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A dual role of H4K16 acetylation in the
establishment of yeast silent chromatin
Mariano Oppikofer
1
, Stephanie Kueng
1
,
Fabrizio Martino
2
, Szabolcs Soeroes
3
,
Susan M Hancock
2
, Jason W Chin
2
,
Wolfgang Fischle
3
and Susan M Gasser
1,
*
1
Friedrich Miescher Institute for Biomedical Research, Basel,
Switzerland;
2
Medical Research Council Laboratory of Molecular
Biology, Cambridge, UK and
3
Max Planck Institute for Biophysical
Chemistry, Go
¨ttingen, Germany
Discrete regions of the eukaryotic genome assume herita-
ble chromatin structure that is refractory to transcription.
In budding yeast, silent chromatin is characterized by the
binding of the Silent Information Regulatory (Sir) proteins
to unmodified nucleosomes. Using an in vitro reconstitu-
tion assay, which allows us to load Sir proteins onto arrays
of regularly spaced nucleosomes, we have examined the
impact of specific histone modifications on Sir protein
binding and linker DNA accessibility. Two typical marks
for active chromatin, H3K79
me
and H4K16
ac
decrease the
affinity of Sir3 for chromatin, yet only H4K16
ac
affects
chromatin structure, as measured by nuclease accessibility.
Surprisingly, we found that the Sir2-4 subcomplex, unlike
Sir3, has higher affinity for chromatin carrying H4K16
ac
.
NAD-dependent deacetylation of H4K16
ac
promotes bind-
ing of the SIR holocomplex but not of the Sir2-4 hetero-
dimer. This function of H4K16
ac
cannot be substituted
by H3K56
ac
. We conclude that acetylated H4K16 has a
dual role in silencing: it recruits Sir2-4 and repels Sir3.
Moreover, the deacetylation of H4K16
ac
by Sir2 actively
promotes the high-affinity binding of the SIR holocomplex.
The EMBO Journal (2011) 30, 2610–2621. doi:10.1038/
emboj.2011.170; Published online 10 June 2011
Subject Categories: chromatin & transcription
Keywords: H4K16; histone deacetylation; methylation; silent
chromatin; SIR complex
Introduction
Heterochromatin is a heritable, specialized chromatin struc-
ture that silences discrete regions in eukaryotic genomes.
Among other features, gene silencing within heterochromatic
regions is thought to involve compaction of the chromatin
fibre in order to structurally limit DNA accessibility. In
budding yeast, silent chromatin requires the binding of
Silent Information Regulatory (Sir) proteins to unmodified
nucleosomes. Work of many laboratories has identified Sir2,
Sir3 and Sir4 proteins as the core components of silent
chromatin at telomeres and silent mating type loci (Rine
and Herskowitz, 1987; reviewed in Rusche et al (2003)).
Two-hybrid and protein binding studies suggested that they
form a complex, with Sir4 being a scaffold protein that
bridges between Sir2 and Sir3 (Moazed et al, 1997; Strahl-
Bolsinger et al, 1997; Rudner et al, 2005; Cubizolles et al,
2006). Although initial attempts to purify the Sir proteins
from yeast yielded only an Sir2-4 heterodimer (Ghidelli et al,
2001; Hoppe et al, 2002), a stable Sir2-Sir3-Sir4 heterotrimer
with 1:1:1 stoichiometry (hereafter SIR complex) was purified
from insect cells (Cubizolles et al, 2006). A fourth Sir protein,
Sir1, is important for the establishment of silencing at the
silent mating type loci, but is not required for repression at
telomeres (Pillus and Rine, 1989; Aparicio et al, 1991).
Sir proteins do not bind DNA in a sequence-specific
manner, yet zones of silencing are restricted to specific
domains in the yeast genome. To achieve targeted silencing,
Sir proteins are recruited by bifunctional DNA binding
factors, such as Rap1, Abf1 and Orc1, which bind yeast
silencer elements. The SIR complex then spreads from this
nucleation site for 3–20 kb, depe nding on the abundance and
balance of available Sir proteins (reviewed in Gasser and
Cockell (2001) and Rusche et al (2003)). SIR complex asso-
ciation decreases the ability of enzymes, like DNA methylases
or endonucleases, to access the DNA (Gottschling, 1992; Loo
and Rine, 1994). Transcription in these regions is repressed,
most likely by reducing RNA polymerase II occupancy at
promoters (Chen and Widom, 2005; Lynch and Rusche,
2009), although other studies suggest that Sir protein binding
interferes with RNA polymerase II elongation (Sekinger and
Gross, 2001; Gao and Gross, 2008).
All three Sir proteins, Sir2, Sir3 and Sir4, are essential for
transcriptional repression. Sir3 and Sir4 are primarily thought
to be structural proteins of silent chromatin (Gasser and
Cockell, 2001). SIR3 arose from a duplication of the ORC1
gene, with which it shares an N-terminal BAH domain and a
related AAA þATPase domain (Hickman and Rusche, 2010).
Sir4 is found only in related ascomycetes species, while Sir2 is
a NAD-dependent histone deacetylase conserved from bacteria
to man. Its enzymatic activity is required for gene silencing
(Tanny et al, 1999; Imai et al, 2000; Smith et al, 2000).
The key substrate of Sir2 is histone H4 acetylated on lysine
16 (H4K16
ac
; Imai et al, 2000; Smith et al, 2000; Borra et al,
2004). This mark is found on transcriptionally active chro-
matin in most species and marks early firing origins in yeast
and flies (Kimura et al, 2002; Suka et al, 2002; Schwaiger
et al, 2010). It has been shown that unmodified H4K16
promotes compaction of the chromatin fibre in vitro and
in vivo (Smith et al, 2003; Shogren-Knaak et al, 2006;
Robinson et al, 2008). Consistently, in budding yeast
H4K16
ac
is found throughout the genome except at silent
loci (Suka et al, 2001; Smith et al, 2003).
Recombinant fragments of Sir3 and Sir4 were shown to
bind to the histone H4 tail in vitro, in a manner sensitive to
Received: 17 December 2010; accepted: 28 April 2011; published
online: 10 June 2011
*Corresponding author. Friedrich Miescher Institute for Biomedical
Research, Maulbeerstrasse 66, 4058 Basel, Switzerland.
Tel.: þ41 61 697 5025; Fax: þ41 61 697 3976;
E-mail: susan.gasser@fmi.ch
The EMBO Journal (2011) 30, 2610–2621 |
&
2011 European Molecular Biology Organization |All Rights Reserved 0261-4189/11
www.embojournal.org
The EMBO Journal VOL 30 |NO 13 |2011 &2011 European Molecular Biology Organization
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mutations near K16 and to lysine acetylation (Hecht et al,
1995; Carmen et al, 2002). Using recombinant proteins
and nucleosomal substrates, it was found that the H4K16A
mutation can decrease binding of Sir3 in vitro (Johnson
et al, 2009), while mutations of H4K16 to glycine or gluta-
mate, and to a lesser extent arginine, diminished mating
efficiency in vivo (Johnson et al, 1990; Megee et al, 1990;
Park and Szostak, 1990). Finally it was shown that the
H4K16G phenotype could be suppressed by a compen-
satory mutation in Sir3, suggesting that Sir3 contacts
the H4 tail in an acetylation-sensitive manner (Johnson
et al, 1990).
In yeast, H4K16 is acetylated primarily by the histone
acetyltransferase (HAT) Sas2 (Kimura et al, 2002; Suka
et al, 2002) and secondarily by the essential HAT Esa1,
which also targets H4K5 and H4K12 (Suka et al,2001,
2002; Chang and Pillus, 2009). Similar to conservative muta-
tions in H4K16, the deletion of SAS2 impairs repression of a
reporter gene at telomeres or the HML locus, although the
same mutation favours silencing of a reporter at HMR,
which has much stronger silencer elements (Reifsnyder
et al, 1996; Ehrenhofer-Murray et al, 1997; Meijsing and
Ehrenhofer-Murray, 2001). However, the rate of Sir3 recruit-
ment to HMR was slower in cells that lack Sas2 (Katan-
Khaykovich and Struhl, 2005), as was the establishment of
silencing at HML (Osborne et al, 2009). This, together with
the observation that the catalytic activity of Sir2 is required
for silencing (Tanny et al, 1999; Imai et al, 2000; Smith et al,
2000; Yang and Kirchmaier, 2006; Yang et al, 2008a), suggests
that Sir-mediated deacetylation of H4K16
ac
might have an
active role in the formation of silent chromatin.
The H4K16
ac
mark is also required for efficient methylation
of lysine 79 on the histone H3 (H3K79
me
) by the methyltrans-
ferase Dot1 (Altaf et al, 2007). In vivo H3K79
me
appears to
impair the spreading of the Sir proteins, and is thought to act
by reducing association of Sir3 to chromatin (Ng et al, 2002,
2003; van Leeuwen et al, 2002; Altaf et al, 2007; Onishi et al,
2007). Consistently, a recent study with recombinant proteins
has suggested that both Sir3 and the SIR holocomplex have
lower affinities for reconstituted chromatin bearing H3K79
me
(Martino et al, 2009). This shows that, in addition to the
histone tails, the Sir3 protein also interacts with the nucleoso-
mal core, a property that has been assigned both to the N-
terminal BAH domain (Onishi et al, 2007; Buchberger et al,
2008; Norris et al, 2008; Sampath et al, 2009) as well as the
Sir3 C-terminal region (Altaf et al, 2007).
A further histone modification that interferes with SIR-
mediated repression is the acetylation of K56 on histone H3.
In budding yeast, H3K56
ac
is deposited by Rtt109 during
S phase before the loading of the histones onto DNA, and
therefore serves as a marker for newly assembled nucleo-
somes (Hyland et al, 2005; Masumoto et al, 2005; Han
et al, 2007; Li et al, 2008). A large number of studies have
also linked H3K56
ac
to gene transcription from yeast to man
(Xu et al, 2005, 2007; Schneider et al, 2006; Williams et al,
2008; Yang et al, 2008b; Michishita et al, 2009; Xie et al,
2009). In yeast, amino-acid substitutions at H3K56 severely
disrupt silencing without completely displacing the SIR com-
plex (Xu et al, 2007; Yang et al, 2008b). Moreover, elimination
of the histone deacetylases responsible for removal of
H3K56
ac
, Hst3 and Hst4, disrupts SIR-mediated repression
as well (Yang et al, 2008b). A recent report has shown that
acetylation of H3K56 favours transcriptional elongation
through yeast heterochromatin, generating speculation that
H3K56
ac
promotes the displacement of the Sir proteins (Varv
et al, 2010). However, there is as yet no direct evidence that
the affinity of Sir proteins for nucleosomes is lowered by
H3K56
ac
.
To gain insight into the role played by these histone
modifications in the assembly of silent chromatin, we recon-
stituted SIR-bound chromatin in vitro using nucleosomes
homogeneously modified on only one residue. Our system
recapitulates many of the characteristics of silent chromatin
(Martino et al, 2009) and allows us to probe both Sir protein
binding and accessibility of the linker DNA to micrococcal
nuclease (MNase). We find that both H3K79
me
and H4K16
ac
decrease the affinity of Sir3 for chromatin, while only
H4K16
ac
has an effect on MNase accessibility. Surprisingly,
we found that Sir2-4 prefers to bind to chromatin acetylated
on H4K16. The binding of Sir2-4, in presence of NAD and
Sir3, leads to the removal of the H4K16
ac
mark and couples
stable binding of the Sir2-3-4 complex with a significant
decrease in linker DNA accessibility. On the other hand,
H3K56
ac
slightly increases MNase accessibility and reduces
the interaction of chromatin with the SIR complex.
Importantly, we find that H3K56
ac
is not a substrate for
Sir2-mediated deacetylation. We, thus, show how the anti-
silencing properties of different histone modifications differ-
entially affect silent chromatin. Of particular interest are the
two contradictory roles played by H4K16
ac
, which reduces
the binding of Sir3 and favours the recruitment of Sir2-4. The
acetylation and deacetylation of H4K16 thus appear to or-
chestrate the sequential binding of Sir proteins in order to
establish a stable silent chromatin.
Results
The H4K16
ac
mark differentially affects the binding of
Sir2-4 and Sir3 to chromatin
It is generally accepted that the H4K16
ac
mark plays an
important role in silent chromatin by preventing the ectopic
spread of the Sir proteins from the non-acetylated silent
domains (Kimura et al, 2002; Suka et al, 2002; Millar et al,
2004; Yang et al, 2008a). However, accumulating evidence
suggests that not only the absence of the acetyl mark but its
Sir2-dependent removal may be required for efficient estab-
lishment of silencing (Liou et al, 2005; Yang and Kirchmaier,
2006; Yang et al, 2008a; Martino et al, 2009; Osborne et al,
2009). To shed light on this matter, we analysed in detail the
binding of SIR subcomplexes to nucleosomal arrays bearing a
fully acetylated H4K16.
Nucleosomes were reconstituted with recombinant his-
tones that were either unmodified or fully acetylated on
H4K16. These were generated by expressing a truncated
version of H4 and adding the N-terminal tail by native
chemical ligation (NCL) using a synthetic peptide (Shogren-
Knaak et al, 2006; Supplementary Figure S1). Nucleosomal
arrays were then reconstituted by salt dialysis using a DNA
template containing repeated arrays of a 167-bp histone
octamer positioning sequence (Widom 601) as described
previously (Huynh et al, 2005; Martino et al, 2009).
Recombinant Sir proteins were purified from insect cells
(Figure 2F; Cubizolles et al, 2006; Martino et al, 2009).
Dual role of H4K16
ac
in yeast silencing
M Oppikofer et al
&2011 European Molecular Biology Organization The EMBO Journal VOL 30 |NO 13 |2011 2611
We first compared acetylated and non-acetylated arrays of
a 6mer of nucleosomes by monitoring the accessibility of
linker DNA to MNase in the absence of Sir proteins. Previous
studies have shown that acetylation of H4K16 inhibits chro-
matin compaction both in vitro and in vivo (Shogren-Knaak
et al, 2006; Robinson et al, 2008). By challenging this
chromatin with increasing amounts of MNase, we found
that H4K16
ac
enhances linker DNA accessibility of a chroma-
tin template as short as six nucleosomes (Figure 1A).
Sir3 has been reported to be more susceptible than Sir4 to
modifications on histone tails (Carmen et al, 2002; Johnson
et al, 2009). Therefore, we examined first the effect of
H4K16
ac
on the binding of Sir3 to nucleosomal arrays.
Increasing amounts of Sir3 were titrated onto unmodified or
H4K16
ac
arrays of nucleosomes. The binding was analysed by
gel shift and quantified by scoring the loss of the unbound
6mer. In agreement with previous studies (Carmen et al,
2002; Johnson et al, 2009), we found that H4K16
ac
reduces
the binding affinity of Sir3 to an in vitro reconstituted
nucleosomal array by roughly two-fold (Figure 1B). In con-
trast, the binding affinity of the Sir2-4 heterodimer to chro-
matin was increased nearly two-fold by the presence of the
H4K16
ac
mark (Figure 1C). Superficially, this appears to
contradict the fact that silent chromatin is depleted for this
mark, although it is consistent with the notion that H4K16
ac
is a key substrate of Sir2-4 (see also Johnson et al (2009)).
Therefore, we decided to perform competition experiments in
order to reinforce this observation. The binding of increasing
amounts of Sir2-4 to an unmodified Cy5-labelled array was
competed with a four-fold excess of either unlabelled unmo-
dified or unlabelled H4K16
ac
chromatin. Confirming our
previous results, H4K16
ac
chromatin competed roughly two-
fold more efficiently for the binding of Sir2-4 compared with
unmodified chromatin (Figure 1D).
We previously showed that our recombinant Sir2-4 has
efficient histone deacetylase activity in the presence of its co-
factor NAD (Cubizolles et al, 2006). We reasoned that if
Sir2-4 bound H4K16
ac
with higher affinity because it is a
preferred substrate of Sir2, then the complex should have
less affinity once H4K16
ac
had been deacetylated. To test this,
we quantified the binding of the Sir2-4 heterodimer to
H4K16
ac
chromatin in the absence or presence of NAD.
Confirming our hypothesis, Sir2-4 bound more readily to
acetylated chromatin and less readily following deacetylation
(Figure 1E). Surprisingly, this shows an enhanced affinity
of Sir2-4 for acetylated H4K16, while the opposite is true
for Sir3.
Sir2-dependent deacetylation of H4K16
ac
stabilizes the
association of Sir2-3-4 to chromatin
Removal of H4K16
ac
through the catalytic activity of Sir2 has
been reported to be important for silencing (Johnson et al,
1990; Suka et al, 2001, 2002; Carmen et al, 2002; Kimura et al,
2002). However, it is not clear whether this is due exclusively
Figure 1 Acetylation of H4K16 decreases the binding affinity of
Sir3 but favours the loading of Sir2-4 onto chromatin. (A) Equally
saturated 6mer of 601 nucleosomes with either unmodified or
acetylated H4K16 was digested with increasing amount of MNase,
as indicated. After protein digestion, the denatured DNA was
separated by electrophoresis and visualized by SYBR
s
Safe stain-
ing. The bands showed by an arrow (6-, 5-, 4- and 3mers) were
quantified and normalized to input. The histograms show the ratio
between the amount of intact 6-3mers of H4K16
ac
over unmodified
chromatin for the indicated MNase titration point. The Sir3 protein
(B) or the Sir2-4 heterodimer (C) were titrated into a constant
amount of unmodified or H4K16
ac
6mer of 601 nucleosomes.
Samples were separated by native agarose gel electrophoresis and
visualized by SYBR
s
Safe staining. (D) The binding of increasing
amounts of Sir2-4 to 8 nM of unmodified Cy5-labelled 6mer of
nucleosomes (indicated by the arrowhead) was competed with
32 nM of either unlabelled unmodified or unlabelled H4K16
ac
6mer of nucleosomes. Cy5 fluorescence was used to monitor the
binding of Sir2-4 to the unmodified labelled chromatin. The asterisk
indicates a Cy5-labelled contaminant DNA. (E) The Sir2-4 hetero-
dimer was titrated into a constant amount of H4K16
ac
6mer of 601
nucleosomes. Deacetylation is allowed by the addition of 150 mM
NAD where indicated. Samples were separated and visualized as in
(B,C). The images are representative of at least three independent
experiments, quantifications show the mean value±s.e.m. of the %
of unbound chromatin compared with the input.
Dual role of H4K16
ac
in yeast silencing
M Oppikofer et al
The EMBO Journal VOL 30 |NO 13 |2011 &2011 European Molecular Biology Organization2612
to the generation of unmodified H4K16 or whether it addi-
tionally involves a conformational change coupled to O-
acetyl-ADP-ribose (O-AADPR) production (Liou et al, 2005;
Martino et al, 2009). To gain insight into the molecular
consequences of H4K16
ac
deacetylation on the establishment
of silencing, we compared the binding of the SIR complex
with unmodified or H4K16
ac
chromatin in the presence or
absence of NAD. We first examined the effect of H4K16
ac
on
the binding of the Sir2-3-4 heterotrimer in absence of NAD
(Figure 2A) and found that, similar to Sir2-4 (Figure 1C) but
in a less pronounced manner, the SIR holocomplex bound
slightly better to acetylated chromatin. We then confirmed
that our purified Sir2-3-4 complex was able to efficiently
deacetylate H4K16
ac
within chromatin in the presence of
NAD (Figure 2E), as shown previously for chemically acety-
lated histone octamers (Cubizolles et al, 2006). In the follow-
ing experiments, the term ‘deacetylated chromatin’ will be
used whenever H4K16
ac
marks were actively removed by Sir2
in the presence of NAD, to distinguish it from chromatin
assembled from unmodified histones.
We next compared the binding affinity of Sir2-3-4 with
unmodified or H4K16
ac
chromatin in the presence of NAD.
We found that the active removal of the H4K16
ac
mark
increased the binding affinity of the SIR complex to chroma-
tin by roughly two-fold (Figure 2B). This effect is not caused
by NAD alone as enhanced binding was not observed when
H4K16
ac
chromatin was replaced with unmodified chromatin
(Supplementary Figure S2A). Indeed, in absence of Sir2,
Figure 2 Sir2-dependent deacetylation of H4K16
ac
stabilizes the association of Sir2-3-4 to chromatin. The Sir2-3-4 complex (A,B) or the
catalytically dead Sir2cd-3-4 mutant (C,D) was titrated into a constant amount of unmodified or H4K16
ac
6mer of 601 nucleosomes. Where
indicated, 150 mM NAD was added to the samples. Scatter plot quantifications show the mean value±s.e.m. of the % of unbound chromatin
compared with the input for at least three experiments. (E) Reconstituted chromatin fully acetylated on H4K16 was subjected to NAD-
dependent deacetylation in presence of a 2.5-fold molar excess of the Sir2-3-4 complex or the Sir2cd-3-4 mutant. The acetylation state was then
determined by immunoblotting using acetylation mark-specific antibodies and H3 for loading. (F) Two micrograms of the indicated Sir protein
were denatured in sample buffer and run on a 4–12% NuPAGEsNovexsBis-Tris Gel.
Dual role of H4K16
ac
in yeast silencing
M Oppikofer et al
&2011 European Molecular Biology Organization The EMBO Journal VOL 30 |NO 13 |2011 2613
NAD does not affect the acetylation state of chromatin
(Supplementary Figure S2B). This shows that the binding
affinity of the SIR complex for deacetylated chromatin is
higher compared with chromatin assembled from unmodified
histones.
In order to reinforce this finding, we tested whether the
deacetylase activity of Sir2 itself was required for the en-
hanced binding of acetylated template in the presence of
NAD. We generated a catalytic inactive Sir2 (Sir2cd) by
introducing the point mutation N345A, which maps to the
nucleotide binding motif (Rossman fold). This mutation
disrupts Sir2 enzymatic activity in vitro and in vivo (Imai
et al, 2000; Armstrong et al, 2002). The N345A substitution,
however, did not affect the stability of Sir2 or its interaction
with Sir3 and Sir4 and we were able to purify the mutated
Sir2cd-3-4 from insect cells with the same efficiency as for the
Sir2-3-4 complex (Figure 2F). We could furthermore confirm
that this mutant did not retain significant deacetylase activity
(Figure 2E).
In order to confirm that the Sir2cd-3-4 mutant was still able
to recognize its substrate, we monitored the binding of Sir2cd-
3-4 to unmodified or H4K16
ac
chromatinintheabsenceof
NAD. We found that, like Sir2-3-4, Sir2cd-3-4 has a slight
preference for H4K16
ac
chromatin (compare Figure 2A and C).
We then monitored the loading of the Sir2cd-3-4 mutant onto
unmodified or H4K16
ac
chromatin in the presence of NAD.
Unlike the Sir2-3-4 complex, the catalytic dead Sir2cd-3-4
showed the same slight preference for the H4K16
ac
chromatin,
as it did in the absence of NAD (compare Figure 2C and D).
This data reinforce our observation that the deacetylation
reaction has a positive role on the loading of the SIR complex
onto chromatin (Figure 2B). Given that the deacetylation of
H4K16
ac
chromatin reduced the binding affinity of the Sir2-4
heterodimer (Figure 1E), these results suggest that Sir3 and the
deacetylation of H4K16
ac
by Sir2 jointly promote the binding of
the SIR holocomplex to chromatin.
The Sir3 protein and Sir2-dependent deacetylation of
H4K16
ac
are both required to decrease nuclease
accessibility of the linker DNA
SIR complex bound chromatin is thought to have a more
compact structure in vivo as it is less accessible to enzy-
matic attack (Gottschling, 1992; Loo and Rine, 1994). The SIR
complex could compact chromatin in two ways: first by
deacetylating H4K16, and second by binding to chromatin.
We observed that loading of the SIR complex onto unmodi-
fied chromatin greatly reduces the accessibility of the linker
DNA to MNase and the restriction enzyme AvaIin vitro,
consistent with a direct role for binding (Supplementary
Figure S3A and B; Martino et al, 2009). In order to test the
impact of H4K16
ac
deacetylation on compaction in vitro,we
first incubated H4K16
ac
chromatin with the Sir2-4 subcom-
plex in the presence or absence of NAD and then challenged
it with increasing amounts of MNase. We found that the
presence of NAD did not significantly change the accessibility
of the linker DNA (Figure 3A). Since acetylation of chro-
matin usually results in greater accessibility (Figure 1A), this
result is likely a combination of changed accessibility due to
removal of H4K16
ac
and reduced binding affinity of Sir2-4 for
deacetylated chromatin (Figure 1E).
We then performed the same experiment but replaced Sir2-
4 with the SIR holocomplex. The concentration of Sir2-3-4
used resulted in a complete upshift of both acetylated and
unmodified chromatin in a binding assay, ruling out differ-
ential accessibility due to incomplete ligand occupancy.
Interestingly, we observed that in the presence of NAD the
linker DNA was more protected from MNase than SIR-bound
Figure 3 Sir3 is required to translate the Sir2-dependent deacetyla-
tion of H4K16
ac
into a decrease of nuclease accessibility of the linker
DNA. Unmodified or H4K16
ac
6mer of 601 nucleosomes (50 nM)
was incubated with the indicated amount of Sir2-4 (A), Sir2-3-4
(B,Dand E) or Sir2cd-3-4 (C) and was challenged with increasing
amounts of MNase. Where indicated, SIR-bound chromatin was
supplemented with 150 mM NAD and incubated for 15 min at 301C
before MNase digestion. Deproteinated samples were separated by
electrophoresis and the amount of intact 6mer DNA (black arrow)
was quantified and normalized to input. Quantification of at least
three experiments was used to generate the vertical bar charts, data
represent mean value±s.e.m.
Dual role of H4K16
ac
in yeast silencing
M Oppikofer et al
The EMBO Journal VOL 30 |NO 13 |2011 &2011 European Molecular Biology Organization2614
chromatin in the absence of NAD (Figure 3B). Importantly,
the addition of NAD did not change the protection of linker
DNA of an array bound by the catalytic inactive Sir2cd-3-4
(Figure 3C). The same analysis in the absence of Sir3 did
not increase protection against MNase attack (Figure 3A),
arguing that the protective effect of NAD-dependent deacety-
lation of H4K16
ac
by Sir2 requires Sir3 (compare Figures 3A
and B). Finally, the increased linker DNA protection observed
in Figure 3B was not caused by the NAD molecule per se,as
no NAD-dependent differences were scored for linker DNA
accessibility when Sir proteins were bound to unmodified
chromatin (Supplementary Figure S3C). These results sug-
gested that the deacetylation of H4K16
ac
by the SIR complex
promotes linker DNA protection.
When comparing unmodified with acetylated chromatin
bound by Sir2-3-4 in the absence of NAD, we found that the
linker DNA is slightly more protected (Figure 3D), indicating
that at least some of the protection observed in Figure 3B is
due to loss of H4K16
ac
per se. However, given our previous
observation that deacetylated chromatin is bound with higher
affinity than unmodified chromatin (Figure 2B), we decided
to explore the possibility that the deacetylation reaction itself
may also contribute to the increased linker DNA protection
observed in Figure 3B. Therefore, we compared the linker
DNA accessibility of unmodified and H4K16
ac
chromatin in
the presence of Sir2-3-4 and NAD. We found that deacetylated
chromatin is reproducibly more protected from MNase attack
compared with chromatin assembled from unmodified his-
tones (Figure 3E). Together, these results show that both Sir2-
dependent deacetylation of H4K16
ac
and Sir3 are required to
decrease the nuclease accessibility of linker DNA, which
presumably reflects the tighter binding of the SIR holocom-
plex to chromatin. In addition, there may be a conformational
change that enhances linker DNA protection.
H3K56
ac
loosens Sir protein binding to chromatin,
slightly increasing linker DNA accessibility
To ask if our observation for H4K16
ac
can be generalized to
other acetylation marks we tested the effects of H3K56
acetylation, which is found on newly assembled nucleosomes
in S phase. Since there are contradictory reports about which
enzyme deacetylates H3K56
ac
(Xu et al, 2007; Yang et al,
2008b) we first tested if Sir2 can deacetylate H3K56
ac
as
suggested earlier. We incubated H3K56
ac
chromatin with
SIR complex in the presence or absence of NAD. Chromatin
homogenously acetylated at H3K56 was obtained by purify-
ing acetylated H3 from E. coli using an expanded genetic code
strategy (Neumann et al, 2008). Probing the histones with
H3K56
ac
antibodies after incubation showed that, unlike for
H4K16
ac
, the level of H3K56 acetylation remained unchanged
(Figure 4B). We conclude that H3K56
ac
is not a substrate of
the NAD-dependent deacetylase activity of Sir2. This sup-
ports previous work reporting that two Sir2-related enzymes,
Hst3 and Hst4, are required for H3K56
ac
deacetylation in vivo
(Celic et al, 2006; Maas et al, 2006; Yang and Kirchmaier,
2006) and suggests that Hst3 and Hst4 are the exclusive
deacetylases for this residue.
To address whether acetylation of H3K56 has an effect on
Sir protein loading, we compared the binding of the SIR
holocomplex with unmodified and H3K56
ac
chromatin. We
found that H3K56
ac
reduces the affinity of the SIR holocom-
plex for chromatin by roughly two-fold (Figure 4C). The
binding affinity of the Sir2-4 heterodimer was also reduced
in presence of the H3K56
ac
mark (Figure 4D), while the
binding affinity of the Sir3 protein alone was mostly un-
changed (Figure 4E). To explore whether the slight affinity
decrease observed here for the SIR complex could be respon-
sible for the silencing defects seen in vivo, we investigated
whether the SIR complex efficiently protects linker DNA in
chromatin bearing the H3K56
ac
mark. The acetylation on
H3K56 per se has been shown to increase transient unwrap-
ping of the DNA from the histone octamer but not to change
the higher order structure of a 61mer nucleosomal array
(Neumann et al, 2009). Consistently we show, by means of
an MNase digestion assay, a slight increase in linker DNA
accessibility for the chromatin bearing H3K56
ac
over the
unmodified control (Figure 4F). Subsequently, after adding
the SIR complex in saturating concentrations (Supplementary
Figure S4A), the H3K56
ac
chromatin continued to show
slightly higher linker DNA accessibility as compared with
unmodified chromatin (Figure 4G). This is consistent with a
previous in vivo study indicating that H3K56
ac
chromatin
is more sensitive to DNA methylation by an ectopically
expressed bacterial dam methylase (Xu et al, 2007).
Moreover, H3K56 point mutations disrupted silencing at
telomeres without affecting Sir protein spreading (Xu et al,
2007). We conclude that H3K56
ac
does not have a role similar
to that of H4K16
ac
, neither in the recruitment of Sir2-4, nor by
being a substrate for Sir2.
Methylation of H3K79 by Dot1 neither increases linker
DNA accessibility nor reduces Sir2-4 loading
Another mark associated with active chromatin in yeast is
methylation of lysine 79 of histone H3 (H3K79). This methy-
lation is exclusively catalysed by Dot1 and is thought to act as
a boundary for the inappropriate spreading of the Sir proteins
on chromatin (van Leeuwen et al, 2002; Frederiks et al, 2008;
Martino et al, 2009; Verzijlbergen et al, 2009). Moreover,
in vitro studies showed that interaction of the Sir3 protein with
histone peptides was sensitive to the methylation of H3K79
(Altaf et al, 2007; Onishi et al, 2007). Previous work from our
laboratory showed that we can make use of recombinant Dot1
in order to methylate reconstituted nucleosomal arrays in vitro
(Martino et al, 2009). We have previously shown that even
partial methylation of H3K79 decreases the binding affinity of
both the SIR complex and the Sir3 protein alone to chromatin
(Martino et al, 2009). We now provide further evidence that the
lowered affinity indeed affects Sir3 binding, since the Sir2-4
heterodimer associates with unmodified and H3K79
me
chroma-
tin with nearly equal affinity (Figure 5A), while Sir3 clearly
prefers unmodified chromatin (Figure 5B).
We then decided to test whether H3K79
me
also impacts the
structure of SIR-bound or SIR-depleted chromatin. To exam-
ine the potential impact of H3K79
me
on linker DNA protec-
tion, we challenged in vitro methylated chromatin lacking Sir
proteins with increasing amounts of MNase. Unlike the case
for H3K56
ac
, the accessibility of the linker DNA in the
absence of SIR complex was unaffected by H3K79
me
(Figure 5C). However, in the presence of substoichiometric
amounts of Sir2-3-4 (Supplementary Figure S4B), the acces-
sibility of the linker DNA was higher for H3K79
me
chromatin
than for unmodified chromatin (Figure 5D), consistent with
notion that better SIR complex binding enhances linker DNA
protection. When we added additional Sir2-3-4 such that
Dual role of H4K16
ac
in yeast silencing
M Oppikofer et al
&2011 European Molecular Biology Organization The EMBO Journal VOL 30 |NO 13 |2011 2615
we score an equal degree of binding on both substrates
(Supplementary Figure S4C), we observed no difference in
accessibility of linker DNA (Figure 5E). This suggests that
H3K79
me
neither changes chromatin structure nor prevents
the SIR complex from compacting it, but decreases the
affinity of the SIR complex for chromatin. Thus, it antago-
nizes silencing through a mechanism distinct from H3K56
ac
.
Discussion
Silent chromatin in Saccharomyces cerevisiae is the best
studied system of heterochromatic gene silencing, yet we
still do not fully understand the molecular mechanisms of
its assembly and the role of histone modifications in this
process. In vitro binding analysis between Sir protein
domains and histone peptides have been informative, yet
they only reflect a small part of the chromatin template.
To examine the molecular basis of SIR-dependent silen-
cing, we have established a fully recombinant system that
recapitulates key features of silent chromatin in budding
yeast (Martino et al, 2009). Here, we extend this system to
examine how histone modifications participate in the forma-
tion of stable silent and active domains.
We show here that the H4K16
ac
mark has both a positive
and a negative role in SIR binding in a sequential manner (see
Figure 6). Importantly, we show that H4K16
ac
decreases the
binding affinity of Sir3, but, in contrast, promotes the asso-
ciation of the Sir2-4 heterodimer to chromatin. Even the
binding affinity of the SIR holocomplex is slightly increased
by the presence of H4K16
ac
in the absence of NAD (see also
Johnson et al (2009)). This result, while initially counter-
intuitive, helps elucidate the dual role of H4K16
ac
in hetero-
chromatin formation. On one hand, H4K16
ac
prevents the
dispersion of its key ligand, Sir3, into euchromatic chromatin.
On the other hand, the high affinity of Sir2-4 for H4K16
ac
may
help nucleate silent chromatin, since it is likely in yeast that
the targeted nucleosomes are acetylated before SIR complex
loading.
In support of this dual role, it was shown that the sub-
stitution of H4K16 by not only an acetyl-mimicking residue,
but also unacetylatable amino acids, disrupts silencing at
telomeres and mating type loci (Johnson et al, 1990, 1992;
Megee et al, 1990; Park and Szostak, 1990; Aparicio et al,
1991; Millar et al, 2004). Moreover, the deletion of SAS2,
which encodes the HAT responsible for most H4K16
ac
in
yeast, impaired the repression of reporter genes at certain
loci, such as TelVIIL or within the HML locus (Reifsnyder
et al, 1996; Meijsing and Ehrenhofer-Murray, 2001) and led to
the spreading of Sir proteins into subtelomeric regions that
usually lack SIR-mediated repression (Kimura et al, 2002;
Figure 4 H3K56
ac
decreases Sir protein binding affinity and slightly increases linker DNA accessibility. (A) Cartoon representation of the
nucleosome core particle (NCP147; Davey et al, 2002) highlighting the position of H3K56 (black) at the entry/exit point of the DNA around the
histone octamer. (B) Reconstituted chromatin fully acetylated on H3K56 was subjected to NAD-dependent deacetylation in presence of a 2.5-
fold molar excess of the SIR complex. The acetylation state was then determined by immunoblotting using acetylation mark-specific antibodies
and H3 for loading. The SIR complex (C), Sir2-4 heterodimer (D) or Sir3 (E) were titrated into a constant amount of unmodified or H3K56
ac
6mer of 601 nucleosomes. Samples were analysed as in Figure 2. Unmodified or H3K56
ac
6mer of nucleosomes were challenged with an
increasing amount of MNase in absence (F) or presence (G) of the SIR complex. The 6mer, 5mer and 4mer bands (F) or the band corresponding
to the intact 6mer alone (G), shown by black arrows, were quantified and normalized to the input. The histograms show the ratio between the
amounts of quantified DNA from H4K16
ac
chromatin over unmodified for the indicated MNase titration point±s.e.m.
Dual role of H4K16
ac
in yeast silencing
M Oppikofer et al
The EMBO Journal VOL 30 |NO 13 |2011 &2011 European Molecular Biology Organization2616
Suka et al, 2002). In contrast, the repression of a reporter at
HMR was enhanced, probably because this locus has much
stronger silencers, which dominate over an indiscriminate
spreading of Sirs (Ehrenhofer-Murray et al, 1997). Impor-
tantly, both the kinetics of Sir3 recruitment to HMR and the
establishment of silencing at HML were slower in cells that
lack the H4K16-specific HAT, Sas2 (Katan-Khaykovich and
Struhl, 2005; Osborne et al, 2009), suggesting a positive role
for H4K16 acetylation. Collectively, these results support the
model that Sas2-mediated acetylation of H4K16 has more
than one role in silent chromatin formation (see also Zou and
Bi (2008)). Reporter context appears to determine which
role is rate limiting: the recruitment of Sir2-4, or the assembly
and propagation of the Sir3-containing holocomplex along
nucleosomes.
Sequential assembly of nuclease-resistant SIR-bound
chromatin requires H4K16
ac
deacetylation
In vivo the absence of H4K16
ac
from silent chromatin suggests
that it is removed by Sir2 as soon as the SIR complex is
loaded. Moreover, it was shown that in the absence of Sir2
catalytic activity, H4K16
ac
prevents the formation of silent
domains (Yang and Kirchmaier, 2006). On the other hand,
as mentioned above, even conservative substitutions at
H4K16 decrease silencing efficiency at HML and at telomeres
(Johnson et al, 1990; Meijsing and Ehrenhofer-Murray, 2001;
Yang and Kirchmaier, 2006). This supports the notion that not
only the recruitment of Sir2-4 by H4K16
ac
, but the deacetyla-
tion reaction itself helps to seed repression (Johnson et al,
1992; Imai et al, 2000; Millar et al, 2004; Liou et al, 2005;
Yang et al, 2008a; Martino et al, 2009).
Why is the deacetylation reaction important for silencing?
The recapitulation of these steps in vitro helped us to address
this question. Indeed, by adding NAD to SIR-bound H4K16
ac
chromatin, we catalysed deacetylation of H4K16 and in-
creased the binding affinity of the SIR holocomplex to chro-
matin. This shows in a well-defined recombinant system
that removal of the single H4K16
ac
mark by Sir2 increases
SIR complex binding. Even more importantly, we found that
the linker DNA was better protected from MNase digestion
when the SIR complex was assembled on chromatin in the
presence of H4K16
ac
and NAD, as compared with its being
loaded onto unmodified chromatin. This protection nicely
mimics the DNA shielding observed in SIR-silenced chroma-
tin regions in vivo (Gottschling, 1992; Loo and Rine, 1994;
Xu et al, 2007) and argues that the SIR complex may associate
with chromatin in more than one conformation. It was
previously proposed that a by-product of Sir2 NAD-
dependent deacetylation, O-AADPR, might trigger a confor-
mational change of the SIR–chromatin complex to favour
repression (Liou et al, 2005; Onishi et al, 2007; Martino et al,
2009).
Importantly, the deacetylation-dependent increase in affi-
nity of the SIR holocomplex for chromatin, and the increase
in linker DNA protection, depends crucially on the presence
of Sir3. This is consistent with previous results which showed
that exogenously added O-AADPR enhances the binding of
both Sir3 and the SIR holocomplex to chromatin (Martino
et al, 2009). A further study showed that addition of an excess
of acetylated peptides and NAD to SIR–chromatin assemblies
generated a structure that appeared more compact by elec-
tron microscopy (Johnson et al, 2009), perhaps reflecting a
conformational change in the SIR complex (Liou et al, 2005).
Nevertheless, O-AADPR is probably neither absolutely req-
uired for SIR complex loading nor for silencing, since repres-
sion can be achieved in a strain devoid of NAD-dependent
deacetylases if an ectopic HDAC is fused to Sir3 (Chou et al,
2008) or if Sir3 is overexpressed in a H4K16R background
(Yang and Kirchmaier, 2006). Taken together, our data sup-
port a scenario in which the sequential loading of Sir2-4
Figure 5 Methylation of H3K79 by Dot1 affects neither linker DNA
accessibility nor Sir2-4 loading onto chromatin. The Sir2-4 hetero-
dimer (A) or Sir3 (B) was titrated into a constant amount of
unmodified or H3K79
me
6mer of nucleosomes. Samples were ana-
lysed as in Figure 2. Unmodified or H3K79
me
chromatin were
challenged with an increasing amount of MNase in absence (C)or
presence (D,E) of the indicated amount of the SIR complex. The
6mer DNA band alone (C,E) or the 3mer to 6mer bands (D), shown
by black arrows, were quantified and normalized to the input. The
histograms show the ratio between the amount of quantified DNA
from H3K79
me
over unmodified chromatin for the indicated MNase
titration point.
Dual role of H4K16
ac
in yeast silencing
M Oppikofer et al
&2011 European Molecular Biology Organization The EMBO Journal VOL 30 |NO 13 |2011 2617
onto nucleosomes containing H4K16
ac
, its NAD-dependent
deacetylation and the loading of Sir3, sequentially promote a
stable assembly that protects linker DNA from exogenous
factors (Figure 6).
Boundary formation and reduction of SIR holocomplex
affinity by histone modifications
The observation that H4K16
ac
might provide a boundary for
heterochromatin spreading (Kimura et al, 2002; Suka et al,
2002) seems counterintuitive given the results described
above. However, H4K16
ac
does affect other processes beyond
SIR complex association to chromatin, most notably, the
recruitment of the histone methyltransferase Dot1 to chro-
matin (Altaf et al, 2007). Given that Sir2-4 preferentially
binds chromatin carrying H4K16
ac
, we propose that Dot1
competes with the recruitment of Sir2-4, and not as proposed
earlier, with Sir3 (Altaf et al, 2007). On the other hand, the
anti-silencing role of the methylation mark itself, H3K79
me
,is
most likely a reflection of reduced interaction between Sir3
and methylated chromatin (Ng et al, 2002, 2003; van
Leeuwen et al, 2002; Altaf et al, 2007; Onishi et al,2007;
Martino et al, 2009). Consistently, we found that nucleo-
somes bearing H3K79
me
neither affect the binding of the
Sir2-4 heterodimer, nor was there an inherent change in
structural properties of H3K79
me
-containing chromatin. This
is consistent with crystallographic analyses which argue that
H3K79
me
does not alter the structure of the nucleosome
(Lu et al, 2008). Given that no enzyme has been found so
far that removes the H3K79
me
mark, depletion of this mark
may depend on histone eviction or on sequential dilution
through rounds of DNA replication. Its slow removal renders
H3K79
me
a more stable barrier to the spreading of the SIR
complex than H4K16
ac
, which instead recruits Sir2-4 and
promotes the spread of repression (Figure 6).
The third histone mark correlated with active chromatin in
yeast is the acetylation of H3 on K56. In contrast with
H3K79
me
, H3K56
ac
is clearly subject to active deacetylation.
Here, we show that Sir2 is unable to remove the H3K56
ac
mark in vitro, which indirectly supports previous work
showing that H3K56
ac
is primarily deacetylated by two Sir2-
related enzymes: Hst3 and Hst4 (Celic et al, 2006; Maas et al,
2006). Indeed, Sir-mediated repression cannot be established
in the absence of these two enzymes, although Sir proteins
still bind telomeres in an hst3Dhst4Dmutant (Yang et al,
2008b). Consistent with our work, this suggests that the
H3K56
ac
mark does not completely block SIR–chromatin
interaction.
How then does H3K56
ac
impair the formation of silent
chromatin? Using our in vitro system, we found that H3K56
ac
affects both the affinity with which SIR complexes bind
chromatin and the formation of a chromatin structure that
is less accessible to MNase attack. The observed drop in
affinity of SIR holocomplex for chromatin agrees with an
in vivo study, which suggested that H3K56
ac
facilitates Sir
protein displacement and RNA polymerase II elongation
within heterochromatin regions (Varv et al, 2010). Our
Figure 6 Combinatorial histone modifications distinguish silent and active chromatin regions. (A) Outline of the role played by different
histone modifications on Sir protein loading and chromatin structure. (B) The Sir proteins are recruited onto chromatin by protein–protein
interactions and bind tightly unmodified nucleosomes driving gene silencing. Spreading of the SIR complex is promoted by H4K16
ac
that
recruits the Sir2-4 heterodimer yet prevents the ectopic spreading of Sir3 alone. The NAD-dependent deacetylation reaction of H4K16
ac
by Sir2
generates a high-affinity binding substrate for Sir3 and the synthesis of O-AADPR favours the tight association of the SIR complex to
unmodified nucleosomes. H3K56
ac
and H3K79
me
generate a boundary to the spreading of the SIR complex mainly by reducing the binding
affinity of the Sir2-4 heterodimer and the Sir3 protein, respectively. The acetylation of H3K56 and H4K16 also enhance the accessibility of the
chromatin fibre, unlike methylation of H3K79.
Dual role of H4K16
ac
in yeast silencing
M Oppikofer et al
The EMBO Journal VOL 30 |NO 13 |2011 &2011 European Molecular Biology Organization2618
observation that H3K56
ac
increases accessibility of the
linker DNA is consistent with an increase in spontaneous
(but transient) unwrapping of the DNA from the histone
octamer, which may reflect the position of H3K56 at the
entry/exit point of the nucleosomal DNA (Figure 4A;
Neumann et al, 2009). It is striking that even SIR-saturated
arrays showed increased linker DNA accessibility in the
presence of H3K56
ac
, indicating that SIR binding cannot
overcome the effect of H3K56
ac
on nucleosomal structure.
Although it is unclear why this modification reduces SIR
complex binding, this and the increased linker DNA exposure
are likely to account for the anti-silencing effect of the
H3K56
ac
mark.
To conclude, we propose that the euchromatic mark
H4K16
ac
is required for the formation of both active and
silent chromatin. The process of creating stable silent and
active states is not an one-step event, but requires positive
feedback loops. H4K16
ac
may be the starting point for silent
domains, which are reinforced by the Sir2 deacetylation
reaction and possibly the generation of O-AADPR, and active
domains, where it promotes H3K79 methylation. These inter-
dependent pathways are conserved throughout evolution and
mathematical modelling clearly shows that such networks
are required to establish a stable binary switch (Dodd et al,
2007; Mukhopadhyay et al, 2010). Here, we have demon-
strated that H4K16
ac
is actively implicated in the establish-
ment of yeast silent chromatin, being the first histone mark
shown to recruit Sir proteins to chromatin.
Materials and methods
SIR purification and chromatin reconstitution
In vitro reconstitution of SIR-bound chromatin was carried out
essentially as described (Cubizolles et al, 2006; Martino et al, 2009).
Briefly, the Sir proteins were expressed in sf21 insect cells with
baculoviruses generated using BD BaculoGoldTM, BD-Biosciences.
Co-infection was used to produce the Sir2-3-4 complex, the catalytic
dead Sir2cd-3-4 and the Sir2-4 heterodimer, a single infection was
used to produce the Sir3 protein alone (Cubizolles et al, 2006).
Recombinant X. laevis histones were use to reconstitute histone
octamers as described previously (Luger et al, 1997). Chromatin
was assembled in vitro by adding increasing amounts of purified
histone octamer to a constant amount of DNA arrays containing six
601-Widom positioning elements separated by 20 bp of linker DNA,
referred as 601-167-6mer (Lowary and Widom, 1998). An unspecific
DNA sequence of 147 bp (referred as ‘competitor’ on the figures)
was added to the mix in order to bind the excess of histone
octamers subsequent to the saturation of the 601-167-6mer (Huynh
et al, 2005). Cy5-labelled 601-167-6mer was generated by filling the
50overhang-ends of an EcoRI site with Klenow enzyme (NEB,
accordingly to manufacturer’s instruction), using d-CTP-Cy5 (GE
Healthcare). The free nucleotides were then separated from the
Cy5-labelled array using small Bio-spin columns (Bio-Rad). DNA
and histones were mixed in 40 ml of buffer A (10 mM TEA pH 7.4
and 1 mM EDTA) and 2 M NaCl on ice, and chromatin was
reconstituted by step dialysis in buffer A containing 1.2, 1, 0.8 or
0.6 M NaCl for 2 h at 41C and in buffer A overnight (Lee and
Narlikar, 2001). The 601-167-6mer was routinely prepared at a final
nucleosomal concentration of 10
6
M. Increasing amounts of Sir
proteins were added to the 601-167-6mer diluted to 5 10
8
Mor
2.5 10
8
M in 10mM TEA pH 8, 25 mM NaCl, 0.05% Tween-20 on
ice and after 10min incubation the samples were fixed with
0.0025% glutaraldehyde for 10 min on ice. The fixation yields
slightly sharper bands but the results are very similar without
fixation. When chromatin deacetylation was coupled to Sir protein
loading, SIR-bound chromatin was incubated with or without
150 mM NAD for 15 min at 301C before incubation on ice for a
10 min fixation as above. The samples were routinely run at 80 V for
90 min at 41C in a 0.7% agarose gel 0.2 TB: 18 mM Tris, 18 mM
Boric acid. The gel was soaked for 20 min in 1 SYBRsSafe and
the DNA was visualized in a Typhoon 9400 scanner.
Preparation of the histone modifications
Methylation of H3K79 was carried out on reconstituted chromatin
as described before (Martino et al, 2009). Briefly, 0.8 pmol of
recombinant Dot1 was incubated with 8 pmol of reconstituted 601-
167-6mer in 25 mM Tris pH 7.9, 20 mM NaCl, 0.4mM EDTA, with or
without 160 pmol of S-adenosylmethionine (SAM) at 301C for
30 min, then 160 pmol of SAM was added and the reaction was
continued for 30 min. Mass spectrometry analysis showed that
H3K79 is mono-, di- and, to a lesser extent, tri-methylated on at
least 50% of the available K79 residues (Frederiks et al, 2008;
Martino et al, 2009). The chromatin was then stored at 41C.
Homogeneous acetylated histone H3 at the lysine 56 was
obtained using an aminoacyl-tRNA synthetase and tRNA
CUA
pair
created by directed evolution in E. coli (Neumann et al, 2008). An
unmodified control was prepared in parallel. Histone octamers were
assembled as described previously (Luger et al, 1997) and kept at
41C before chromatin reconstitution.
Full acetylation of H4K16 was obtained by NCL as described
previously (Shogren-Knaak et al, 2006). Briefly, the H4 N-terminal
peptide containing residues 1–22 and acetylated lysine at position
16 was synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl)-
based solid-phase synthesis and activated at the C-terminus by
thioesterification. Subsequently, the globular X. laevis H4D1-
22,R23C was ligated to the activated H4 peptide and the ligation
product was purified as described previously (Shogren-Knaak
et al, 2006). Identity and purity of the histones were verified by
SDS–PAGE as well as ESI-MS (Supplementary Figure S1). Histone
octamers were assembled as described previously (Luger et al,
1997) and kept at 41C before chromatin reconstitution.
MNase digestion assay
MNase digestion was carried out in 20 ml of 10 mM TEA pH 8,
1.5 mM CaCl
2
, 25 mM NaCl, 0.05% Tween-20. In all, 1 pmol of
601-167-6mer was digested with increasing amounts of MNase, as
detailed in the figures, for 12 min on ice. The digestion was stopped
by adding 10 mM EGTA, proteins were removed by proteinase K
digestion for 15 min at 301C and the samples were run at 65 V for
60 min in a 1.2% agarose gel 1 TBE: 90 mM Tris, 90 mM Boric
acid, 2 mM EDTA. Digestion of SIR-bound chromatin was performed
on 601-167-6mer pre-incubated with the indicated amount of Sir
proteins for 10 min on ice. MNase digestion of deacetylated
chromatin was performed on 601-167-6mer incubated with
0.66 pmol of Sir2-3-4, Sir2cd-3-4 or Sir2-4 for 15 min at 301C with
or without 150 mM NAD and recovered on ice. Concerning the Sir2-
3-4 complex, similar results were obtained by incubating the
nucleosomal array with 0.66 pmol of Sir2-4 at first and adding
0.66 pmol of Sir3 before the recovery on ice. In order to strengthen
our observations, different batches of modified and unmodified
chromatins were compared.
Deacetylation reaction
Deacetylation of 2 pmol of reconstituted chromatin was performed
in 30 ml of 25 mM Tris pH 8, 50 mM NaCl in presence of 5 pmol of
the Sir2-3-4 complex, the Sir2cd-3-4 mutant or Sir2-4 and 150 mM
NAD for 30 min at 301C and stopped by addition of 4 Laemmli
buffer. Similar results were obtained in 25 mM Tris pH 8, 137 mM
NaCl, 2.7 mM KCl and 1 mM MgCl
2
. The acetylation state was
determined by immunoblotting using acetylation mark-specific
antibodies (anti-H3K56
ac
Upstate #07-677, anti-H4K16
ac
Serotec
AHP417) and H3 for loading (anti-H3 Abcam ab1791-100).
Supplementary data
Supplementary data are available at The EMBO Journal Online
(http://www.embojournal.org).
Acknowledgements
We would like to thank the Gasser laboratory and in particular
Helder Ferreira for discussion and support as well as Simon Lattman
for assistance in analysing the data. We thank Heinz Neumann
for providing a preliminary batch of H3K56
ac
histone octamers.
The Gasser laboratory is supported by the Novartis Research
Foundation and the EU network Nucleosome 4D. SK was supported
Dual role of H4K16
ac
in yeast silencing
M Oppikofer et al
&2011 European Molecular Biology Organization The EMBO Journal VOL 30 |NO 13 |2011 2619
by an EMBO long-term fellowship and an FWF Schroedinger
fellowship.
Author contributions: MO, SK and SMG designed the experiments
and interpreted results. MO performed the experiments. SK and
FM contributed reagents. SS and WF contributed the H4K16
ac
histone octamers. SMH and JC contributed the H3K56
ac
histone
octamers. MO, SK and SMG wrote the manuscript. SMG supervised
the work.
Conflict of interest
The authors declare that they have no conflict of interest.
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