Mediator influences telomeric silencing and cellular life span.
ABSTRACT The Mediator complex is required for the regulated transcription of nearly all RNA polymerase II-dependent genes. Here we demonstrate a new role for Mediator which appears to be separate from its function as a transcriptional coactivator. Mediator associates directly with heterochromatin at telomeres and influences the exact boundary between active and inactive chromatin. Loss of the Mediator Med5 subunit or mutations in Med7 cause a depletion of the complex from regions located near subtelomeric X elements, which leads to a change in the balance between the Sir2 and Sas2 proteins. These changes in turn result in increased levels of H4K16 acetylation near telomeres and in desilencing of subtelomeric genes. Increases in H4K16 acetylation have been observed at telomeres in aging cells. In agreement with this observation, we found that the loss of MED5 leads to shortening of the Saccharomyces cerevisiae (budding yeast) replicative life span.
- [show abstract] [hide abstract]
ABSTRACT: The Saccharomyces cerevisiae Mediator is a 25-subunit complex that facilitates both transcriptional activation and repression. Structural and functional studies have divided Mediator subunits into four distinct modules. The Head, Middle, and Tail modules form the core functional Mediator complex, whereas a fourth, the Cyc-C module, is variably associated with the core. By purifying Mediator from a strain lacking the Med19(Rox3) subunit, we have found that a complex missing only the Med19(Rox3) subunit can be isolated under mild conditions. Additionally, we have established that the entire Middle module is released when the Deltamed19(rox3) Mediator is purified under more stringent conditions. In contrast to most models of the modular structure of Mediator, we show that release of the Middle module in the Deltamed19(rox3) Mediator leaves a stable complex made up solely of Head and Tail subunits. Both the intact and Head-Tail Deltamed19(rox3) Mediator complexes have defects in enhanced basal transcription, enhanced TFIIH phosphorylation of the CTD, as well as binding of RNA Pol II and the CTD. The largely intact Deltamed19(rox3) complex facilitates activated transcription at levels similar to the wild type Mediator. In the absence of the Middle module, however, the Deltamed19(rox3) Mediator is unable to facilitate activated transcription. Although the Middle module is unnecessary for holding the Head and Tail modules together, it is required for the complex to function as a conduit between activators and the core transcription machinery.Journal of Biological Chemistry 03/2007; 282(8):5551-9. · 4.65 Impact Factor
Article: The mediator complex.Advances in protein chemistry 02/2004; 67:43-65. · 3.20 Impact Factor
Article: Yeast cell synchronization.Methods in molecular biology (Clifton, N.J.) 02/2004; 241:55-76.
MOLECULAR AND CELLULAR BIOLOGY, June 2011, p. 2413–2421
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 31, No. 12
Mediator Influences Telomeric Silencing and Cellular Life Span?
Xuefeng Zhu,1* Beidong Liu,2,3Jonas O. P. Carlsten,1Jenny Beve,1Thomas Nystro ¨m,2
Lawrence C. Myers,4and Claes M. Gustafsson1,5*
Department of Medical Biochemistry and Cell Biology, University of Gothenburg, SE-405 30 Go ¨teborg, Sweden1; Department of
Cell and Molecular Biology, University of Gothenburg, Medicinaregatan 9C, SE-413 90 Go ¨teborg, Sweden2; Department of
Life Science and Engineering, Harbin Institute of Technology, Harbin 150001, China3; Department of Biochemistry,
Dartmouth Medical School, Hanover, New Hampshire 037554; and Max Planck Institute for Biology of
Ageing, Gleueler Str. 50a, D-50931 Cologne, Germany5
Received 9 February 2011/Accepted 29 March 2011
The Mediator complex is required for the regulated transcription of nearly all RNA polymerase II-dependent
genes. Here we demonstrate a new role for Mediator which appears to be separate from its function as a
transcriptional coactivator. Mediator associates directly with heterochromatin at telomeres and influences the
exact boundary between active and inactive chromatin. Loss of the Mediator Med5 subunit or mutations in
Med7 cause a depletion of the complex from regions located near subtelomeric X elements, which leads to a
change in the balance between the Sir2 and Sas2 proteins. These changes in turn result in increased levels of
H4K16 acetylation near telomeres and in desilencing of subtelomeric genes. Increases in H4K16 acetylation
have been observed at telomeres in aging cells. In agreement with this observation, we found that the loss of
MED5 leads to shortening of the Saccharomyces cerevisiae (budding yeast) replicative life span.
In Saccharomyces cerevisiae (budding yeast), telomeric DNA
consists of imperfect tandem repeats of the consensus se-
quence (TG1–3)n, with a combined length of about 300 nucle-
otides (21). Next to the telomere is the subtelomeric region,
which often contains two types of repeats, the Y? and X ele-
ments. The Y? elements are between 4 and 12 kb long and are
located next to the telomeres at many chromosome ends (26).
The sizes of X elements vary, but they always contain a
“core X” repeat region that is found at nearly all telomeres.
Depending on how the Y? and X elements are distributed, S.
cerevisiae chromosome ends can be divided into X and X-Y?
types (22, 23).
Genes situated close to telomeres undergo reversible silenc-
ing, a phenomenon that has been termed the telomere position
effect (TPE) (28). This effect was first observed when a re-
porter gene was inserted next to a telomeric TG1–3tract of an
artificial telomere. TPE can also be observed at native yeast
telomeres, but the phenomenon appears to be a bit more
complex at these locations, since TPE varies between telo-
meres and in different strain backgrounds (29, 40, 41). The
molecular basis of TPE is believed to be the Rap1, Ku, and Sir
protein-mediated spreading of heterochromatin-like structures
from the telomeric DNA inwards, which represses genes lo-
cated in the subtelomeric region (42). According to this model,
the Rap1/Ku/Sir structures are formed at telomeres and prop-
agate toward the subtelomeres via interactions between the Sir
proteins and histone tails. Sir2 is an active histone deacetylase
that removes the acetyl group on lysine 16 of histone H4
(H4K16), which allows Sir3 and Sir4 to bind the nonacetylated
histone tails (11, 35).
As mentioned, TPE varies between individual chromosome
ends, and the exact repeat structure of the subtelomeric region
may in fact influence the spread of heterochromatin. Y? ele-
ments counteract the spread of Sir proteins, and the Y? regions
display high levels of H4K16 acetylation. Furthermore, Y?
elements are highly enriched in nucleosomes and are transcrip-
tionally active. In contrast, even X elements situated at some
distance from the telomeric ends appear to be repressed tran-
scriptionally. The X elements lack a defined nucleosomal
structure, are bound by Sir proteins, and have very low levels of
H4K16 acetylation (45). At some telomeres, an actively tran-
scribed Y? element may even separate the telomeric repeat
region from a repressed X element bound by Sir proteins.
Deacetylation of H4K16 by Sir2 stimulates the spread of the
Rap1/Ku/Sir structures, whereas Sas2, an H4K16-acetylating
enzyme, antagonizes this process (36). The opposing effects of
Sir2 and Sas2 generate a gradient of H4K16 acetylation, which
in turn marks the boundary between active and silenced chro-
matin near telomeres. How the balance between Sir2 and Sas2
is regulated is not understood in detail, but a recent report
implicated the SAGA subunit Ada2 as a possible regulator of
this process. Ada2 was shown to bind telomeric chromatin and
the silencing protein Sir2 in vivo, and loss of ADA2 increased
the levels of Sir2 and Sir3 in subtelomeric regions, concomitant
with decreased H4K16 acetylation (13).
The SIR2 gene has also been identified as a major determi-
nant of the replicative life span in budding yeast (18). Inacti-
vation of SIR2 reduces the yeast life span, whereas an increase
in SIR2 dosage extends the life span. The Sir2 protein re-
presses homologous recombination within ribosomal DNA re-
peats, which reduces the formation of extrachromosomal ribo-
somal DNA circles and directly reduces the pace of aging in
yeast (33). Longevity in yeast might also be regulated by
H4K16 acetylation and the chromatin state at telomeres, since
* Corresponding author. Mailing address: Department of Medical
Biochemistry and Cell Biology, University of Gothenburg, Medicin-
aregatan 9A, SE-405 30 Go ¨teborg, Sweden. Phone for Xuefeng Zhu:
46 31 7863276. Fax: 46 31 41 61 08. E-mail: firstname.lastname@example.org
.se. Phone for Claes M. Gustafsson: 46 31 7863826. Fax: 46 31 41 61 08.
?Published ahead of print on 11 April 2011.
there is an age-associated decrease in Sir2 protein occupancy
at telomeres in replicatively old yeast cells (7). This change
leads to an increase in H4K16 acetylation and causes compro-
mised transcriptional silencing in the subtelomeric region. In-
terestingly, Sas2 appears to antagonize the effects of Sir2 on
chromatin and life span, suggesting that the exact boundary
between active and inactive chromatin may directly influence
the replicative life span in budding yeast (7).
Mediator is an evolutionarily conserved coregulator complex
required for transcription of almost all RNA polymerase II
(Pol II)-dependent genes (5). One function of this multiprotein
complex is to serve as a functional bridge between gene-spe-
cific regulatory proteins bound to upstream elements and the
general transcription machinery bound at the promoter. Sev-
eral pieces of evidence also link Mediator to the maintenance
of telomeric heterochromatin. Deletion of genes encoding Me-
diator components (MED1, MED15, MED18, and MED20)
leads to shortening of telomere repeat lengths (2). In addition,
deletion of MED15 suppresses silencing defects in a rap1 mu-
tant strain and restores telomere repeat length to near wild-
type (wt) levels (37).
We previously observed that purified Mediator interacts di-
rectly with reconstituted mononucleosomes (20). Here we
demonstrate that Mediator associates with heterochromatin at
telomeres and regulates the balance between Sir2 and Sas2.
This appears to be a completely new role for the Mediator
complex that is distinct from its role as a transcriptional co-
regulator. Loss of the Med5 component of Mediator causes
decreased Mediator occupancy and a defect in the heterochro-
matin block, which in turn leads to impaired telomeric silenc-
ing. The observed effects appear to be functionally significant,
since the change of the boundary between active and inactive
chromatin at telomeres in the med5? strain is associated with
a shortening of the replicative life span.
MATERIALS AND METHODS
ChIP. Chromatin immunoprecipitation (ChIP) assays were performed as pre-
viously described (46). Briefly, we cultured yeast cells to an optical density at 600
nm (OD600) of 0.6 to 0.8, followed by cross-linking by incubation with 1%
formaldehyde for 10 min at room temperature. The reactions were quenched
with 125 mM glycine, and the cells were spun down for 5 min at 3,000 rpm and
4°C. Pellets were washed twice in cold phosphate-buffered saline (PBS) and
resuspended in 400 ?l cold lysis buffer with protease inhibitors (Roche). The
fixed cells were lysed with 400 ?l glass beads (Sigma-Aldrich), using a Fast-
Prep-24 machine (MP Biomedicals). The extracts were sonicated to obtain chro-
matin fragments of about 250 bp (Bioruptor UCD-200; Diagenode). Antibodies
used for chromatin immunoprecipitation were against histone H4 (ab2423; Ab-
cam), the C-terminal domain of RNA polymerase II (4H8; Abcam), acetylated
H4K16 (ab61240; Abcam), Med1 (a gift from Stefan Bjo ¨rklund, Umeå Univer-
sity, Sweden), Sir2 (ab4626; Abcam), and Sas2 (a gift from Jerry L. Workman,
Stowers Institute for Medical Research). After overnight incubation with anti-
body, 30 ?l of protein A Sepharose slurry (GE Healthcare) was added to the
reaction mixtures, followed by incubation for 1 h. After being washed and eluted,
samples were treated with proteinase K and the cross-link was reversed by
overnight incubation at 65°C. DNA was purified by phenol-chloroform extrac-
tion, ethanol precipitation, and incubation with RNase A. The purified DNA was
used for real-time PCR analysis (Bio-Rad). The names and sequences of the
primers used are available at on request. Quantifications were performed using
real-time PCR software (Bio-Rad) and Excel (Microsoft); ratios of IP/input are
depicted in the figures after subtracting ratios obtained with a nonantibody
Global gene expression profiles and data analysis. Expression profiling data
were obtained from a previously published study (39). Moving average analysis
was carried out with Microsoft Excel and Access (Microsoft) as previously de-
scribed (43). For each coding gene, the distance to the nearest chromosome end
was calculated based on the translation start site (ATG).
Yeast strains. Saccharomyces cerevisiae strains used in this study are listed at
Electrophoretic mobility shift assays. The Mediator complex was purified
from yeast as previously described (25). Flag-tagged Sir3 protein was overex-
pressed and purified from yeast as described previously (27). To test binding of
Mediator to32P-labeled mononucleosomes, we performed electrophoretic mo-
bility shift assays as previously described (9, 20, 44), with the following modifi-
cations. Binding reaction mixtures (12 ?l) comprised a buffer containing 10 mM
Tris-Cl (pH 8.0), 5% glycerol, 50 mM NaCl, 0.1 mg/ml bovine serum albumin
(BSA), 1 mM dithiothreitol (DTT), and 0.16 nM nucleosomes, with or without
Sir3 or Mediator complex at the final concentrations indicated in the figure
Life span analysis. To determine the replicative life span, wild-type (BY4741)
and med5? cells were grown to mid-log phase (OD600? 0.5) at 30°C and plated
on yeast extract-peptone-dextrose (YPD) medium. Experiments were performed
with 64 virgin cells per plate. Plates were incubated at 30°C during working hours
and kept at 4°C overnight. Daughter cells generated by each individual mother
were removed and counted by a micromanipulator (Singer Instruments), and the
life span was calculated as described previously (15). Statistical assessment of life
span differences was determined using the Wilcoxon rank sum test.
Mediator mutant strains affect telomeric silencing. Compo-
nents of the Cdk8 module of Mediator have been shown to
repress transcription of many genes, and genome-wide analysis
demonstrated that this effect was independent of gene local-
ization relative to the telomere (Fig. 1A and data not shown).
In contrast, deletion of the MED16 gene caused a specific
increase in the transcription of genes located in subtelomeric
regions (Fig. 1A). We previously reported that Mediator binds
near the telomere region (34), and the effect of the med16?
mutation that we now observed could therefore indicate that
Mediator directly regulates transcription activity in the subte-
lomeric region. To investigate this possibility, we used a URA3
marker inserted next to the (TG1–3)nrepeat region of telomere
7L (tel7L) (34). In wt cells, this URA3 marker is subjected to
telomeric silencing, but deletion of genes regulating the telo-
meric structure, e.g., SIR2, has previously been shown to acti-
vate expression of this telomeric marker gene. We used this
approach to investigate effects in the med16? strain. We also
investigated effects of MED5 and MED15 deletions. The Med5
protein forms a distinct Mediator submodule together with the
Med16 protein. The Med15 protein is also associated with
Med16 in the tail module of the Mediator complex but forms
its own subcomplex with two other Mediator components,
Med2 and Med3. The med15? strain displayed a slow-growing
phenotype, but we could not observe significant effects on
URA3 gene expression in this strain (Fig. 1B). In contrast, loss
of MED16 caused a silencing defect, demonstrating that the
Med16 protein may be required for telomeric silencing. Sur-
prisingly, the med16? strain was also able to survive in the
presence of 5-fluoroorotic acid (5-FOA), a substrate that
should kill cells expressing URA3. This observation suggested
that loss of MED16 might cause a metastable situation, with
fluctuation between an active and a repressed state for the
URA3 gene, similar to the incomplete suppression of the his4-
912? gene construct by deletion of MED16 (14). Heterochro-
matin silencing occurs not only at telomeres but also at the
mating type loci. In S. cerevisiae, a cells respond to the ? cell
mating pheromone (the ? factor) by growing a projection
known as a shmoo. To investigate if the med16? cells gener-
2414 ZHU ET AL.MOL. CELL. BIOL.
ated a metastable chromatin phenotype at mating type loci, we
investigated shmoo formation after ? factor treatment of
MATa cells (8). Among wild-type cells, 79% (n ? 320) re-
sponded to ? factor to activate shmoo formation, whereas only
46% of med16? cells (n ? 518) displayed shmoo formation.
Therefore, med16? cells display a metastable phenotype at
both telomeres and mating type loci. Even though we found
this effect very interesting, it made further analysis of subtelo-
meric silencing in the med16? strain difficult.
The med5? strain also displayed a subtelomeric silencing
defect, but in contrast to the case for the med16? strain, the
phenotype appeared to be stable, since the strain was unable to
grow on 5-FOA (Fig. 1B). Real-time quantitative PCR con-
firmed this observation and revealed upregulation of URA3
transcription in med5? cells similar to that seen in the sir2?
strain (Fig. 1C). Deletion of MED5 affects very few genes, and
it was therefore difficult to observe statistically significant
changes between different chromatin regions. We could con-
clude, however, that the frequency of genes affected in the
med5? strain was slightly higher near telomeres than at other
regions, further supporting a role for the Med5 protein in
subtelomeric silencing (data not shown). Telomeres have a
distinct chromosome conformation which can block binding of
Pol II and other transcription factors. We performed a ChIP
assay to monitor Pol II occupancy at the promoter and coding
regions of the telomeric URA3 gene. In agreement with our
observation of increased gene transcription, we found in-
creased Pol II occupancy at both the promoter and coding
regions of the gene (Fig. 1D). The tel7L-URA3 strain lacks the
PPR1 gene, which encodes an activator of URA3 gene tran-
FIG. 1. Deletion of Mediator components disrupts transcription silencing at telomeres. (A) Moving average analysis of gene expression changes
in med16? and med12? mutant strains. The distance from a gene to the telomere was calculated based on the position of the translation start site.
The window size is 150. (B) Fivefold dilutions of the indicated deletion strains were spotted on complete medium, on synthetic complete medium
lacking uracil (SC?URA), and on synthetic complete medium containing 5-FOA (SC ? 5-FOA). All strains (UCC3505, CGC209, CGC210,
CGC211, and CGC212) contained the URA3 reporter gene inserted at tel7L. (C) Real-time PCR analysis shows fold changes of tel7L URA3 gene
transcription in strains UCC3505, CGC210, and CGC209. (D) A ChIP assay using the 4H8 antibody followed by real-time PCR analysis shows Pol
II occupancy at URA3 promoter and coding regions of the indicated strains. Background values were calculated using a no-antibody control. Error
bars indicate standard deviations based on experiments from at least three independent cultures. (E) The X core elements of telomeres show
silencing defects in med5? cells. The positions (1 to 4) of URA3 gene insertions near tel11L are indicated. Silencing assays were performed with
wild-type and med5? cells bearing the URA3 gene inserted at positions 1 to 4. The extent of silencing is expressed as the fraction of cells resistant
to 5-FOA (n ? 4). Error bars shows standard deviations. (Schematic diagram adapted from reference 7 with permission of the publisher.)
VOL. 31, 2011MEDIATOR AND SUBTELOMERIC SILENCING2415
scription. The absence of the Ppr1 protein may increase the
distance over which telomeric silencing of URA3 is observed
and therefore create somewhat artificial effects. We therefore
also investigated silencing in strains with the PPR1 gene intact
in which the URA3 marker had been inserted at different
locations of the native yeast telomere 11L (29). In wt cells,
URA3 transcription was strongly repressed in the subtelomeric
X element, located close to the telomeric repeat region,
whereas URA3 located further downstream of the telomere
end displayed gradually weaker repression (Fig. 1E). In the
med5? cells, we observed derepression at the X element as
monitored by decreased 5-FOA resistance, demonstrating a
role for Med5 in repression of transcription at native telo-
meres, particularly at X elements.
Reduced Mediator complex occupancy in Med5 mutant.
Previously published genome-wide studies have demonstrated
the presence of Mediator in subtelomeric regions (1, 10).
Given the observed effects of the med5? mutation on telomeric
silencing, we decided to test whether deletion of MED5 leads
to reduced Mediator complex occupancy at this location. To
monitor Mediator occupancy, we performed ChIP analysis
with an antibody against the Mediator component Med1 (Fig.
2). For our analysis, we used 10 primer pairs detecting genomic
regions situated at various distances (0.9 to 19 kb) from the
TG1–3repeats of tel7L (Fig. 2A). We observed a distinct peak
of Mediator occupancy near the X element (primer pairs 1 and
2), whereas Mediator occupancy was lower at more distant
positions (Fig. 2B). Mediator occupancy at the peak position
near the telomeric end (primer pairs 1 and 2) was significantly
reduced in the med5? mutant strain, whereas other sites were
The subtelomeric region of tel7L contains an X element
(positions ?781 to ?35) situated directly adjacent to the TG1–3
repeats, but very similar X elements are also present at other
chromosome ends. Therefore, we could not construct a primer
pair that would specifically amplify the X element at tel7L. We
could, however, use primer pairs against unique sequences
within the X and Y? elements in the subtelomeric region of
tel5R (7). For comparison, we used primers detecting other
chromosome regions, i.e., the ACT1 promoter (active euchro-
matin), NTS1 and RDN58 (transcribed ribosomal DNA), and
HML-?1 (mating type locus). We noted that Mediator occu-
pancy was distinctly different between X and Y? elements in
wild-type cells, with the levels of Mediator occupancy being
about 10- to 15-fold higher at the Y? elements than at X
elements (Fig. 2C). In fact, we observed nearly no Mediator
binding to the tel5R X element, suggesting that Mediator is
more or less absent from this region. Interestingly, loss of
Med5 led to a reduction of Mediator at Y? elements but did
not affect the other genomic locations investigated. Our data
for Mediator occupancy at telomeres C5R and C7L therefore
suggested that Mediator is located in genomic regions border-
ing chromosome ends and X elements, e.g., in the Y?-element
FIG. 2. Mediator complex occupancy near telomeres. (A) Schematic diagram of primer pairs detecting positions at various distances (0.9 kb
to 20 kb) from tel7L. (B) Mediator occupancy at tel7L. ChIP analyses were performed with material from wt (BY4741) and med5? mutant
(CGC117) strains. Mediator occupancy was monitored using an antibody against the Med1 protein, followed by real-time PCR quantification.
Error bars indicate standard deviations for at least three independent cultures. (C) Mediator occupancy at different genomic loci, including
euchromatin (ACT1), ribosomal DNA (NTS1 and RDN58), a mating type locus (HML-?1), and subtelomeric regions (the X and Y? elements of
telomere 5R). X elements include X core (XC) positions and X repeat (XR) positions. The analysis was performed as described for panel B.**,
P ? 0.05.
2416ZHU ET AL.MOL. CELL. BIOL.
region, but that the complex is absent from the X element
Loss of Med5 changes Sir2 and Sas2 protein occupancy at X
elements. Both TG1–3repeat regions and X elements lack a
defined nucleosome structure and instead are bound by Sir
proteins. The opposing effects of Sir2 and Sas2 generate a
gradient of H4K16 acetylation, which in turn marks the bound-
ary between active and silenced chromatin near telomeres.
Given its genomic location and its requirement for subtelo-
meric silencing, we wondered if Mediator could directly con-
tribute to the formation of this border. Indeed, deletion of
MED5 caused a significant reduction of Sir2 levels at the X
element in both telomeres 5R and 7L (Fig. 3A and B). The
observed changes were not due to an overall effect on Sir2
levels, since a Western blot analysis of whole-cell extracts dem-
onstrated that the global amounts of the Sir2 protein remained
unchanged in the Med5 mutant strain (Fig. 4A).
Given the effects on Sir2 occupancy, we also monitored
binding of Sas2, the dominant H4K16 acetyltransferase, which
has been shown to antagonize Sir2 in telomeric regions (16,
36). In this experiment, we observed a significant increase in
the level of Sas2 occupancy in med5? mutant cells at X ele-
ments (Fig. 3C and D), whereas Sas2 occupancy at other re-
gions remained unchanged. Our results therefore demon-
strated that the Mediator complex could affect the balance
between Sir2 and Sas2 in subtelomeric regions.
Loss of Med5 leads to increased H4K16 acetylation levels in
the subtelomeric region. To investigate the functional conse-
quences of the observed changes in Sir2 and Sas2 occupancy,
we investigated if deletion of MED5 could influence levels of
H4K16 acetylation and the genomic distribution of this mod-
ification. Total levels of H4K16 acetylation were monitored by
immunoblotting with whole-cell extracts and remained un-
changed in the Mediator mutant strains investigated (Fig. 4A).
We also monitored the levels of the Sas2 proteins, which re-
mained constant in the wt and mutant cell extracts, similar to
the case for Sir2.
We next investigated if loss of MED5 could induce changes
in histone occupancy and H4K16 acetylation at different
genomic locations. In wt cells, histone H4 occupancy is very
low at X elements, and we observed a slight but consistent
increase of H4 at these locations in the med5? mutant strain
(Fig. 4B). No other gross alterations in histone H4 occupancy
were observed. We next monitored the levels of H4K16 acet-
ylation normalized to total histone H4 occupancy and noticed
a dramatic increase in H4K16 acetylation levels at the X core
element of tel5R. We also noted a significant increase at the
neighboring X repeat element (Fig. 4B). No other genomic
regions were affected.
We next examined the effects of the MED5 gene deletion on
the C7L chromosome end that lacks a conserved Y? element
but contains an X element situated next to the telomeric repeat
region. In wt cells, we found that histone H4 occupancy and
H4K16 acetylation levels were at their lowest close to the X
element and telomere end (Fig. 4C). In the med5? strain, we
observed a strong increase of H4K16 acetylation near the telo-
mere end by use of primer pairs 1 and 2, whereas the other
locations were unaffected. Since primer pairs 1 and 2 are lo-
cated near the boundary of the X element in tel7L, this obser-
vation supported the idea that loss of Med5 leads to increased
levels of H4K16 acetylation near X elements. Similar to our
observations at the tel5R X element, we observed a slight
increase in H4 occupancy near the tel7L X element in the
med5? mutant strain.
Our data therefore demonstrate that Mediator mutations
lead to decreased Sir2 occupancy, which is linked to increased
Sas2 and H4K16 acetylation levels at X elements. Mediator
appears to cause this effect by binding to genomic regions
FIG. 3. Sir2 and Sas2 protein occupancy changes in med5? mutant cells. (A and B) Sir2 occupancy was determined by ChIP assays with wt
(BY4741) and med5? (CGC117) mutant cells, using the primers shown in Fig. 2. (C and D) Sas2 occupancy was determined as described for panels
A and B. Data shown are averages for at least three independent experiments. Error bars represent standard deviations.**, P ? 0.05.
VOL. 31, 2011 MEDIATOR AND SUBTELOMERIC SILENCING2417
adjacent to X elements but not to the X element itself (Fig.
2C). We next investigated if other Mediator mutations could
influence the exact boundary between active and inactive chro-
matin. We found that a temperature-sensitive mutation in the
gene encoding Med7 caused a dramatic change in H4K16
acetylation and in the distribution of Sas2 and Sir proteins
(data not shown). We analyzed the tel7L region by using 10
different primer pairs as described above. In the Med7 mutant
strain (med7-163), the levels of H4K16 acetylation and Sas2
were increased about 3-fold at positions close to the telomere,
whereas Sir3 occupancy was decreased ?2-fold at the same
positions upon a shift from the permissive to the nonpermissive
temperature. The changes observed in the med7-163 strain
were therefore similar to but even more dramatic than those
observed in the med5? mutant strain.
Sir3 and Mediator compete for binding to nucleosomes. As
demonstrated here, both Mediator and Sir proteins bind to
subtelomeric regions. Our ChIP data suggested mutually ex-
clusive targeting of Mediator and Sir proteins and that Medi-
ator is not a stoichiometric component of heterochromatin
akin to the Sir proteins. We therefore wanted to investigate if
Mediator and Sir3 could co-occupy the same nucleosomes in
vitro. To investigate this phenomenon in vitro, we followed
previously published protocols and purified Mediator to near
homogeneity (see Fig. 2C in reference 3). We preincubated
purified Mediator with reconstituted nucleosomes and ana-
lyzed complex formation in gel retardation experiments (Fig.
5). We noticed the formation of a Mediator-nucleosome com-
plex (Fig. 5A), as previously reported (20). To further verify
the identity of this complex, we also used a Mediator complex,
purified from ?med2 cells, that lacks the Med2, Med3, and
Med15 subunits (24). We incubated the smaller ?med2 Medi-
ator with mononucleosomes and compared the results with
those obtained for the wild-type Mediator complex. Indeed,
the ?med2 mutant Mediator bound mononucleosomes with an
affinity comparable to that observed with wild-type Mediator
(Fig. 5A), but the shifted band was lower due to the decreased
molecular weight of the mutant Mediator.
Next, we investigated if Mediator could compete with Sir3
for binding to mononucleosomes. We first created Sir3-nucleo-
some complexes with different concentrations of Sir3. We
noted the formation of a distinct complex between Sir3 and
mononucleosomes (Fig. 5B, lanes 3, 5, and 7). We added
Mediator to free mononucleosomes or to mononucleosomes
that had been preincubated for 10 min with the indicated
concentrations of Sir3. We noted that Mediator could compete
with Sir3 for binding to mononucleosomes in a manner that
depended on the concentrations of Sir3 and Mediator (Fig. 5B,
lanes 4, 6, 8, and 9). Interestingly, it appeared that Mediator
bound to nucleosomes more strongly than did Sir3, since much
lower concentrations of Mediator (than of Sir3) were necessary
to shift mononucleosomes and to outcompete Sir3 for mono-
nucleosome binding (Fig. 5B).
Deletion of MED5 shortens replicative life span. H4K16
acetylation has been implicated in the regulation of cellular
life span in S. cerevisiae (7). In old yeast cells, the total
protein level of Sir2 was decreased, which in turn led to an
increase in H4K16 acetylation and to compromised subte-
lomeric transcriptional silencing. Since we had observed an
effect of Mediator on the balance between Sir2 and Sas2, we
FIG. 4. H4K16 acetylation levels change in med5? mutant cells.
(A) Western analyses revealed that the overall levels of Sir2, Sas2,
H4K16 acetylation, and H4 remained unchanged in a med5? strain
(CGC117) compared to a wt strain (BY4741). The Sir2 and Sas2
proteins are indicated with black arrows. (B) ChIP analysis of histone
H4 levels and H4K16 acetylation in med5? and wt cells. The genomic
locations are indicated in Fig. 2. (C) ChIP analysis of histone H4 and
H4K16 acetylation levels near telomere 7L. H4K16 acetylation and
histone H4 occupancy were normalized to the input. H4K16 acetyla-
tion was also normalized to the histone H4 level. Error bars indicate
standard deviations calculated for at least three independent cultures.
**, P ? 0.05.
2418ZHU ET AL.MOL. CELL. BIOL.
decided to examine the effect of a MED5 deletion on rep-
licative life span. In agreement with the idea that Med5 may
be a key factor in the regulation of subtelomeric H4K16
acetylation patterns, we observed an approximately 20%
decrease of the replicative life span in the med5? mutant
compared to that for wt cells (Fig. 6).
In this work, we demonstrate that Mediator associates with
telomeric regions and influences the exact boundary between
active and inactive chromatin. We suggest that Mediator in-
fluences the delicate balance that exists between Sir2 and Sas2.
The opposing effects of these enzymes are believed to generate
a gradient of H4K16 acetylation, which in turn marks the
boundary between active and silenced chromatin near telo-
meres. Our data suggest that Mediator may interact directly
with the subtelomeric region and physically regulate this bal-
ance. Mutations that lead to a loss of Mediator from this
region cause a decrease of subtelomeric silencing and a shift in
the H4K16 acetylation gradient.
Our findings are distinctly different from those presented in
a recent report that identified the SAGA subunit Ada2 as a
possible regulator of the Sir2-Sas2 balance at telomeres (13).
Ada2 was shown to bind telomeric chromatin and the silencing
protein Sir2 in vivo, and loss of ADA2 caused a spread of Sir2
and Sir3 into subtelomeric regions and decreased histone
H4K16 acetylation. Interestingly, a series of publications dem-
onstrated a close link between the SAGA complex and Medi-
ator (17, 31). The two protein complexes appear to be required
for stepwise activation of transcription at many promoters. In
addition, recruitment of Mediator and the SAGA complexes
by Gcn4 has been shown to be interdependent (30, 38). Fur-
thermore, the SAGA complex makes physical contacts with
Mediator, and mutations/deletions of many genes encoding
SAGA components (including ADA2) synthetically interact
with mutations/deletions of Mediator-encoding genes (6, 19).
In our studies, we observed a dramatic increase of H4K16
acetylation at the conserved X core element and also noticed a
slight increase of histone H4 levels. The function of this ele-
ment is not well understood, but in wt cells it is depleted of
nucleosomes and instead bound by typical telomeric proteins,
e.g., Sir2 and Rap1 (45). Based on our findings, we suggest that
Mediator helps to set up the boundary between X elements
bound by Sir proteins and surrounding regions bound by Sas2.
In support of this notion, we found that deletion of MED5 or
temperature-sensitive mutations in MED7 could disturb the
balance between Sir2 and Sas2, consequently allowing nucleo-
somes acetylated at H4K16 to “leak” into nearby X elements.
How Mediator is recruited to X element border regions is
not yet clear, but one factor could be the modification status of
the histones, since Mediator can interact with histone H4 N-
terminal tail peptides and H4K16 acetylation has a strong
negative effect on this interaction (X. Zhu et al., submitted for
publication). Even if Mediator and Sir3 can both bind to
deacetylated H4 N-terminal tails and nucleosomes in vitro, they
FIG. 5. The Mediator complex and the Sir3 protein compete for
binding to mononucleosomes. Electrophoretic mobility shift assays
investigated the binding of wild-type and mutant Mediator complexes
to32P-labeled mononucleosomes. Each panel shows the result of an
individual experiment where all samples were run in the same gel.
Mediator/Nuc, Mediator-mononucleosome cocomplex; Sir3p/Nuc,
Sir3-mononucleosome cocomplex; well, material that was unable to
enter the gel. (A) Wild-type and ?med2 Mediator complexes bind to
mononucleosomes with comparable affinities. Different concentrations
of Mediator were incubated with an invariant concentration of mono-
nucleosomes (0.16 nM), and the reaction mixture was loaded onto the
gel. (B) Various concentrations of Sir3 (as shown in the figure) were
incubated with32P-labeled reconstituted mononucleosomes (0.16 nM)
for 10 min, after which Mediator (at the concentrations specified in the
figure) was added to the binding reaction mixtures, followed by incu-
bation for another 10 min.
FIG. 6. The med5? strain displays a shorter replicative life span
than that of the wild-type strain (for BY4741, median replicative life
span ? 24.5 generations; for med5? strain, median replicative life
span ? 20.5 generations; P ? 0.029). Life spans were determined by
counting the total number of daughters generated by each mother cell.
Experiments were performed with 64 virgin cells per plate.
VOL. 31, 2011MEDIATOR AND SUBTELOMERIC SILENCING 2419
cannot do so simultaneously. Instead, our experiments suggest
that they are mutually exclusive, supporting the idea that Me-
diator may function as a border element. But Mediator blocks
not only Sir3 binding but also spreading of Sas2, which may
explain why the loss of Mediator does not lead to increased
Sir3 occupancy but instead to a spreading of Sas2 and H4K16
acetylation into X elements. In support of this idea, overex-
pression of the Sas2 protein causes lower occupancy of the Sir2
protein and higher H4K16 acetylation levels in telomeric re-
gions (32). Interestingly, the increase of H4K16 acetylation
seen in aging cells is also especially pronounced within X
Med5 is structurally located at the interface of the tail and
middle modules of Mediator, and a med5? mutant strain dis-
plays increased respiration and mitochondrial activity (4). This
result could potentially be coupled to our observation of a
shortening life span in med5? deletion cells, since impaired
respiratory chain function has been linked to premature aging
in many organisms. However, we determined the life span of
another Mediator tail component mutant strain, the med16?
strain, that also displayed a similar increase in respiratory
activity. We failed to observe changes in replicative life span in
the med16? strain, arguing against changed mitochondrial ac-
tivity being the reason for aging in the med5? mutant cells
(data not shown). The Med5 protein has also been identified as
an active histone acetyltransferase, but its substrate specificity
remains unknown (12). We do, however, find it unlikely that
Med5 directly acetylates H4K16, since the observed increase in
H4K16 acetylation levels was associated with a decrease of
Mediator in the subtelomeric region. In addition, we cannot
rule out the possibility that Med5 acts in concert with other
components of the Mediator complex. We previously reported
that Mediator interacts directly with Med16 and forms a spe-
cific Mediator subcomplex in the tail module of the Mediator
complex. Previous reports have demonstrated that loss of
MED16 is associated with global alterations in chromatin ac-
cessibility (1, 46), and as demonstrated here, deletion of
MED16 also affects subtelomeric silencing. It is therefore pos-
sible that Med5 and Med16 together form a Mediator submod-
ule that affects chromatin structure at specific genomic loca-
tions. Med16 (in contrast to Med5) is conserved in evolution,
and Mediator may therefore affect chromatin structure in
higher eukaryotes as well.
A large number of studies have identified the Mediator
complex as the component of the transcription preinitiation
complex required to stimulate Pol II-dependent transcription.
Genome-wide occupancy studies have slightly modified this
view, however, and demonstrated distinctly different patterns
for Mediator and Pol II (1, 46). Mediator is present at many
regions throughout the subtelomeric regions, whereas Pol II is
depleted from these locations. Furthermore, Mediator is also
enriched at other heterochromatin regions, such as the centro-
mere (10). Our observations suggest that Mediator could con-
tribute to the formation of a border between active and inac-
tive chromatin regions. In support of this notion, loss of Med5
leads to a pronounced change in histone occupancy and
H4K16 acetylation levels in X elements but does not change
Mediator occupancy within the X elements. Instead, loss of
Med5 affects Mediator occupancy at adjacent regions border-
ing the X elements. It is tempting to speculate that Mediator
may have a similar function at some gene regulatory regions
and that a role for Mediator in defining the border between
active and inactive chromatin may be a recurring theme at
many genomic locations.
We thank Jerry Workman for providing antibodies against Sas2 and
Stefan Bjo ¨rklund for antibodies against Med1. We thank Danesh
Moazed for the Sir3-Flag yeast expression plasmid. We also thank
Edward J. Louis for providing a yeast strain bearing the tel11L URA3
This work was supported by grants to C.M.G. from the Swedish
Research Council, the Swedish Cancer Society, the European Re-
search Council, and the IngaBritt and Arne Lundberg Research Foun-
dation. This work was also supported by NIH grant GM62483 (to
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