 gene has been shown to be
efficiently repressed at HMR and allows very sensitive
detection of weak silencing defects by a simple nonselective colony
color assay. Although two different SIR3 deletion-insertion mutations (removing amino acids 108 to 945 or nearly the entire open
reading frame [ORF], amino acids 1 to 972) caused strong derepression
of all three ADE2 reporters, the Sir3p(456-479) deletion showed a strong silencing defect only in the
hmrA::ADE2 strain (data not shown),
consistent with the results obtained in the TRP1 reporter
strains. Taken together, these data suggest that loss of Sir3p binding
to the carboxyl terminus of Rap1p causes a partial silencing defect at
HMR, but only when the HMR-E silencer lacks a
functional ORC binding site.
Deletion of Rap1p interaction domain of Sir3p impairs telomeric
silencing.
At telomeres, multiple Rap1p binding sites are found
within the terminal TG1-3 repeats (16,
33), where the Rap1 protein is involved in regulation of both
telomere structure and telomeric silencing (8, 27, 28, 32, 41,
47). We showed previously that mutations of the Rap1p carboxyl
terminus that impair Sir3p binding also impair telomeric repression
(47). If the Sir3p mutants described above were unable to
bind to native Rap1p in vivo, one would predict that they should cause
a telomeric silencing defect.
We tested this idea using a standard telomeric silencing assay in which
the URA3 gene is placed immediately adjacent to a telomere
created at the ADH4 locus (20). In these cells,
the telomeric URA3 reporter gene is subject to a variegated
form of silencing that results in the repression of the gene in 
50%
of the cells in a culture, which can be quantified by measuring the ability of the cells to grow in the presence of 5-FOA, which kills cells expressing URA3. We created a sir3
derivative of this strain and transformed the cells with plasmids
carrying wild-type SIR3, two alleles encoding a deletion of
the Rap1p interaction domain [Sir3p(456-479) and
Sir3p(440-480)], and one allele with a mutation that does not
cause an impairment of Rap1p interaction [Sir3p(333-357)].
As shown in Table 3, growth in medium
containing 5-FOA is impaired in cells expressing Sir3p(456-479)
and Sir3p(440-480) compared with cells expressing wild-type
Sir3p. About 45% of the cells expressing wild-type Sir3p are able to
grow in the presence of 5-FOA. On the other hand, only 27 and 26% of
the total cell population are able to grow in the same medium for cells
expressing Sir3p(456-479) and Sir3p(440-480),
respectively, resulting in a decrease of about 40 to 43% compared with
the wild type. In contrast, the control mutant Sir3p(333-357)
gives wild-type levels of growth in 5-FOA of about 50%, demonstrating
the specificity of the deletion of the Rap1p interaction domain in
telomeric repression. No significant differences were measured between
wild-type and mutant alleles of SIR3 in growth in the
absence of uracil.
These data indicate that, as was the case for native silencing at
HMR, loss of a single direct contact between Rap1p and Sir3p causes a relatively weak impairment of telomeric silencing compared to
the effect of deleting the carboxy-terminal Sir3p-interacting domain of
Rap1p (28, 47). One possible explanation for these results
is that the Sir3p mutations that we created cause only a partial loss
of Rap1p-Sir3p binding in vivo and thus do not significantly compromise
the ability of Rap1p to recruit Sir3p to the chromosome. An
alternative, though not mutually exclusive, possibility is that the
carboxyl terminus of Rap1p possesses additional mechanisms to recruit a
functional Sir complex. For example, Orc1p has been shown to play an
important role in transcriptional silencing (3, 14, 44)
and has extensive regions of similarity with Sir3p over the full length
of the protein (4). We thus used the two-hybrid system to
ask whether the Rap1p carboxyl terminus might also be able to interact
with Orc1p, which might in turn recruit the Sir2/3/4 complex through
its ability to interact with Sir1p (60). However, we found
that a GAD/Orc1p(5-914) hybrid, capable
of interacting with LexA/Sir1p, failed to interact with either
LexA/Rap1p(635-827) or LexA/Rap1p(679-827) (data not
shown). This result suggests that Orc1p may not interact with the
carboxyl terminus of Rap1p and may be unable to substitute for Sir3p in Sir complex recruitment. Another possible explanation of the relatively weak silencing effect caused by a Rap1p/Sir3p interaction defect is
that Rap1p interacts directly with Sir4p, which can itself associate
with Sir3p. This possibility is addressed below.
Sir4p binds to Rap1p in vitro and interacts with Rap1p carboxyl
terminus in vivo in the absence of endogenous Sir or Rif1
proteins.
We had previously shown that the carboxyl terminus of
Sir4p can interact with Rap1p in a two-hybrid assay, but were unable to
determine whether or not this interaction is direct
(47). To address this question, we first used a GST
pulldown assay. The same GST/Rap1p(562-827) fusion and binding
conditions used in the analysis of the Rap1p-Sir3p interaction were
used with two different 35S-labeled fragments of
Sir4p [Sir4p(737-1358) and Sir4p(737-840)]. As shown in
Fig. 5, the Sir4p(737-1358) fragment
can interact with GST/Rap1p(562-827), whereas the shorter
Sir4p(737-840) fragment cannot bind specifically to Rap1p even
when larger amounts of the 35S-labeled protein
are used. These data indicate that functionally important parts of
Sir4p and Rap1p can interact directly in vitro or at least without the
assistance of other yeast proteins.
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FIG. 5.
Sir4p carboxyl terminus binds to GST/Rap1p(562-827)
in vitro. See Materials and Methods for details of the binding assay.
Right, in vitro-produced labeled carboxy-terminal fragments of
Sir4p(839-1358) and Sir4p(839-1275) (from a separate gel)
before addition to GST/Rap1p(562-827) or GST-agarose beads. Left,
material bound to the GST/Rap1p beads in the Sir4p(839-1358) lane
has the same mobility as the higher-molecular-weight translation
product in the right panel. Lane M, size standards.
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To determine whether Rap1p and Sir4p might associate directly in vivo,
we extended our previous two-hybrid analysis in which an interaction
between GAD/Sir4p(1205-1358) and
LexA/Rap1p(635-827) but not shorter Rap1p fusions (amino-terminal
endpoints at positions 647, 653, 655, 667, 679, and 691) was detected
(47). Reasoning that a GAD/Sir4p
construct expressing a larger fragment of the carboxyl terminus of
Sir4p might be able to interact more strongly with Rap1p, we
constructed and tested a GAD/Sir4p(839-1358)
fusion with the same series of LexA/Rap1p constructs. As shown in Fig. 6 (first and third columns), LexA/Rap1p
fusions with amino-terminal endpoints between positions 647 and 679 can
weakly but specifically interact with
GAD/Sir4p(839-1358). These five LexA/Rap1p
fusions are also able to interact with
GAD/Sir3p(307-978) and
GAD/Rif1p(1614-1916) in two-hybrid assays
(47). In contrast, LexA/Rap1p(691-827) does not show
a significant difference in -galactosidase signal with
GAD/Sir4p(839-1358) compared to the
GAD-alone control. This last LexA/Rap1p fusion is
unable to interact with either GAD/Sir3p or
GAD/Rif1p but expresses a protein of the
expected size and in amounts comparable to those produced by the other
LexA/Rap1p constructs (data not shown). As a control for the
Rap1p-Sir4p interaction, we tested
GAD/Sir4p(839-1358) with a LexA/Adh1p
fusion. The last two rows of Fig. 6 show that
GAD/Sir4p(839-1358) does not interact
significantly above background with either LexA/Lamin or LexA/Adh1p,
suggesting that the Rap1p-Sir4p interaction is specific.
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FIG. 6.
Interaction of
GAD/Sir4p(839-1358),
GAD/Sir4p(839-1275), and GAD
with a series of LexA/Rap1p hybrids using the two-hybrid system.
LexA/Rap1p hybrids with different amino-terminal Rap1p fusion endpoints
and either a wild-type Rap1p carboxyl terminus (647-827, 653-827,
655-827, 667-827, 679-827, and 691-827) or a linker insertion
mutation at amino acid position 825 (653-825*) were assayed as before.
The LexA/Lamin and LexA/Adh1p hybrids were included as negative
controls.
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Based on the results shown above, the possibility remains that the in
vivo Sir4p-Rap1p interaction is indirect, mediated, for example, by an
interaction between these two proteins and Sir3p (47) or
another silencing protein(s). To determine whether the Rap1p-Sir4p
two-hybrid interaction requires the function of the endogenous Sir
proteins or Rif1p, we tested all of the interactions reported above in
strains containing mutations in SIR1, SIR2, SIR3, SIR4, or RIF1. None of the
interactions are abolished in the mutant strains (data not shown),
supporting the idea that Rap1p and Sir4p interact directly in vivo and
that this interaction does not require a functional Sir2/3/4 complex.
Silencing-defective mutations in Rap1p or Sir4p abolish the
two-hybrid interaction between the two proteins.
Truncation of
Sir4p at position 1237 in the sir4-42 mutant causes a
silencing defect at HM loci and telomeres (25).
To determine whether this phenotype might result from an inability of
the truncated protein to interact with Rap1p, we created a
GAD/Sir4p(839-1275) fusion and tested it for
the ability to interact with the carboxyl terminus of Rap1p. As shown
in the middle column of Fig. 6, deletion of the last 83 amino acids of
Sir4p completely abolishes the two-hybrid interaction with all
LexA/Rap1p fusions. This result demonstrates that the interaction of
GAD/Sir4p with LexA/Rap1p is dependent on Sir4p
sequences of the GAD hybrid and suggests that a
short carboxy-terminal domain of Sir4p required for silencing may also be necessary for its physical association with Rap1p in vivo.
To ask whether the converse is also true, we analyzed a
silencing-defective Rap1p mutation for its effect on the two-hybrid interaction with Sir4p. As shown in Fig. 6, the incorporation of a
small linker insertion mutation at position 825 of Rap1p [Rap1p(825*)], which results in the addition of five amino acids at the carboxyl terminus of the protein, completely abolishes the
ability of the LexA/Rap1p(653-825*) fusion to interact with GAD/Sir4p(839-1358). Since
LexA/Rap1p(653-825*) is much less severely impaired in its
interactions with both Sir3p and Rif1p compared to the wild type (1.6- and 3.8-fold decreases, respectively [47]), the complete
absence of an interaction with
GAD/Sir4p(839-1358) is unlikely to be due to
decreased expression or stability of the LexA/Rap1p fusion protein.
Taken together, these data are consistent with the idea that a direct
Rap1p-Sir4p interaction plays an important role in silencing.
Increased gene dosage of SIR4 but not
SIR1 improves silencing in sir3 mutants.
The gene dosage of both SIR1 and
SIR4 can have profound effects on HMR
silencing. For example, elevated gene dosage of SIR1 can
suppress various silencing mutations (29, 55, 57), whereas increased SIR4 dosage can improve or disrupt silencing
depending on the genetic background and the number of added copies of
the gene (39, 42, 50, 57, 58). One possible mechanism for an improvement in silencing with increased SIR1 or
SIR4 dosage is increased availability of these proteins
at the silencers, which might act to strengthen the formation of a
complex that nucleates heterochromatin assembly.
We reasoned that the decreased silencing seen at
hmrA::TRP1 in SIR3 mutants
defective in Rap1p binding might be explained by the combined loss of
two independent interactions, one between the Sir2/3/4p complex and
Rap1p and the other between the Sir2/3/4 complex and Sir1p/ORC
(60). The loss of the first interaction would be the
result of the SIR3 mutation, whereas the loss of the second
would be explained by the deletion of the ORC binding site (the
silencer A element). We therefore asked whether increased SIR1 or SIR4 dosage might improve silencing under
these mutant conditions. As shown in Fig.
7, one or two extra copies of
SIR4 can strongly suppress the silencing defect exhibited by
SIR3 mutants that are defective in binding to Rap1p. The
partial derepression of hmrA::TRP1 in the
presence of Sir3p(456-479) or Sir3p(440-480) is
completely reversed by the addition of one extra copy of
SIR4 (Fig. 7, rows 2 and 3 of top panels). In contrast, much
higher gene dosage of SIR1 (present on a 2µm plasmid) has
no effect under these conditions (rows 2 and 3 of bottom panels). These
results are consistent with the hypothesis that Sir4p interacts with
the carboxyl terminus of Rap1p and contributes to the recruitment of
the Sir complex to the HMR-E silencer. In addition, the data suggest that Sir1p may act at the HMR-E silencer only when a
strong ORC binding site is present (10, 60).
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FIG. 7.
Extra dosage of SIR4 suppresses the
silencing defect caused by mutation of the ARS consensus
sequence at the HMR-E silencer in combination with the
deletion of the Rap1p interaction domain of Sir3p. Cells expressing
different SIR3 alleles were transformed with a
low-copy-number CEN plasmid containing the
SIR4 gene (top panels) or a high-copy-number 2µm
plasmid containing SIR1 (bottom panels). The assays were
performed as described for Fig. 3.
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DISCUSSION |
Specific Rap1p interaction domain of Sir3p.
We have identified
a minimal Rap1p interaction domain of Sir3p between amino acids 455 and
481 that, by several criteria, appears to play an important and
specific role in the initiation of silencing. First, this short region
is sufficient to mediate a two-hybrid interaction with the carboxyl
terminus of Rap1p yet is incapable of interacting with either Sir3p or
Sir4p. Second, deletion of amino acids 456 to 479 in Sir3p completely
abolishes its association with Rap1p in the two-hybrid assay but does
not affect its Sir3p or Sir4p interactions. Third, deletion of the 456 to 479 region impairs Sir3p binding to the carboxyl terminus of Rap1p
in vitro. Fourth, deletion of this small region of Sir3p causes a
complete loss of silencing at HMR when the initiation of
repression is dependent on targeting of the carboxyl terminus of Rap1p,
but not when it is initiated by either Sir3p or Sir4p targeting.
It is worth noting that the Rap1p interaction domain of Sir3p
identified here maps well upstream on the linear protein sequence of
regions required for self-association, Sir4p binding, or histone binding. A Sir3p interaction with the amino-terminal tails of histones
H4 and H3 requires regions between amino acids 623 and 762 and 799 and
910 of Sir3p and is unaffected by deletion of the Rap1p interaction
domain defined here (23). Similarly, we have also mapped
Sir3p sequences required for self-association and Sir4p binding to two
separate regions, both of which are carboxy-terminal to the Rap1p
interaction domain (unpublished results), and within a broad region
defined by Park and colleagues (49). The picture that
emerges from these studies is that Sir3p may be able to interact simultaneously with several different proteins (Rap1p, Sir4p, and
histones H3 and H4). The possible significance of this observation is
discussed below.
Rap1p carboxy-terminal silencing domain can interact directly and
independently with both Sir3p and Sir4p.
Previous studies showed
that Rap1p and Sir4p coimmunoprecipitate from yeast extracts (11,
24, 56) and interact in the two-hybrid system (47).
However, none of these studies were able to address the question of
whether Rap1p and Sir4p interact directly and, if so, whether their
association is important for silencing. Here we present the first
demonstration that Rap1p and Sir4p can bind to each other directly in
vitro in the absence of other yeast proteins and that their in vivo
interaction in a two-hybrid assay is independent of SIR
function. These findings suggest that the Rap1p-Sir4p association seen
in large chromatin complexes is due, at least in part, to direct
interactions that contribute to the stability of these complexes. As
discussed in detail below, genetic data reported here indicate that
direct binding between Rap1p and Sir4p is important in vivo for silencing.
On the basis of the results presented here, we propose that Sir3p and
Sir4p interact directly and independently with partially overlapping
regions of the carboxyl terminus of Rap1p. Although both Sir
interactions require an intact Rap1p carboxyl terminus, the
amino-terminal boundaries of the Rap1p sequences required for Sir3p and
Sir4p association differ. A relatively small carboxy-terminal fragment
(amino acids 679 to 827) interacts strongly with Sir3p but only weakly
with Sir4p, which requires sequences upstream of position 679 for
stronger Rap1p binding. This increase is specific to Sir4p and thus
unlikely to be a trivial consequence of increased protein stability,
because these larger Rap1p hybrids actually interact less well with
Sir3p (47). The importance of Rap1p sequences upstream of
679 for silencing is underscored by the observation that
GBD/Rap1p hybrids containing these additional sequences initiate repression much more efficiently than does GBD/Rap1(679-827) (8). It is
worth pointing out that more amino-terminal sequences of Rap1p might
also contribute to Sir protein binding, particularly for the case
of Sir4p. However, LexA/Rap1p hybrids containing such sequences,
which include the Rap1p DNA-binding domain, do not work in the
two-hybrid system.
Given the above considerations, we favor the idea that Sir3p and Sir4p
simultaneously contact the Rap1p carboxyl terminus in vivo. Although
our experiments do not address this question directly, the fact that
Sir3p and Sir4p can interact with each other (47) through
regions not required for their interactions with Rap1p (our unpublished
data) suggests that these two Sir proteins form a unique ternary
complex with Rap1p that is stabilized by interactions among all three
proteins. An alternative possibility that we cannot rule out at present
is that Rap1p-Sir3p and Rap1p-Sir4p interactions are mutually
exclusive, so that individual Rap1p molecules interact with one or the
other Sir protein in forming a Rap1/Sir complex.
Cooperativity in Sir complex recruitment by Rap1p.
The
apparent ability of Rap1p to interact independently with both Sir3p and
Sir4p and the ability of these two proteins to interact with themselves
and each other (9, 47) suggest a cooperative and redundant
mechanism for recruitment of the Sir complex by Rap1p. This notion is
strongly supported by the genetic studies described here. Thus,
although a small region of Sir3p(455-481) is required (by both in
vitro and in vivo criteria) for an interaction with Rap1p,
SIR3 mutants lacking this specific domain display only a
weak silencing defect even when the silencer being tested is totally
dependent on Rap1p. Significantly, however, when such mutants are
tested in a targeted silencing system (where Sir4p recruitment by
GBD/Rap1p may be poor), they are severely
silencing defective. The simplest interpretation of these results is
that the Rap1p-Sir3p interaction that we have characterized is
important but not essential for Rap1p's action at either
HMR or a telomere. The same appears to hold for the
Rap1p-Sir4p interaction, which is specifically abolished by a linker
insertion mutation very near the carboxyl terminus of Rap1p with
relatively little effect on TPE (47). In contrast, the
loss of a Rap1p interaction with both Sir3p and Sir4p (due to Rap1p
truncations at amino acids positions 716, 703, and 695) causes a
complete TPE defect (28, 47).
The idea that Sir3p and Sir4p cooperate in recruitment to silencers and
telomeres by Rap1p is further supported by the observation that
increased SIR4 gene dosage significantly improves silencing in cells carrying a deletion of the Rap1p-interacting domain of Sir3p. Finally, the idea that Sir3p can interact independently with
Rap1p in vivo is strongly supported by coimmunoprecipitation studies
using an antigen-tagged version of Sir3p (24), which showed that mutation of SIR4 reduces but does not abolish
Rap1p binding to Sir3p.
In addition to the Rap1p-Sir protein interactions described here, a
large number of other protein-protein interactions have been implicated
in either the establishment or maintenance of silencing at either
HM mating type loci or telomeres (e.g., ORC-Sir1p, Yku70p-Sir4p, Sir1p-Sir4p, Sir3p-Sir3p, Sir4p-Sir4p, Sir4p-Sir2p, Sir3p-H3, Sir3p-H4, Sir4p-H3, and Sir4p-H4) (9, 11, 24, 31,
45-47, 60). Additional protein-protein interactions (e.g., Rap1-Rif1p, Rap1p-Rif2p, and Sif2p-Sir4p) appear to downregulate silencing at telomeres (12, 22, 28, 61). At present it is
unclear why the recruitment and assembly of Sir proteins at HM loci and telomeres involve such a complex network of
interactions. One possibility is that this complexity is necessitated
by the tight regulation of Sir2/3/4-mediated silencing, which is
particularly stable at HM mating type loci, less so at
telomeres, and excluded from most other chromosomal sites
(62). This "hierarchy" of silencing (1)
has been linked to the different complexity of the silencers
themselves, and the present work lends further support to this idea.
For example, we found that mutation of the Rap1p interaction domain of
Sir3p has a more severe effect at an HMR-E silencer lacking
the ORC binding site (A element) than at a silencer lacking the Abf1
site (B element). These data indicate that the different silencer
elements make independent and quantitatively different contributions to
the strength of the silencer. This conclusion is supported by evidence
that the silencer A element recruits Sir1p and, indirectly, Sir4p
through the ORC (60), whereas Abf1p appears to act by
recruiting Sir3p (P. Moretti and D. Shore, unpublished data).
Structural role for Rap1p in silent chromatin?
Initial
molecular models suggested that silencer- or telomere-binding proteins
(e.g., Rap1p, ORC, Abf1p, and Yku70/80) initiate silencing by
recruiting a Sir2/3/4 complex to the chromosome, but do not participate
directly in the subsequent "spreading" of this complex along
adjacent nucleosomal DNA (21, 35, 36). However, several
studies using chromatin immunoprecipitation (ChIP) have now shown that
Rap1p and Yku80p are bound at distances of 2 to 4 kb from a telomere
end, together with the Sir2/3/4 proteins (13, 24, 43, 56).
These results are surprising in light of the fact that the
TG1-3 repeat tracts (which constitute the telomeric Rap1p
DNA-binding sites) extend only about 300 to 400 bp from the chromosome
end and that Yku protein is generally thought to interact with the end
of the TG1-3 repeat tract, based on its in vitro
preference for DNA ends or duplex/single-strand junctions. The ChIP
results have thus been interpreted to mean that the telomere repeat
tract folds back on more internal nucleosomal regions through Sir-Sir
interactions between these two domains (13, 17, 56), thus
associating repeat tract-bound Rap1p indirectly with distal heterochromatin.
Our results suggest an additional explanation for this finding.
Specifically, we propose that Rap1p might contribute directly to the
stability of silent chromatin through simultaneous interactions with
the Sir2/3/4 complex and (nonspecific) DNA sites. According to this
model, Rap1p spreads together with the Sir2/3/4 complex by virtue of
its ability to bind cooperatively to Sir3p and Sir4p and to nonspecific
DNA sites. The finding that Sir3p may be able to simultaneously contact
both Rap1p and histone tails raises the intriguing possibility that the
Sir complex can promote the stable coassociation of Rap1p and
nucleosomes on silent regions. This model might help to better explain
two puzzling observations regarding telomeric heterochromatin. First,
coimmunoprecipitation experiments with antibodies against Sir3p show
that interactions with Rap1p and histone H4 are surprisingly
interdependent, so that Sir3p-Rap1p binding appears to be lost in
strains containing H4 amino-terminal tail mutations
(23). These data are difficult to reconcile with a simple
version of the telomere "fold-back" model but are
consistent with the proposal that the bulk of Rap1p-Sir3p interactions
at telomeres actually occurs within the silent chromatin itself through
cooperative interactions between histones and Sir3/4 proteins on the
one hand and DNA-bound Rap1p and Sir3/4 on the other. Second, indirect
immunofluorescence experiments have revealed a significant loss of
punctate Rap1p staining in strains carrying either SIR3 or
SIR4 mutations (48), which cannot be explained by a loss of telomere clustering (18). This result would
be predicted if one assumes again that a large pool of
telomere-associated Rap1p is not bound directly to the
TG1-3 repeat tracts but is instead held there by
nonspecific binding and cooperative interactions with the Sir complex
(17). It may prove difficult, however, to distinguish
experimentally between different models to explain the spreading of
Rap1p in telomeric heterochromatin. Furthermore, none of these models
are mutually exclusive. Perhaps a good test of the idea that Rap1p is a
structural component of the silent chromatin would be to determine if
Rap1p also spreads together with the Sir complex at HM
silent mating type loci, where a telomere fold-back model would not apply.
While our manuscript was under revision, a whole-genome analysis of
both Rap1p and Sir protein binding in vivo was published (30). Data presented in that paper indicate considerable
spreading of Rap1p (together with Sir2p, Sir3p, and Sir4p in most
cases) to sequences outside of those bounded by the E and I silencers at both HML and HMR. As pointed out above, this
observation is consistent with our model but difficult to accommodate
in the context of a fold-back-type model for Rap1p spreading outside of
its high-affinity binding sites.
We thank members of the Shore laboratory, in particular Stephen
Buck and Stéphane Marcand, for helpful comments throughout the
course of this study. We are particularly grateful to Saul Silverstein
for continuous support. We also thank Nicholas Roggli for help with the figures.
This work was supported by grants from the National Institutes of
Health (GM-40094), the American Cancer Society (VM-62A), the
Swiss National Science Foundation, and by funds from the Canton of Geneva.
| 1.
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Aparicio, O. M.,
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Abstract
Introduction
Present address: Department of Molecular and Human Genetics, Baylor
College of Medicine, Houston, TX 77030.
