Article

Subtelomeric Elements Influence But Do Not Determine Silencing Levels at Saccharomyces cerevisiae Telomeres

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
Genetics (Impact Factor: 5.96). 01/2008; 177(4):2541-6. DOI: 10.1534/genetics.107.079806
Source: PubMed

ABSTRACT

In Saccharomyces cerevisiae, genes placed near telomeres are transcriptionally repressed (telomere position effect, TPE). Although telomeric DNA sequence is the same at all chromosome ends, the subtelomeric elements (STEs) and level of TPE vary from telomere to telomere. We tested whether STEs determine TPE levels. STEs contributed to TPE, as deleting the X element from the VI-R telomere modestly decreased silencing at this telomere. However, STEs were not the major determinant of TPE levels, as inserting the VI-R X element at the truncated VII-L telomere did not increase TPE. These data suggest that the TPE levels of individual telomeres are dependent on some aspect of chromosome context.

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Available from: Michelle A Mondoux, Aug 14, 2014
Copyright Ó 2007 by the Genetics Society of America
DOI: 10.1534/genetics.107.079806
Note
Subtelomeric Elements Influence But Do Not Determine Silencing
Levels at Saccharomyces cerevisiae Telomeres
Michelle A. Mondoux and Virginia A. Zakian
1
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544
Manuscript received August 2, 2007
Accepted for publication October 5, 2007
ABSTRACT
In Saccharomyces cerevisiae, genes placed near telomeres are transcriptionally repressed (telomere position
effect, TPE). Although telomeric DNA sequence is the same at all chromosome ends, the subtelomeric
elements (STEs) and level of TPE vary from telomere to telomere. We tested whether STEs determine TPE
levels. STEs contributed to TPE, as deleting the X element from the VI-R telomere modestly decreased
silencing at this telomere. However, STEs were not the major determinant of TPE levels, as inserting the
VI-R X element at the truncated VII-L telomere did not increase TPE. These data suggest that the TPE
levels of individual telomeres are dependent on some aspect of chromosome context.
I
N Saccharomyces cerevisiae and other organisms, in-
cluding humans, genes placed near telomeres are
transcriptionally repressed, a phenomenon termed
telomere position effect (TPE; reviewed in Mondoux
and Zakian 2005). There are at least six telomere-
associated proteins that are essential for TPE in yeast:
Rap1p, a sequence-specific telomeric DNA binding
protein, Sir2p, Sir3p, Sir4p, and the Ku heterodimeric
complex.Allof theseproteinsexceptKu arealso required
for silencing at the two silent mating-type (HM) loci.
In most organisms, including S. cerevisiae,middlere-
petitive subtelomeric elements (STEs) are found immedi-
atelyproximaltothesimplesequenceTG
1–3
telomere
repeats. In yeast, there are two types of STEs, X and Y9
(reviewed in Louis 1995). X is heterogeneous, ranging in
size from 300 bp to 3 kb, but each X element contains a
‘‘c o r e - X ’’ r e p ea t th a t is f o u nd a t essentially all telomeres.
The core X consists of an autonomously replicating
sequence (ARS) consensus sequence (ACS), which can
bind the multisubunit origin recognition complex (ORC),
and the transcription factor Abf1p. ORC and Abf1p are
both important for silencing at the HM loci (Diffley and
Stillman 1989; Kurtz and Shore 1991; reviewed in
Haber 1998). Most X elements also contain antisilencing
X-combinatorial repeats (XCRs) that have recognition
sites for Reb1p, a transcription factor (Morrow et al.
1989; Wang et al. 1990) and Tbf1p, an essential protein of
unknown function (Brigati et al. 1993). Sites bound by
Tbf1p act as heterochromatin boundaries that keep silenc-
ing from spreading to more internal parts of the chromo-
some (Fourel et al. 1999, 2001). The combination, number,
and arrangement of XCRs vary from telomere to telo-
mere. In contrast to the ubiquitous X element, the Y9
element is found at only one-half to two-thirds of yeast
telomeres. The Y9 element also has binding sites for
Reb1p and Tbf1p. When present, Y9 is distal to X and is
found in up to four tandem copies (Chan and Tye 1983).
Although there are only two classes of STEs, the X and Y9
elements are sufficiently diverse that each of the 32 yeast
telomeres can be thought of as having a distinct identity.
TPE wa s discovered by placing reporter genes im-
mediately adjacent to the telomeric TG
1–3
tract, which
generates telomeres that lack both the X and Y9
elements (Gottschling et al. 1990). Two methods are
used to study TPE at native yeast telomeres: (1) inserting
a reporter gene into the X-ACS, keeping the Y 9 and X
elements largely intact (Fourel et al. 1999; Pryde and
Louis 1999; hereafter referred to as ‘native’ telomeres)
and (2) observing RNA levels of subtelomeric genes that
are naturally near chromosome ends (Vega-Palas et al.
1997, 2000; Wyrick et al. 1999, 2001; Barton and
Kabac k 2006). A subset of native telomeres and all trun-
cated telomeres that have been tested exhibit TPE (for
summary of silencing status of individual telomeres, see
http://www.molbio1.princeton.edu/labs/zakian/assets/
2007-08-mondoux-phenotypes.pdf). However, in the
same strain background, the level of TPE can vary sub-
stantially from telomere to telomere: at some telo-
meres, telomere-adjacent genes are repressed in a small
percentage and at others in 100% of cells. Thus, in
1
Corresponding author: Department of Molecular Biology , Lewis Thomas
Laboratory , Princeton University, Princeton, NJ 08544.
E-mail: vzakian@princeton.e du
Genetics 177: 2541–2546 (December 2007)
Page 1
addition to having different subtelomeric structures,
telomeres exhibit differ ent levels of TPE.
In addition to varied TPE levels, requirements for
silencing proteins are different at different telomeres.
Sir1p acts in the establishment of silencing at the HM
loci (Pillus and Rine 1989) via binding to ORC (Foss
et al. 1993; Triolo and Sternglanz 1996). Sir1p is not
necessary for TPE at truncated telomeres (Aparicio
et al. 1991) . In contrast, TPE is reduced at the native
XI-L telomere in a sir1D strain (Fourel et al. 1999;
Pryde and Louis 1999). In addition, if Sir1p is tethered
to the truncated VII-L telomere, silencing increases
(Chien et al. 1993). Sir1p could promote silencing at
native telomeres by assoc iating with ORC bound to the
X-ACS (Wyrick et al. 2001; Xu et al. 2006). Since Sir1p
interacts with Sir4p (Triolo and Sternglanz 1996), its
binding can recruit other silencing proteins to the
telomere. Mutation of the X-ACS or its Abf1p binding
site reduces TPE at native telomere XI-L (Pryde and
Louis 1999). In addition, the core X element can im-
prove silencing at a weakened HM locus and can coun-
teract the antisilencing effects of XCRs when both are
integrated at the truncated VII-L telomere (Lebrun
et al. 2001). Given that the subtelomeric DNA can
recruit different trans-acting factors to telomeres, the
sequence composition of the subtelomere might de-
termine the silencing profile of individual telomeres.
This model is not unique to yeast, because subtelomeric
regions of Drosophila and human chromosomes are
also di verse, and there are differences in TPE pheno-
types between Drosophila telomeres with different
subtelomeric sequences (S hanower et al. 2005).
TodeterminetheeffectsofSTEsonTPE,wecompared
silencing at the truncated VII-L telomere (Gottschling
et al. 1990) to silencing at the VI -R telom ere, which has the
minimal subtelomeric core X element, but no XCRs or Y9
elements. TPE is biologically relevant at the VI-R telo-
mere, as YFR057w, the uncharacterized ORF that is the
closest unique sequence to the VI-R telomere, is tran-
scriptionally silenced in wild-type strains, and is expressed
upon deletion of the Sir proteins (Vega-Palas et al. 2000).
At VI-R, URA3 was integrated into the subtelomeric X
element in a manner that largely maintains its structure
(as in Pryde and Louis 1999; Figure 1A). We then
Figure 1.—Telomere structure and TPE analy-
sis at the truncated VII-L and VI-R telomeres. (A)
The VI-R subtelomere contains a 380-bp ‘core X’
element (shaded), containing an ARS consensus
sequence (ACS; circle) and Abf1p binding site
(diamond). The URA3 TPE reporter was intro-
duced at the VI-R telomere X-ACS in a manner
analogous to the creation of native TPE reporters
described in Pryde and Louis (1999). PCR pri-
mers whose 5 9 ends corresponded to VI-R
X-element sequence surrounding the X-ACS
were used to amplify URA3 from ADH4UCAIV,
the same plasmid used to create the truncated
VII-L telomere reporter (Gottschling et al.
1990). Unlike the system that truncated the
VII-L telomere at ADH4 (solid), the ‘native’
VI-R TPE reporter preserves the subtelomeric
structure. The closest upstream gene is YFR057w
(striped box). Both strains also contain upstream
lac operator arrays for visualization studies
(Mondoux et al. 2007). All strains used in this
study were constructed in the YPH background
(ura3-52 lys2-801 ade2-101 trp1-D63 his3-D200
leu2-D1; Sikorski and Hieter 1989) and grown
at 30° in yeast complete (YC) synthetic medium
and plates (Zakian and Scott 1982). All TPE re-
porter strains were verified for correct integra-
tion by Southern blotting and pulsed-field gel
electrophoresis. SIR1 was deleted in each strain
background using a PCR-mediated knockout that
eliminated the complete open reading frame, re-
placing it with a hygromycin resistance cassette
(Goldstein and McCusker 1999). (B) TPE as-
says. Tenfold serial dilutions of the VI-R, trun-
cated VII-L, and sir1D versions of these strains
were plated onto 1 Ura or 5-FOA plates to assay silencing and photographed after 3 days’ growth. TPE is higher at VI-R compared
to truncated VII-L and is not dependent on Sir1p at either telomere. (C) Quantitation of TPE. 1Ura-grown cells were plated onto
1Ura and 5-FOA plates and colonies counted after 3 days of growth. The percentage of total cells (1Ura) that grew on 5-FOA plates
is represented as % TPE. Error bars represent standard deviations. TPE at VI-R is significantly higher than TPE at VII-L by Student’s
t-test (P , 7 3 10
5
). There was no significant difference in TPE at either the VI-R or VII-L telomere in the absence of Sir1p.
2542 M. A. Mondoux and V. A. Zakian
Page 2
determined the TPE phenotypes of these marked trun-
cated VII-L and native VI-R strains.
In our strain, the URA3 gene was silenced at the
truncated VII-L telomere in 15% of yeast complete
(YC)-grown cells (14.2 6 7.9%; Figure 1C). Although this
average TPE level was lower than the average reported for
truncated VII-L in some studies (e.g.,Gottschling et al.
1990; Hediger et al. 2002), these analyses were in
different strain backgrounds and/or under different
growth conditions. Moreover, our average value is within
the range of TPE values for VII-L in earlier studies (e.g.,
3–78% TPE for YC-grown cells; Hediger et al. 2002), and
the TPE value reported here was very similar to the TPE
level for haploid YC-grown PPR1 cells in the same strain
background (Tham et al. 2001).
In contrast, in the same strain background, the URA3
gene at the native VI-R telomere was silenced in 85%
of the cells (84.0 6 6.8%; Figure 1C). Thus, the VI-R
telomere had a higher level of TPE than the VII-L
telomere, despite the fact that the URA3 transcription
start site was 328 bp farther away from the telomere at
VI-R compared to VII-L (Figure 1A), and TPE levels are
known to decrease exponentially with distance at trun-
cated telomeres (Gottschling et al. 1990; Renauld
et al. 1993). The level of TPE at the native VI-R telomere
is among the highest reported for a yeast telomere.
Virtually all X elements have an Abf1p binding site
and an ACS that can bind ORC and thereby recruit
Sir1p to the telomere. Although Abf1p binding at
telomeres has not been tested, the VI-R X-ACS is bound
by ORC (Xu et al. 2006). We therefore predicted that
the VI-R X element is responsible for the high TPE
phenotype of this telomere. We also predicted that the
VI-R X element would be able to confer a strong TPE
phenotype on another chromosome end.
As expected (Aparicio et al. 1991), we observed no
significant difference in TPE at the truncated VII-L
telomere in the absence of SIR1 (19.5 6 9.1%; Figure
1C). However, there was also no significant difference in
TPE levels at the native VI-R telomere in the absence of
SIR1 (80.2 6 7.2%). This Sir1p independence is in
contrast to what is seen at the XI-L telomere (Pryde
and Louis 1999), but consistent with expression of
YFR057w, the telomere proximal ORF on VI-R, which
shows wild-type TPE in a sir1D strain by Northern
analysis (Vega-Palas et al. 2000). Thus, the very high
level of silencing seen at the VI-R telomere is not due to
a Sir1p-dependent mechanism that is absent at the
truncated VII-L telomere.
The X element plays a role in TPE at telomere XI-L
that is distinct from the recruitment of Sir1p, as
mutating the X-ACS or the Abf1p binding site results
in a larger (10–100-fold) decrease in TPE (Pryde
and
Louis 1999). Since deleting SIR1 did not reduce TPE at
the native VI-R telomere, we dec ided to eliminate the X
element. We designed primers that eliminated X from
the VI-R telomere and replaced it with URA3, leaving
behind the last 56 bp of the X element but removing all
known binding and regulatory sites, including the X-
ACS and the Abf1p binding site (strain VI-R koX; Figure
2A). URA3 then serves as the TPE reporter, with the
transcription start site at approximately the same
distance from the telomere as it is at the truncated
VII-L telomere. Surprisingly, deleting the X element did
not have a large effect on TPE at VI-R (Figure 2B), as
silencing was decreased less than twofold in its absence
(52.2 6 18.7%; Figure 2C). Therefore, Abf1p binding
did not appear to make a major contribution to TPE,
nor did ORC binding via the X-ACS site. Either the X
element is largely dispensable for TPE at VI-R or there is
some unidentified activity in the final 56 bp of the X
element left at the VI-R telomere that contributes pos-
itively to TPE.
We next did th e reciprocal experiment, asking
whether the X element could confer a high level of
TPE at the truncated VII-L telomere. We wanted to
clone the VI-R X element specifically, so as to directly
compare its function at the VI-R telomere with its
potential function at the truncated VII-L telomere.
Previous studies used X elements cloned from different
telomeres (II-R and XI-L) and inserted fragments of
these X elements between the URA3 reporter and the
truncated VII-L telomere (Fourel et al. 1999). Both of
these elements contain XCRs, which the VI-R X element
lacks. The VI-R X element and URA3 TPE reporte r were
cloned via genomic PCR from the native VI-R telomere
strain and integrated at the tru ncated VII-L telomere
(truncated VII-L 1 X; Figure 2A). We sequenced the
cloned VI-R X element and found that it was virtually
identical to the sequence of the VI-R X in the Saccha-
romyces Genome Database (SGD), differing at only
3 bp, none of which was in the ACS or Abf1p binding
sites (data not shown). Thus, the transcription start site
and sequence and spacing of regulatory sites were
identical betwe en the telomeres in the native VI-R and
truncated VII-L 1 X strains.
The insertion of the VI-R X element at the truncated
VII-L telomere did not increase TPE at this telomere.
Rather, the TPE level was actually decreased approxi-
mately threefold relative to the truncated VII-L telo-
mere (4.3 6 1.9%; Figure 2C). This decrease was
probably due to the increased distance of the URA3
transcription start site from the telomere end. Since this
construct included the 56-bp fragment of the VI-R X
element that was left behind in the X-element knockout
at VI-R (Figure 2A), neither this fragment nor the entire
X element from telomere VI-R was sufficient to confer a
higher level of TPE on the VII-L telomere.
We propose that the different responses by the VI-R
and XI-L telomeres to genetic perturbations are ex-
plained by differences in STE content. Although the X
elements at both telomeres appear to bind ORC to
similar extents (Xu et al. 2006), antisilencing XCR
elements are present at the XI-L telomere but not at
Note 2543
Page 3
the VI-R telomere. Perhaps the core X contributes sub-
stantively to TPE only at telomeres that contain XCR
elements because it acts by countering their negative
effects.
Since Sir1p had no effect on silencing at the VI-R
telomere in a wild-type strain, despite the telomere’s
ability to bind ORC, we next asked whether Sir1p could
contribute to TPE at the native VI-R telomere if TPE
were compromised. Overexpressing the C terminus of
Sir4p (S4-CT) greatly reduces TPE at truncated telo-
mere VII-L as well as HM silencing (Marshall et al.
1987). S4-CT interacts with itself (Chien et al. 1991),
Sir2p (Strahl-Bolsinger et al. 1997), Sir3p (Moazed
et al. 1997), Rap1p (Moretti et al. 1994), and yKu70p
(Tsukamoto et al. 1997). Sir4p recruits Sir2p and Sir3p
to the telomere (Bourns et al. 1998; Luo et al. 2002), so
the reduction in TPE observed with S4-CT overexpres-
sion presumably occurs via the titration of other
silencing factors away from telomeres. TPE at native
telomere VI-R was reduced 20-fold in the presence of
S4-CT (Figure 3A). In the absence of SIR1, TPE was
reduced an additional 3.5-fold in the S4-CT strain
(Figure 3B). Thus, Sir1p does affect TPE at the
X-bearing VI-R telomere, but this contribution is only
seen when silencing proteins are limiting.
Although deletion of the core-X element slightly
decreased TPE levels at the VI-R telomere, TPE was
nonetheless higher at VI-R koX than at either truncated
VII-L (Figure 2C) or XI-L with a mutant X element
(Pryde and Louis 1999). Therefore, we next examined
whether, like Sir1p, the X element would make a larger
contribution to the TPE status of the VI-R telomere in
the prese nce of excess S4-CT. Deletion of the VI-R X
element resulted in an 8.5-fold reduction in TPE when
silencing was compromised (VI-R/VI-R koX 1 S4-CT;
Figure 3B). In contrast, the X element did not enhance
TPE at the VII-L telomere even in the presence of S4-CT
(Figure 3A).
Thus, our data suggest that the role of core X in
silencing increases when TPE is compromised by the
limitation of Sir proteins, just as it plays a role in th e
presence of XCRs. Both Sir1p and the VI-R X element
can contribute to silencing at the VI-R telomere, but
their effects are minimal (koX) or undetectable (sir1D)
unless silencing is compromised by overexpression of
the carboxyl terminus of Sir4p. If the X element at the
VI-R telomere recruits Sir1p and other silencing pro-
teins, this recruitment pathway must be redundant in a
wild-type cell, becoming important only when silencing
proteins are limiting. However, at the truncated VII-L
Figure 2.—The X element requires chromo-
somal context to function. (A) Construction of
VI-R koX and VII-L 1 X telomere reporter
strains. The VI-R koX TPE reporter was created
via integration of the URA3 gene flanked by an
upstream segment of unique DNA (YFR057w,
striped box) and the final 56 bp of the X element
(crosshatched box), knocking out most of the X
element, including the X-ACS and Abf1p binding
site. The VII-L 1 X TPE reporter was created via
truncation of the VII-L telomere seed at an up-
stream segment of unique DNA (ADH4 gene,
solid). The VI-R X element was cloned using an
upstream primer with the same sequence as the
primer used to integrate URA3 at the X-ACS
and a downstream primer with the same se-
quence used to knockout the VI-R X element.
This PCR product replaced the URA3 fragment
in the VII-L truncation plasmid, pADH4UCAIV
(Gottschling et al. 1990). The transcription
start site (arrow), X-ACS (circle), and Abf1p bind-
ing site (diamond) are positioned identically to
their locations in the VI-R strain (see Figure
1A). (B) TPE assays. Tenfold serial dilutions were
plated as in Figure 1B. TPE appeared unchanged
in the absence of the X element at VI-R and was
not enhanced by the presence of the X element
on truncated VII-L. (C) Quantitation of TPE.
TPE at VI-R was reduced (P , 0.02) in the ab-
sence of the X element, but was still significantly
higher than TPE at VII-L (P , 0.002), which also
lacks the X element. The addition of the X ele-
ment to the truncated VII-L telomere did not
increase the level of TPE at that telomere.
2544 M. A. Mondoux and V. A. Zakian
Page 4
telomere, which has a much low er inherent level of
silencing than the VI-R telomere, neither the VI-R X
element nor Sir1p improved its TPE phenoty pe, even
when silencing was compromised by S4-CT expression
(Figure 3A). In addition, in the same strain two different
truncated telomeres, neither of which has STEs, can
have quite different levels of TPE (3% vs. 30%;
Gottschling et al. 1990). We conclude that there must
be an aspect (or aspects) of an individual chromosome
end, other than the identity of its STEs, that is a major
determinant of its TPE level.
What aspects of chromosome identity could contribute
to TPE? One possibility is that control of TPE might act in
trans, through higher-order chromatin structure, chromo-
some dynamics, or nuclear localization. Elsewhere we
demonstrate that the truncated VII-L and native VI-R
telomeres localize similarly to both the nuclear periphery
and to pools of silencing proteins (Mondoux et al. 2007,
accompanying article, this issue). Thus, differential local-
ization of the two telomeres to these structures does not
explain their different TPE phenotypes. In addition, there
is no difference in the level of telomere binding of
proteins that promote (Rap1p, yKu80p) or antagonize
(Rif1p, Rif2p) TPE between the truncated VII-L and native
VI-R telomeres (Sabourin et al. 2007). We also do not
observe any difference in replication timing between the
two telomeres (Sabourin et al. 2007).
Another model for the different TPE levels at
different chromosome ends is the presence of proximal
cis-acting sequences that either promote or repress TPE.
Heterochromatin formation is thought to originate at
the telomere and spread inward, with distance of spread
determined by the concentration of available Sir3p
(Renauld et al. 1993) and the activity of protosilencers
and antisilencers in the subtelomeric reg ion (Fourel
et al. 1999, 2001; Pryde and Louis 1999). Although
there are no known differences in the distal chromatin
structure of these two telomeres that explain their
different TPE phenotypes, it is possible that there are
differences in the proximal chromatin structure that
influence TPE. Asymmetric nucleosome spacing on
either side of the HML-I and HMR-E silencers correlates
with the preferential association of the Sir proteins
to one side (Zou et al. 2006). The establishment of
asymmetry precedes the formation of heterochromatin
and is dependent on ORC and Abf1p (Zou et al. 2006).
It is also possible that the distance of heterochromatin
spread, and therefore level of TPE, depends on other
proximal elements, which could include the identity
and transcriptional activity of nearby genes. For exam-
ple, the level of silencing at the VI-R X-ACS may be high
in part because of the repression of the proximal gene,
YFR057w (Wyrick et al. 1999; Vega-Palas et al. 2000).
We thank David Shore and Rolf Sternglanz for strains and plasmids,
and Ed Louis, Jane Phillips, and Michelle Sabourin for helpful
discussions and advice. We also thank James Broach, Paul Schedl,
and Eugenia Xu for providing suggestions on the manuscript. This
work was supported by grants from the National Institutes of Health
to V.A.Z. and a National Science Foundation predoctoral fellowship
to M.A.M.
Figure 3.—Sir1p and the X element contrib-
ute to TPE at VI-R when silencing is compro-
mised. (A) TPE assays. Tenfold serial dilutions
were plated to assay silencing. Strains were grown
in 1Ura His medium to maintain the S4-CT
plasmid. TPE is compromised by the overexpres-
sion of the Sir4p C terminus, and this phenotype
was exacerbated in the absence of Sir1p or the
VI-R X element. (B) Quantitation of TPE. Error
bars represent standard deviations. When possi-
ble, standard deviations were calculated sepa-
rately above and below the mean. Values are
presented as ratios of TPE level at the wild-type
VI-R telomere to TPE level at VI-R in the absence
of Sir1p or the X element. When TPE was com-
promised by S4-CT (striped bars), there was a sta-
tistically significant increase in the TPE ratio at
VI-R in the absence of Sir1p, in comparison to
sir1D alone (P , 0.008). When TPE was compro-
mised by SIR4-CT, there was a statistically signifi-
cant increase in the TPE ratio at VI-R koX, in
comparison to the X-element knockout alone
(P , 0.04).
Note 2545
Page 5
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Communicating editor: F. Winston
2546 M. A. Mondoux and V. A. Zakian
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    • "These results seem to rule out a critical role for tethering in TPE (Ferreira et al. 2011). This conclusion is consistent with experiments indicating that TPE and tethering are separable phenotypes (Tham et al. 2001; Mondoux and Zakian 2007). However, this conclusion is still surprising , given numerous examples in diverse organisms for a connection between the nuclear periphery, heterochromatin formation, and gene silencing. "
    [Show abstract] [Hide abstract] ABSTRACT: The mechanisms that maintain the stability of chromosome ends have broad impact on genome integrity in all eukaryotes. Budding yeast is a premier organism for telomere studies. Many fundamental concepts of telomere and telomerase function were first established in yeast and then extended to other organisms. We present a comprehensive review of yeast telomere biology that covers capping, replication, recombination, and transcription. We think of it as yeast telomeres--soup to nuts.
    Full-text · Article · Aug 2012 · Genetics
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    • "The recent study by Takahashi et al. (63) pointed out that a modified telomere could differ from a natural one. We then examined the association of 13Myc-tagged Sir2 with natural telomeres VIIL, VIR and IIIR in wild-type and Mediator mutant strains (64,65) (Figure 4A–C). The natural telomere VIIL (n7L) contains a ∼0.75 kb X element adjacent to the TG1-3 repeats at the chromosome end (Figure 4A). "
    [Show abstract] [Hide abstract] ABSTRACT: Eukaryotic chromosome ends have a DNA–protein complex structure termed telomere. Integrity of telomeres is essential for cell proliferation. Genome-wide screenings for telomere length maintenance genes identified several components of the transcriptional regulator, the Mediator complex. Our work provides evidence that Mediator is involved in telomere length regulation and telomere heterochromatin maintenance. Tail module of Mediator is required for telomere silencing by promoting or stabilizing Sir protein binding and spreading on telomeres. Mediator binds on telomere and may be a component of telomeric chromatin. Our study reveals a specific role of Mediator complex at the heterochromatic telomere and this function is specific to telomeres as it has no effect on the HMR locus.
    Full-text · Article · Sep 2011 · Nucleic Acids Research
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    • "However, a catalytically inactive methyltransferase domain (Dot1 172-582 G401R ) did not disrupt silencing (Figure 2D-E). Different native telomeres and truncated telomeres can show different silencing properties, Sir protein binding, and nucleosome positioning [29,53,54]. To test whether the derepressor activity of Dot1 is a general property or is restricted to the truncated telomere used here, LexA operators and the URA3 gene were introduced at three different native chromosome ends. "
    [Show abstract] [Hide abstract] ABSTRACT: Methylation of histone H3 lysine 79 (H3K79) by Dot1 is highly conserved among species and has been associated with both gene repression and activation. To eliminate indirect effects and examine the direct consequences of Dot1 binding and H3K79 methylation, we investigated the effects of targeting Dot1 to different positions in the yeast genome. Targeting Dot1 did not activate transcription at a euchromatic locus. However, chromatin-bound Dot1 derepressed heterochromatin-mediated gene silencing over a considerable distance. Unexpectedly, Dot1-mediated derepression was established by both a H3K79 methylation-dependent and a methylation-independent mechanism; the latter required the histone acetyltransferase Gcn5. By monitoring the localization of a fluorescently tagged telomere in living cells, we found that the targeting of Dot1, but not its methylation activity, led to the release of a telomere from the repressive environment at the nuclear periphery. This probably contributes to the activity-independent derepression effect of Dot1. Targeting of Dot1 promoted gene expression by antagonizing gene repression through both histone methylation and chromatin relocalization. Our findings show that binding of Dot1 to chromatin can positively affect local gene expression by chromatin rearrangements over a considerable distance.
    Full-text · Article · Feb 2011 · Epigenetics & Chromatin
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