Histone sumoylation is a negative
regulator in Saccharomyces cerevisiae
and shows dynamic interplay with
positive-acting histone modifications
Dafna Nathan,1,5Kristin Ingvarsdottir,1,5David E. Sterner,1,6Gwendolyn R. Bylebyl,2
Milos Dokmanovic,4Jean A. Dorsey,1Kelly A. Whelan,1Mihajlo Krsmanovic,4William S. Lane,3
Pamela B. Meluh4, Erica S. Johnson,2and Shelley L. Berger1,7
1Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, Pennsylvania 19104, USA;2Department of
Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 10107, USA;
3Microchemistry and Proteomics Analysis Facility, Harvard University, Cambridge, Massachusetts 02138, USA;4Molecular
Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
Covalent histone post-translational modifications such as acetylation, methylation, phosphorylation, and
ubiquitylation play pivotal roles in regulating many cellular processes, including transcription, response to
DNA damage, and epigenetic control. Although positive-acting post-translational modifications have been
studied in Saccharomyces cerevisiae, histone modifications that are associated with transcriptional repression
have not been shown to occur in this yeast. Here, we provide evidence that histone sumoylation negatively
regulates transcription in S. cerevisiae. We show that all four core histones are sumoylated and identify
specific sites of sumoylation in histones H2A, H2B, and H4. We demonstrate that histone sumoylation sites
are involved directly in transcriptional repression. Further, while histone sumoylation occurs at all loci tested
throughout the genome, slightly higher levels occur proximal to telomeres. We observe a dynamic interplay
between histone sumoylation and either acetylation or ubiquitylation, where sumoylation serves as a
potential block to these activating modifications. These results indicate that sumoylation is the first negative
histone modification to be identified in S. cerevisiae and further suggest that sumoylation may serve as a
general dynamic mark to oppose transcription.
[Keywords: Histone post-translational modifications; sumoylation; transcription regulation; telomeric
Received August 23, 2005; revised version accepted February 8, 2006.
Covalent post-translational modifications of lysine resi-
dues such as acetylation, methylation, and ubiquityla-
tion play important roles in controlling protein function.
In histone proteins, dynamic lysine acetylation/deacety-
lation was the first modification to be characterized in
detail (Brownell et al. 1996; Taunton et al. 1996), thereby
galvanizing the study of chromatin structure and regu-
lation of DNA-related processes. The histones H2A,
H2B, H3, and H4 comprise the core complex of the
nucleosome, which is the basic repeating unit of chro-
matin in the eukaryotic cell. The nucleosome core
serves as a paradigm for modifications and their effect on
cellular processes such as transcription, repair, and epi-
genetic control. Various modifications of histones affect
interactions within the core unit as well as with the
associated DNA and other factors and complexes (Strahl
and Allis 2000; Berger 2002). Further, certain protein
modules, often occurring within protein complexes, rec-
ognize specific patterns of histone modifications to re-
cruit effector proteins that change chromatin structure
and dynamics (Dhalluin et al. 1999; Bannister et al. 2001;
Lachner et al. 2001). Positive-acting histone modifica-
tions occur universally in eukaryotes and include acety-
lation and phosphorylation, as well as ubiquitylation
(e.g., H2B Lys123 [K123]) and lysine methylation (e.g.,
H3 K4) at certain sites (Berger 2002). In contrast, nega-
tive-acting histone modifications, such as lysine meth-
ylation (including H3 K9, K27, and H4 K20) and H2A
K119 ubiquitylation, occur in Schizosaccharomyces
pombe and metazoans but have not been detected in
Saccharomyces cerevisiae (Martin and Zhang 2005). One
possibility is that the small, gene-rich S. cerevisiae ge-
nome, which is subject to rapid and wide-spread gene
5These authors contributed equally to this work.
6Current address: Progenra, Inc., 271A Great Valley Parkway, Malvern,
PA 19355, USA.
E-MAIL firstname.lastname@example.org; FAX (215) 898-0663.
Article published online ahead of print. Article and publication date are
966GENES & DEVELOPMENT 20:966–976 © 2006 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/06; www.genesdev.org
induction, has chromatin “poised” for transcription and
hence in a constitutively open state.
Recently, it has been shown that histones are sum-
oylated in mammalian cells (Shiio and Eisenman 2003).
SUMO (SMT3 in the yeast S. cerevisiae) is a small ubiq-
uitin-related modifier protein of ∼100 amino acids that is
conserved in all eukaryotes. It shares a similar three-
dimensional structure with ubiquitin and a related, but
separate pathway of activation (E1), conjugation (E2), and
ligation (E3) of its processed form to the targeted lysine
side chain (Johnson 2004). In yeast the heterodimer Aos1/
Uba2 (E1) forms a high-energy thioester bond to activate
SUMO. SUMO is then transferred to Ubc9, the only
known conjugating enzyme (E2) in yeast and higher eu-
karyotes. While Ubc9 is capable of ligating SUMO to its
target, several E3 ligases have been characterized that con-
fer specificity to the sumoylation process. In yeast the
known E3s are Siz1, Siz2 (Nfi1), and Mms21 (Johnson and
Gupta 2001; Takahashi et al. 2001; Zhao and Blobel 2005).
Protein sumoylation results in a variety of effects, in-
cluding changes in cellular localization or stability,
modulation of protein–protein or protein–DNA interac-
tions, or antagonizing other lysine modifications such as
ubiquitylation (Verger et al. 2003; Johnson 2004). Recent
genomic studies aimed at identifying cellular substrates
for sumoylation indicate that many are involved in tran-
scription regulation and include transcription factors,
transcription machinery proteins, and components that
modify chromatin structure (Manza et al. 2004; Wohl-
schlegel et al. 2004; Zhou et al. 2004; Hannich et al.
2005). In most cases, sumoylation of transcriptional ac-
tivators results in transcriptional repression (Gill 2003;
Hay 2005). Many sumoylation sites harbor the sequence
?KXE (where ? is a hydrophobic residue, K is the at-
tachment site lysine, and X represents any amino acid).
Some consensus sites occur in negative regulatory do-
mains of transcription factors (Holmstrom et al. 2003).
Recent studies demonstrate that a few charged and con-
served residues in the SUMO ubiquitin-like fold are es-
sential for its transcriptional repressive properties (Chu-
preta et al. 2005).
In this study we focus on the role of sumoylation of
histones. Histone H4 sumoylation in mammalian cells
has been reported, although attachment sites were not
determined (Shiio and Eisenman 2003). Promoter sum-
oylation was shown to correlate with certain events ac-
companying transcriptional repression, including H3
deacetylation and recruitment of the heterochromatin
protein HP1 (Shiio and Eisenman 2003). However, the
important question remains whether transcriptional re-
pression mediated by the SUMO pathway is linked spe-
cifically to histone sumoylation. In this study we ad-
dressed the role of histone sumoylation in S. cerevisiae.
We detect sumoylation of all four core histones and iden-
tify specific sites of sumoylation in H2A, H2B, and H4.
Importantly, we observe a relationship between histone
sumoylation and transcriptional repression. Our results
further suggest that histone sumoylation may exist in a
dynamic interplay with histone acetylation and ubiqui-
Sumoylation of all core histones within chromatin
SUMO conjugates are broadly distributed on yeast chro-
mosomes as determined by anti-SUMO ChIP, chromatin
fractionation, and chromosome spreads (Biggins et al.
2001; data not shown). To determine the sumoylation
status of the four core histones, we used a shuffle system
in which the endogenous genes were deleted and re-
placed by a plasmid encoding the tagged histone. Immu-
noprecipitation (IP) followed by Western blot (immuno-
blot [IB]) analysis with anti-Flag and anti-SUMO antibod-
ies indicated that all four histones are sumoylated in
vivo (Fig. 1A, left). The anti-SUMO antibody is highly
specific for sumoylated histone in this experiment, as it
does not react with unmodified histone H2B, although
the modified form is a small proportion of the total his-
tone (Fig. 1A, right). At least two sumoylated bands can
be seen, indicating either chain formation or multiple
sumoylation sites. In general, histone sumoylation ac-
counts for a small percentage of total histone (approxi-
mately <5%) (Figs. 1A [right], 2) and is labile under native
conditions. Therefore, IPs were routinely conducted on
denatured cell extracts prepared by trichloroacetic acid
(TCA) precipitation (Ohashi et al. 1982).
Strains defective in the sumoylation process were used
to check their effect on histone sumoylation by trans-
forming the Flag-tagged histone plasmids for ectopic ex-
pression. A temperature-sensitive mutant of UBC9
(ubc9ts, which is partially defective at restrictive tem-
perature) (Schwarz et al. 1998), as well as a double dele-
tion of the E3 ligases SIZ1 and SIZ2 (Johnson and Gupta
2001), cause dramatic reduction of H2B and H4 sumoyla-
tion (Fig. 1B). Deletion of SIZ1 alone did not result in any
blot (IB) analysis of immunoprecipitated (IP) Flag-tagged his-
tones using anti-Flag and anti-SUMO antibodies. Flag-tagged
histones migrate ∼15 kDa. Singly sumoylated histones migrate
between 26 and 37 kDa. (B) Flag IP followed by Western blot (IB)
analysis of histones in sumoylation pathway mutant strains
ubc9ts and siz1?siz2?. Detection was carried out as in A.
In vivo histone sumoylation in yeast. (A) Western
Histone sumoylation is a negative regulator
GENES & DEVELOPMENT 967
changes in histone sumoylation levels (data not shown).
The reduction of histone sumoylation in the mutants is
unlikely to be an indirect effect on the availability of free
SUMO, which is readily detected in whole-cell extracts
of ubc9ts and siz mutant strains (data not shown).
We investigated the chromatin association of sum-
oylated H3 and H4 to determine whether sumoylation
prevents chromatin incorporation. Total histones were
prepared from strains bearing HA-tagged H3 or H4 and
were subjected to fractionation to separate insoluble
chromatin pellet from soluble supernatant (Liang and
Stillman 1997). In a wild-type strain (Fig. 2A) or a strain
bearing a deletion of the SUMO protease Smt4 (Ulp2)
(Fig. 2B), which increases the level of sumolyated his-
tones, sumoylated histones are detected exclusively in
the pellet. The SUMO modification status of the HA-
histones was confirmed using a SUMO antibody (Fig.
2B). In addition, the bands match immunoreactive spe-
cies on anti-H3 and anti-H4 Western blots (data not
shown). Localization of acetylated H3 to the chromatin
pellet serves as a control for the fractionation procedure
(Fig. 2B, bottom). Thus, sumoylated histones occur within
chromatin, suggesting a role in genomic regulation.
H2B and H4 sumoylation sites are located in their
To characterize the sites of sumoylation in histone H2B,
affinity chromatography of His-tagged SUMO was used
to generate samples for tandem mass spectrometry (MS/
MS) analysis. Initial analysis revealed the presence of
H2B and H2A in the samples. Specific endopeptidases
were chosen to generate maximum peptide fragments
containing the branched SUMO moiety from the tar-
geted lysine. These efforts revealed that SUMO is at-
tached to histone H2B at Lys6 or Lys7 (K6/7) in the N-
terminal tail (Fig. 3A) and also showed that one of these
lysines is a novel acetylation site as well. Analysis of a
limited number of peptide fragments (three) indicated
that K6/7 is either singly acetylated or sumoylated, but
no MS/MS spectra were observed where they were si-
multaneously modified. One of these peptide spectra in-
dicated unambiguous acetylation of Lys6. The sequence
surrounding K6/7 bears some similarity to the N-termi-
nal extension of yeast SUMO, which is itself sumoylated
(Bylebyl et al. 2003). In H2B this sequence with sur-
rounding alanines (AEKKPA) is repeated, generating an-
other putative sumoylation/acetylation site at Lys16 and
Lys17 (K16/17). The proteolysis and MS/MS peptide frag-
mentation patterns did not allow for the identification of
this second putative sumoylation site.
Mutation of either K6/7 or K16/17 to alanine resulted
in reduced H2B sumoylation (Fig. 3B, upper panels). Mu-
tating all four K6/7/16/17 to alanine did not result in
further decrease in sumoylation, suggesting that sum-
oylation at one of the repeats may positively influence
sumoylation at the adjacent repeat. Quantitative analy-
sis of the sumoylated forms relative to total H2B indi-
cates that the level of sumoylation decreases by ∼50% in
the mutants compared with wild type (Fig. 3B). Mutating
any other lysine in H2B did not result in significantly
lowered SUMO–H2B levels (data not shown). The re-
sidual sumoylation observed when all four lysines in
these sequences were mutated to alanine also suggests
nonspecific sumoylation or a small population with an
alternative sumoylation site.
Mass spectrometry studies also indicated a sumoyla-
tion site at the C-terminal K126 of histone H2A. How-
ever, substitution of this site (H2A K126R) did not cause
significant changes in H2A sumoylation levels compared
with wild type (data not shown).
In our efforts to identify sumoylation sites in other
histones, we observed that deletion of the N-terminal
tail of histone H4 resulted in a considerable reduction of
sumoylation levels (Fig. 3B, lower panels). This result is
in accordance with previous studies of mammalian his-
tones, which indicated that the H4 tail is a substrate for
sumoylation in vitro (Shiio and Eisenman 2003). Mutat-
ing all five lysines in the N-terminal tail yielded a simi-
lar effect (lowered to 30%–50%; Figure 3B), suggesting
that these are the major sites of sumoylation in H4.
However, mutating any three lysines out of the five did
not reduce H4 sumoylation levels significantly (data not
shown). Thus, it may be that there is a lack of specificity
yeast cells. (A) Western blot analysis using anti-HA antibody of
untagged (vector) fractionated wild-type cells or HA-tagged H3
or H4. Fractions are total (T), soluble (S), or pellet (P). Open
circles indicate HA-H3, closed circles indicate HA-H4, open
arrowheads point to sumoylated HA-H3, and closed arrowheads
point to sumoylated HA-H4. A shorter exposure of the nonsum-
oylated histones is shown below. (B) Similar analysis as in A
except extracts were obtained from a strain bearing a SMT4
(SUMO protease) deletion, which increases the cellular level of
sumoylated histones. HA and SUMO immunoblots are shown,
as well as H3 acetylation (H3ac) immunoblot as a control for
Western blot analysis of histones in fractionated
Nathan et al.
968 GENES & DEVELOPMENT
among these sites. Similar to the H2B multiple mutation
results, residual sumoylation was observed even when
the entire tail of H4 was deleted or all five lysines sub-
stituted, underscoring the apparent redundancy and/or
promiscuity of histone sumoylation. These five N-ter-
minal lysines are major sites of acetylation in H4.
To ensure that the lowered sumoylation states of the
mutant histones H2B and H4 were not due to lower
amounts of total SUMO synthesis/processing, we exam-
ined whole-cell extracts made from these strains and
analyzed by Western with an anti-SUMO antibody.
Compared with wild type, the mutant strains showed
very similar intensities and patterns of sumoylated pro-
teins (Fig. 3C). We therefore conclude that the reduced
histone sumoylation levels seen in the mutants were
indeed the result of eradication of the sumoylation re-
Thus, we identified a subset of histone sumoylation
sites. Overall, it appears that all four core histones are
sumoylated, that there are multiple potential sites
within each histone, and that sumoylation sites are co-
incident with or near known sites of acetylation.
Sumoylation is higher at subtelomeres compared
with more internal regions
We investigated genomic localization of sumoylation as-
sociated with H2B using chromatin IP (ChIP). We were
unable to generate H2B–SUMO-specific antibodies using
branched peptides (containing several SUMO-specific
residues attached to a base of several histone-specific
residues) as antigens. Instead we developed a “ChDIP”
(double-affinity ChIP) method to examine sumoylation,
similar to our previous analysis of ubiquitylation asso-
ciated with H2B (Henry et al. 2003; Kao et al. 2004). We
used a yeast strain carrying a polyhistidine tag on SUMO
and a plasmid bearing Flag-tagged H2B. The ChDIP in-
volved Flag IP followed by elution and nickel column
purification, prior to de-cross-linking and quantitation
by real-time PCR. A portion of the first step of the ChIP
(Flag-H2B) was retained, and results from the second step
(nickel column) were normalized to the Flag-H2B ChIP
signal in order to evaluate the percentage of total H2B
that is sumoylated.
We found H2B-associated sumoylation at many ge-
nomic locations, including the RET2 (Fig. 4A) and GAL1
genes (Fig. 6B, below). We arbitrarily set the ChDIP sig-
nal for RET2 to 1.0 and found comparable levels at nu-
merous genomic locations (data not shown). Interest-
ingly, sequences adjacent to telomeric repeats, such as
the right arm of chromosome VI (Chr VI-R) or the left
arm of chromosome III (Chr III-L) are enriched in H2B-
associated sumoylation by roughly twofold, compared
with other regions in the genome (Fig. 4A, left). Indeed,
there is a gradual decrease in this higher telomeric H2B-
associated sumoylation with ChIP probes more internal
to the chromosome (Fig. 4A, right). This correlation is
opposite to the pattern previously observed for H2B ubiq-
uitylation (Emre et al. 2005).
We created two different strains to serve as controls for
each ChIP step. For the first ChIP step using Flag anti-
body, a strain lacking the tag on H2B was used as a con-
trol, resulting in a nearly undetectable ChDIP signal for
all regions tested (Fig. 4B, left; data not shown). As a
sites in vivo. (A) Regions of sumoylation in histone H2B and H4.
The lysine residues for which sumoylation was confirmed by
MS/MS in histone H2B are shown in bold and underlined let-
ters. Additional putative sumoylation sites in H2B are also un-
derlined. Lysine residues that were mutated to alanine in his-
tone H4 are indicated by underlined letters. (B) Effect of substi-
tution mutations in putative sumoylation sites on in vivo
modification in yeast. Lys6 and Lys7 (6/7), Lys16 and Lys17
(16/17), or all four lysines (6/7 16/17) in H2B were mutated to
alanine and were either shuffled in as the only source of histone
H2B in the cell or transformed and kept as an ectopic copy.
Histone H4 was expressed ectopically, and the entire tail was
deleted (?N), or all five lysines in the H4 tail were mutated to
alanine (5K). Sumoylation levels were detected using Flag IP
followed by Western blot analysis with anti-Flag (bottom) and
anti-SUMO (top) antibodies. The numbers below the SUMO
blots represent quantification of the sumoylation levels com-
pared with the signal in wild-type (WT) strain after normaliza-
tion to the Flag levels in each strain. (C) Whole-cell extracts
from the same strains used in B were analyzed by SDS-PAGE
and immunoblotting with an anti-SUMO antibody. At higher
molecular weight (above the 97-kDa marker), the lanes are
smeared and distinct bands are no longer seen. The H4 wild-
type (WT) lane shows a band ∼33 kDa, which corresponds to the
molecular weight of sumoylated H4, and is greatly reduced in
the H4 mutant lanes. The H2B genes were not on a high-copy
plasmid as were the H4 genes; therefore, the sumoylated form of
H2B is not visible in whole-cell extracts.
Identification and confirmation of histone SUMO
Histone sumoylation is a negative regulator
GENES & DEVELOPMENT969
control for the second SUMO purification step, we pre-
pared an alanine substitution mutant of the four sum-
oylation sites in H2B, yielding K6/7/16/17A. This strain
gave a dramatic decrease in signal intensity compared
with the wild-type strain for all regions tested (Fig. 4B,
right; data not shown), which indicates that the ChDIP
signal largely represents SUMO attached to the tagged
H2B, rather than to other chromatin proteins present at
the same locus. The difference between these controls
(i.e., there is a greater decrease in signal in the no-tag
control than in the mutant H2B control) may represent
other histones that are sumoylated and cross-linked to
H2B. Overall, the ChDIP results indicate histone sumolya-
tion exists at similar levels for many tested regions of the
genome, with slightly higher subtelomeric signals.
Histone sumoylation represses transcription
We examined a possible role of histone sumoylation in
transcriptional repression by comparing gene expression
in the H2B K6/7/16/17A mutant strain (in which H2B
sumoylation is attenuated) to that in wild-type strain.
Under noninducing conditions, basal expression levels of
TRP3, SUC2, and GAL1 are increased between two- and
threefold in the K6/7/16/17A substitution mutant com-
pared with wild type (Fig. 5A). This suggests that the
relatively low basal level of transcription of these three
genes is maintained in part by histone H2B sumoylation.
This effect on transcriptional repression by substitu-
tion mutations in the H2B sumoylation sites is modest.
This might reflect redundancy due to the large number
of sumoylation sites on the histones, or the inability of
the substituted sites to be acetylated to achieve maxi-
mum derepression. We hypothesized that if sumoylation
is indeed repressive to transcription, then direct fusion of
SUMO to the histone should cause more severe and
dominant repression. This approach has been frequently
used to test a role of sumoylation or ubiquitylation of
other factors; e.g., the transcription factor Sp3 (Ross et al.
2002) or GAL4-VP16 (Salghetti et al. 2001). We prepared
Quantitative PCR analysis of TRP3, SUC2, and GAL1 RNA
levels in noninducing (YPD) conditions for wild-type (WT) and
H2B K6/7/16/17A substitution mutant (MUT). (B) Western blot
analysis of whole-cell extracts probed with anti-histone H4 (left)
or anti-histone H2B, anti-Flag, and anti-SUMO antibodies (pan-
els on right). Indicated are the positions of endogenous (bottom),
Flag-tagged histones (middle), and SUMO-fused histones (top).
(C) Quantitative PCR analysis showing transcript levels of
GAL1 for SUMO–H2B (SU-H2B) or Flag-H2B (FL-H2B) fusions
when switching from glucose (Glu) to galactose (Gal) media and
the parallels in histone H4. (D) Quantitative PCR analysis as in
C comparing Flag-H2B and SUMO–H2B with a strain with a
SUMO–H2B fusion containing substitution mutations in
SUMO at Lys37 and Arg46 to glutamic acid (SUMO-mut).
Lower insert shows Western analysis of whole-cell extracts
from SUMO–H2B and SUMO-mut–H2B fusion strains using
anti-SUMO and anti-H2B antibodies.
Sumoylation of histones regulates transcription. (A)
nome. (A) Quantitative PCR analysis of ChDIP in a His-SUMO
Flag-H2B strain. Telomeric end primers are located ∼0.2 kb
away from the telomeric repeats on Chr VI-R or 2.5 kb away
from telomeric repeats of Chr III-L. The right panel shows
ChDIP using primers to regions 0.5, 3.6, and 7.3 kb away from
the telomere end on Chr VI-R. RET2 is located ∼20 kb away
from the Chr VI-R telomere end. (B) Controls for quantitative
PCR analysis of ChDIP samples. All strains have His-tagged
SUMO. Left panel shows a comparison control stain lacking the
Flag-H2B plasmid. Right panel shows a comparison control
strain containing H2B substitution mutant K6/7/16/17A in the
Location of histone sumoylation in the yeast ge-
Nathan et al.
970 GENES & DEVELOPMENT
SUMO–H4 and SUMO–H2B genetic fusions (or Flag-H4
and Flag-H2B as controls) in which the C terminus of
SUMO (or Flag) is fused to the N terminus of histone H4
or histone H2B. This fusion places SUMO close to the
N-terminal residues where it is naturally attached (see
above). The fusion proteins were designed to lack the
natural glycine–glycine motif that forms the isopeptide
bond with the target lysine side chain, and thus cannot
be cleaved by SUMO peptidases. Having a SUMO–his-
tone conjugate as the only source of histone was toxic to
the cell (data not shown), hence the fusions were ex-
pressed ectopically with a high-copy (H4) or low-copy
(H2B) plasmid. Flag- or SUMO–H4 was detected by H4
antibody and was present in similar amounts to each
other and to untagged H4 (Fig. 5B, left). Flag-H2B or
SUMO–H2B could not be detected by the H2B antibody,
and therefore, whole-cell extract was separately probed
with anti-Flag and anti-SUMO antibodies (Fig. 5B, right).
Thus, stable histone fusions were generated with SUMO
close to the endogenous sumoylation site, potentially
mimicking the sumoylated states but not susceptible to
desumoylation like the native conjugates.
We tested GAL1 induction upon switching to galac-
tose media in cells carrying the Flag versus the SUMO–
histone fusion. GAL1 transcription is induced sevenfold
to 10-fold by galactose; the induction is reduced twofold
in the presence of SUMO–H2B and fivefold in the pres-
ence of SUMO–H4 compared with the Flag-histone fu-
sion (Fig. 5C). As a second control, we created a SUMO–
H2B fusion containing substitution mutations in SUMO
that decrease the repressive effects of SUMO (Chupreta
et al. 2005). The fusion containing these mutations com-
pletely reversed the repression seen with SUMO–H2B
(Fig. 5D), and Western analysis indicates that the wild-
type and mutant SUMO–H2B fusions are present at com-
parable levels (Fig. 5D, lower insert). Taken together with
the increased basal transcription exhibited by the substitu-
tion mutations in the natural sumoylation sites in the his-
tones, these results provide further support for a role of
histone sumoylation in transcriptional repression.
Histone sumoylation opposes activating histone
acetylation or ubiquitylation and is low in histone
variant H2AZ compared with H2A
The preceding experiments indicated that the GAL1
gene is under negative control by histone sumoylation.
We hypothesized that histone sumoylation might de-
cline during gene activation when positive histone modi-
fications are induced. To investigate this, we first tested
whether the overall level of histone sumoylation
changes under conditions that affect GAL1 expression.
We examined H2B and H4 sumoylation by IP-Western at
regular intervals following a change in carbon source
from repressing glucose to nonrepressing raffinose or to
inducing galactose. Overall histone sumoylation levels
drop dramatically during the first 5–10 min after a shift
from glucose to raffinose or from glucose to galactose
and then appear to gradually increase to a new “steady-
state” level (Fig. 6A, left and right top panels). The tran-
sient decline in histone sumoylation is not detected in
mock-treated samples “shifted” from glucose to glucose
(Fig. 6A, center top panel) or in samples shifted from raffi-
nose to glucose (data not shown), supporting the view that
it may be related to global gene activation and not to an
artifact of cell manipulation or histone IP procedure.
The finding that sumoylation sites are at, or adjacent
to, acetylation sites in H2B and H4 suggests that histone
sumoylation and acetylation may be in competition. We
examined possible interplay between sumoylation and
acetylation during the change in carbon source. Western
blot analysis (Fig. 6A), as well as ChIP of the GAL1 pro-
moter (Fig. 6B), shows that the histone acetylation pat-
tern partially opposes sumoylation during the time
course in altered carbon source. A reciprocal pattern for
sumoylation/acetylation is also apparent in the switch
from glucose to ethanol/glycerol media, including both
H2B K7 and K16 acetylation (Fig. 6C).
We examined whether reduced sumoylation in ubc9ts
and siz1?siz2? double-deletion strains, which exhibit
and H4 upon changes in carbon source. (A) Flag IP followed by
Western blot analysis of Flag-H2B (left and middle) or Flag-H4
(right) samples taken at the indicated time points during growth
switched from glucose-to-raffinose (Raff, H2B) or glucose-to-ga-
lactose (Gal, H4). Center panels show the control Glu-to-Glu
switch for Flag H2B. Antibodies are as indicated. (B) Real-time
PCR analysis at the GAL1 promoter region of a ChDIP experi-
ment (left) or H2B K16ac ChIP (right) in a His-SUMO Flag-H2B
tagged strain. Samples were taken at the indicated time points
after switching from glucose (YPD) to raffinose. (C) Flag IP fol-
lowed by Western blot analysis of Flag-H2B samples taken at
the indicated time points during glucose (YPD) to ethanol/glyc-
erol (EtOH/Gly) carbon source change. Antibodies are as indi-
Interplay of acetylation–sumoylation in histone H2B
Histone sumoylation is a negative regulator
GENES & DEVELOPMENT971
dramatically lower histone sumoylation compared with
wild type (Fig. 1B), leads to increased histone acetylation.
Western analysis shows higher levels of H3 acetylation
in the ubc9ts strain (Fig. 7A), and ChIP analysis shows
higher gene-associated H3 acetylation in either strain
(Fig. 7B). In addition, cellular levels of H2B sumoylation
are increased in a strain bearing a substitution mutation
at K123 of H2B (Fig. 7C), which is a ubiquitylation site
involved in gene activation (Henry et al. 2003; Kao et al.
2004). Thus, histone sumoylation and activation-linked
histone modifications, including acetylation and ubiqui-
tylation, oppose one another.
The histone variant H2AZ has been localized to het-
erochromatic boundaries that protect euchromatin from
spreading of silencing proteins (Meneghini et al. 2003) or
to promoter regions that poise genes for transcriptional
activation (Santisteban et al. 2000; Guillemette et al.
2005; Raisner et al. 2005). We therefore hypothesized
that a repressive mark, such as sumoylation, may not be
prominent on this histone variant. We noted that H2AZ
exhibits conserved amino acid sequence with canonical
H2A, except near to the end of the C-terminal tail (Fig.
7E), where we had mapped one sumoylation site on H2A
(K126) as described above. Western analysis showed that
the overall level of sumoylation on H2AZ is low com-
pared with sumoylation of H2A (Fig. 7D).
Prior to the present study, the yeast S. cerevisiae ap-
peared to be devoid of typical histone modifications
known to negatively regulate higher eukaryotic ge-
nomes. While eukaryotes ranging from S. pombe to hu-
mans contain negative-acting lysine methylation (Lach-
ner and Jenuwein 2002; Grewal and Moazed 2003; Mar-
tin and Zhang 2005), this class of modification is not
detected in S. cerevisiae. It thus appeared that the S.
cerevisiae genome is “poised” for transcription and may
not be subject to negative regulation through chromatin
modifications (e.g., see White et al. 2001). In apparent
contrast to this point of view, our results suggest that
histone sumoylation has a negative regulatory role in S.
cerevisiae. There appear to be a large number of sum-
oylation sites, with multiple sites on each of the core
histones; we have apparently identified most sites on
H2B and H4, a subset of sites on H2A, while sites on H3
have not yet been identified.
One repressive role we detect for histone sumoylation
is in gene-specific negative regulation, where sumoyla-
tion helps to maintain a low basal level of transcription.
Although there are numerous histone sumoylation sites,
substitution mutation of only four sumoylation acceptor
sites in H2B results in an increase in the uninduced tran-
scriptional level of three genes that are subject to dis-
tinct pathways of inducible regulation (Fig. 5A). It may
be that combinatorial substitutions of more sumoylation
sites on the same histone/different histones will result
in stronger increases in basal transcription. A similar or
even stronger inhibitory effect, on induced GAL1 tran-
scription, correlates with direct fusion of SUMO to his-
tone H2B or H4 (Fig. 5C). Genes representing a wide
range of regulatory pathways were tested in our study
(GAL1 is induced by galactose, SUC2 is induced by su-
crose, and TRP3 is induced by low levels of certain
amino acids) and revealed to be under negative control
by histone sumoylation. These results suggest that the
negative role of histone sumoylation may be quite general.
A second negative regulatory role suggested by our
data is that histone sumoylation may reinforce telomeric
silencing. Telomeric silencing in S. cerevisiae has long
been associated with reduced levels of positive-acting
histone modifications. This includes low acetylation,
maintained through the action of the histone deacetylase
Sir2 (Grunstein 1997; Rusche et al. 2003), and low ubiq-
uitylation and methylation (Bryk et al. 2002; Ng et al.
2003), maintained via the histone deubiquitylase Ubp10,
which also serves to keep methylation levels low (Emre
et al. 2005). We detect slightly higher H2B-associated
sumoylation at telomeres than at more internal chromo-
somal sites or at genes (Fig. 4A). Strikingly, this is the
reverse correlation as previously observed for both H2B-
associated ubiquitylation and H3 methylation (Ng et al.
2003; Emre et al. 2005). Further, we found that telomeric
silencing of URA3 cassette expression (Aparicio et al.
tone mechanisms. (A) Western blot of histones prepared from
wild-type (WT) or ubc9ts strain probed with H3ac (K9ac, K14ac)
antibody (top) or unmodified histone H3 antibody (bottom). (B)
Quantitative PCR analysis of H3ac ChIP of the RET2 gene in
the wild-type (WT), ubc9ts, and siz1?siz2? strains. (C) Western
blot of histones prepared from wild-type (WT) strain or strains
bearing Flag-H2B, Flag-H2B-K123R substitution mutation, or
Flag-H2A. (D) Flag IP followed by Western blot analysis of Flag-
tagged histone H2A and histone variant H2AZ. (E) Alignment of
the C-terminal tails of yeast canonical H2A and variant histone
H2AZ. The sumoylation site is indicated for H2A. Asterisks
indicate identical residues.
Relationship between sumoylation and postive his-
Nathan et al.
972GENES & DEVELOPMENT
1991) is dramatically lowered in the ubc9ts strain (data
not shown), which may indicate a role of histone sum-
oylation in promoting telomeric silencing.
Third, histone sumoylation appears to correlate nega-
tively with positive-acting histone acetylation and ubiq-
uitylation. Reduction of histone sumoylation results in
changes in carbon source correlate with loss of histone
sumoylation in parallel with increased histone acetyla-
tion (Fig. 6). Acetylation occurs broadly in the genome
and can be globally altered by manipulating the levels of
histone acetyltransferases or deacetylases (Vogelauer et
al. 2000; Boudreault et al. 2003). Thus, genome-wide
acetylation and sumoylation, as shown in our study,
may broadly regulate. In addition to alternative sumoyla-
tion/acetylation, substitution mutation of the ubiqui-
tylation site in histone H2B leads to higher H2B sum-
oylation (Fig. 7C) indicating an interplay between these
modifications as well.
Finally, there are lower levels of SUMO on the histone
variant H2AZ compared with canonical H2A (Fig. 7D).
As described above, H2AZ localizes both to genes pro-
moters, potentiating them for activation, and to genes
near silenced regions, protecting them from heterochro-
matic spreading. This observation is reminiscent of re-
cent findings in Drosophila that higher levels of positive-
acting modifications and lower levels of negative-acting
modifications decorate the transcription-linked histone
variant H3.3, compared with the replication-linked ca-
nonical H3.1 (McKittrick et al. 2004). In accordance to
H2AZ correlation with active chromatin, our results of
low sumoylation levels on H2AZ lend further support to
a repressive role for sumoylation.
Thus, we speculate that histone sumoylation may in-
terfere with or counteract activating histone mecha-
nisms. There are several mechanisms that may account
for alternative acetylation and sumoylation. There may
be a direct competition between the modifications since
many of these lysines bear both modifications. Sumoyla-
tion and other lysine modifications occur on the same
residues in other proteins. Proteins that can be either
sumoylated or ubiquitylated include I?B? in the NF-?B
transcription pathway (Desterro et al. 1998), pathogenic
Huntington’s disease protein Huntingtin (Steffan et al.
2004), and the DNA polymerase auxiliary factor, PCNA
(Hoege et al. 2002). Further, sumoylation–deacetylation
interplay has been suggested for the transcription factor
Sp3 (Braun et al. 2001; Ross et al. 2002).
A second possible mechanism is that histone sumoyla-
tion may recruit HDACs (histone deacetylases) to
deacetylate at neighboring sites. This has been demon-
strated for the transcription factor Elk-1, where the sum-
oylated form recruits HDAC-2 to the promoter, resulting
in histone deacetylation and transcriptional repression
(Yang and Sharrocks 2004). The sumoylation machinery
interacts with HDACs, specifically with the HDAC II
protein subfamily (Colombo et al. 2002; David et al.
2002; Kirsh et al. 2002; Verdin et al. 2003; Gregoire and
Yang 2005) and may be part of a regulatory mechanism
that controls their repression activity. Another study
showed that a SUMO–H4 fusion associates with endog-
enous HDAC1 as well as HP1, suggesting that histone
sumoylation is involved in recruitment of deacetylases
(Shiio and Eisenman 2003).
A third possible mechanism would involve activation
of HDACs by sumoylation, which was previously ob-
served (David et al. 2002; Cheng et al. 2004); thus, low-
ered HDAC sumoylation (particularly in the SUMO
pathway mutant strains) may indirectly lead to higher
histone acetylation. However, while HDAC sumoyla-
tion regulates a number of gene regulatory pathways in
mammalian cells, in S. cerevisiae, proteomic analyses of
sumoylated proteins indicate that HDACs are not a ro-
bust target (Wohlschlegel et al. 2004; Zhou et al. 2004;
Hannich et al. 2005), although Hda1 was detected in one
SUMO proteomic analysis (Panse et al. 2004). Our find-
ings that substitution mutations in histone sumoylation
sites increase transcription, and that SUMO fused to his-
tones is repressive to transcription, strengthen the first
two models of direct effect of histone sumoylation
through either competition with acetylation or recruit-
ment of HDACs (i.e., the first and second proposed
mechanisms), rather than an indirect effect through al-
tering HDAC activity (i.e., the third mechanism).
Our study confirms and extends in S. cerevisiae the
initial observation of histone sumoylation made in
mammalian cells. We demonstrate a role for histone
sumoylation in transcriptional repression in S. cerevi-
siae. There is abundant evidence linking sumoylation of
factors with deacetylation, which provides a potential
mechanism to reinforce the antagonism that we detect
between sumoylation and acetylation. The redundancy
or multiplicity of sumoylation sites in histones suggest
that SUMO is attached to a large number of histone ly-
sines and exerts its regulatory effect (either steric block-
ing or recruitment of other factors, such as silencing pro-
teins or HDACs) independent of a specific residue. Thus,
sumoylation appears to be the first evolutionarily con-
served histone modification exhibiting a negative ge-
nomic regulatory role in chromatin.
Materials and methods
Yeast strains and plasmids
Yeast strains used in this study are listed in Table 1. Histone Flag
tagging and amino acid substitution and deletion (QuikChange
kit, Stratagene) of H3 and H4 were performed with pRM204
(Mann and Grunstein 1992) or a high-copy URA3 plasmid
containing Flag H4 (provided by M.A. Osley, Molecular Genet-
ics and Microbiology, University of New Mexico HSC, Albu-
querque, NM). For manipulations of H2A and H2B, plasmids
FB1251 (Hirschhorn et al. 1995) or pRS314 and pRS316 HTB1-
HTA1 (Sikorski and Hieter 1989) were used. The SUMO–H4
fusion plasmid (pRS424) was constructed by first creating a
pRS424-based Flag-H4 plasmid (pTKS791), then replacing the
Flag coding sequence with a PCR-amplified fragment encoding
mature Smt3 (amino acids 1–98; pPM505). To prevent cleavage
of the fusion protein by SUMO proteases, the C-terminal Gly–
Gly motif of Smt3 was mutated to Asp–Val. SUMO–H2B was
cloned by standard methods using SUMO–H4 and Flag-H2B
(RS314) into pRS314. EJ362 expressed a His6-tagged version of
Histone sumoylation is a negative regulator
GENES & DEVELOPMENT973
Smt3 with a His5 sequence (VKPETHHHHHHIN) inserted after
amino acid 22. The His6- and Flag-containing N-terminally trun-
cated version of SMT3 in EJ363 has the N-terminal sequence
MTSHHHHHHMHDYKDDDDKMGST22HINLK. These alleles
were constructed by assembly PCR and transformation into yeast
as described (Johnson and Blobel 1999).
Sumoylated histone H2B purification
EJ363 was grown in 6 L of YPD to an A600of ∼1.0, and cells were
spheroplasted and lysed as described (Li and Hochstrasser 2000)
except that cells were treated with 0.5 mg/mL Zymolyase 20T
(ICN) instead of 100T and disrupted using an Emulsiflex C-5
(Avestin) instead of by sonication. Clarified lysate was bound in
batch overnight at 4°C to ∼50 µL of anti-Flag M2 agarose
(Sigma). Beads were washed several times with 50 mM Tris (pH
8.0), 1 M NaCl, 0.2% Triton X-100, and 2 mM N-ethylma-
leimide. Proteins were eluted at room temperature using 6 M
guanidine HCl, 100 mM Na·PO4, and 10 mM Tris (pH 8.0), and
the eluate was bound in batch to ∼20 µL of Ni-NTA agarose
(Qiagen) for 1 h at room temperature. The Ni-NTA beads were
washed several times with 8 M urea, 50 mM Na·PO4(pH 8.0),
and 0.2% Triton X-100 and were eluted with a small volume of
the same buffer containing 250 mM imidazole. Proteins were
TCA precipitated, separated by SDS-PAGE, and stained with
Coomassie Blue, and bands were excised.
SUMO identification by MS/MS
Based on the predicted C-terminal proteolytic products of
SUMO by endoproteinase GluC (QIGG) and trypsin (EQIGG),
excised protein bands were split in half and subjected to in gel
reduction, carboxyamidomethylation, and separate digestion
with GluC and trypsin. The two digests were pooled immedi-
ately before injection. Peptide sequences were determined using
a 75-µm reverse-phase microcolumn terminating in a custom
nanoelectrospray source (New Objective) directly coupled to an
LCQ DECA XP Plus linear quadrupole ion trap mass spectrom-
eter (Thermo Electron). The instrument was operated in data-
dependent mode fragmenting (relative collision energy, 30%;
isolation width, 2.5 Da; dynamic exclusion) on the four most
abundant ions in each survey scan. Preliminary sequencing of
peptides was facilitated by NCBI nr database correlation with
the algorithm SEQUEST (Eng et al. 1994) and an in-house spec-
trum review workbench, FuzzyIons, and GraphMod, a program
for analyzing the position(s) of a modification within a peptide
sequence for a given MS/MS spectrum. All spectra were manu-
ally inspected for completeness of ion assignments and inten-
sity-based signatures (e.g., neutral loss[es], proline ions, etc.).
Three peptide spectra within histone H2B defined acetylation
and sumoylation at K6 or K7 or both. The N-terminal tryptic
peptide Ac-SSAAEKKPASK unambiguously defined the pres-
ence of an acetylation at Lys6. The GluC-derived peptide
KKPASKAPAE was observed with two independent MS/MS
spectra. In one, the data supported either acetylated K6 or K7. A
second spectrum for this peptide revealed sumoylation at either
of these lysines. Thus, the population was a mix of both acety-
lation and sumoylation, but the modifications were never ob-
served in a single peptide MS/MS spectrum at the same time.
IP and Western blot analysis
Cells (50 mL/IP) were grown to mid-log phase, and pellets were
washed once with 1 mL of 20% TCA and frozen. For IP, cells
were resuspended in 1 mL of 20% TCA and lysed in a mini bead
beater by three repeats of 1.5 min with 2-min intervals on ice.
The lysate was separated from beads and spun down for 10 min
(13,000 rpm, 4°C). TCA was aspirated, and the pellet was resus-
pended in Laemmli buffer (50 mM Tris at pH 6.8, 2% SDS, 10%
glycerol, 2% ?-mercaptoethanol) and boiled for 3 min. Cells
were spun for 10 min at room temperature (13,000 rpm), and the
supernatant was used for IP. Typically, 100 µL were added to
400 µL of IP buffer (50 mM Tris at pH 7.4, 150 mM NaCl, 0.5%
NP-40), and 60 µL of anti-Flag M2-agarose beads from mouse
(Sigma) were added to the mixture. IP was allowed to continue
for 6 h to overnight at 4°C. Beads were washed three times with
IP buffer and eluted 6 h in 40 µL IP buffer containing 2.5 µg of
3xFlag peptide (Sigma). Normally, 8 µL of the eluate was loaded
for WB analysis. The SUMO antibody has been described
(Johnson and Blobel 1999).
In vivo expression analysis
RNA was isolated and DNase treated using RNeasy kit (Qiagen)
according to the manufacturer’s instructions. cDNA was pre-
pared (TaqMan Reverse Transcriptase, Roche) and RT–PCR was
carried out in ABI PRISM 700 sequence detection system (Applied
Biosystems) using either ACT1 or 18S rDNA for normalization.
Typically, 3 mL of cells (in mid-log phase) were used per data
point. Results (average of duplicate or triplicate data points on
plate) are representative of two or three independent experiments.
ChIP experiments and chromatin fractionation
ChIP was performed as described previously (Henry et al. 2003)
except that cells (A600of ∼0.6–0.8) were cross-linked with 1%
Yeast strains used in this study
MATa/MAT?trp1-1/trp1-1 ura3-52/ura3-52 his3-?200/his3-?200 leu2-3,112/leu2-3,112
MAT? ubc9??TRP1 leu2?ubc9Pro-Ser?LEU2
MATa trp1-1 ura3-52 his3-?200 leu2-3,112 lys2-801
MATa His6-Flag-N?21smt3?TRP ulp2??URA3 siz1??LEU2
MATa siz1??LEU2 siz2??TRP1
MATa ura-3-1 leu2-3,-112 his3-11,-15 trp1-1 ade2-1 htb1-1 htb2-1 ssd1,
can1-100 + [Ycp50-HTB1]
MATa his3?200 leu2 ? 1 ura3-52 trp1 ? 63 lys2-128? (hht1-hhf1)??LEU2
(hht2-hhf2)??HIS3 pDM9 (HHT1-HHF1-CEN-URA3)
MAT a hta1-htb1??LEU2, hta2-htb2 ??TRP1, leu2-?1, ura3-52, trp1?63, his3?200,
Finley et al. 1987
Seufert et al. 1995
Dohmen et al. 1995
Johnson and Gupta 2001
Robzyk et al. 2000
FY1716Mann and Grunstein 1992
FY406 Hirschhorn et al. 1995
aDerivative of DF5.
bDerivative of JD52.
Nathan et al.
974GENES & DEVELOPMENT
(v/v) formaldehyde for 5 min. Typically, 10 mg total cellular
protein was used for the ChDIP, and 1 mg for normal ChIP
analysis. For normal ChIP, following lysis and sonication as
described, cells were precleared with protein A sepharose (Am-
ersham) for 1 h prior to overnight precipitation with 1 µL/mg
anti acetyl H3 antibody (Upstate Biotechnology) or anti acetyl
16 H2B (Upstate Biotechnology). For ChDIP after overnight pre-
cipitation (in IP buffer lacking EDTA), Flag elution was per-
formed for 4–6 h, and 90% of the eluate was incubated with 1
column volume (cv) of Ni-NTA Agarose (Qiagen) for 1 h at 4°C.
The slurry was loaded on a small yellow Bio-Rad column,
washed with 4 cv of buffer A (100 mM sodium phosphate at pH
7.5, 150 mM NaCl, 30 mM imidazole) and eluted with 2 cv of
buffer B (100 mM sodium phosphate at pH 7.5, 150 mM NaCl,
250 mM imidazole). All samples were eluted in 100 µL. Samples
were de-cross-linked followed by DNA purification with a PCR
purification kit (Qiagen). All samples were diluted 1:5 prior to
real time PCR analysis. Input was further diluted 1:6 and 10%
Flag input was diluted 1:3. Results were normalized to 10% Flag
input in strains carrying the Flag-histone plasmid. In the control
ChDIP experiments comparing the wild-type Flag-H2B strain
with strains containing either no Flag tag or the Flag-H2B sub-
stitution mutant at K6/7/16/17, values were normalized to total
Chromatin fractionation was as described (Liang and Stillman
1997) except that NEM was added to buffers at a final concen-
tration of 15 or 20 mM.
We thank J.A. Iniguez-Lluhi and G. Moore for valuable discus-
sions. We acknowledge J. Dorsey, T. Sasaki, and R. Heller for
technical assistance. Research was supported by grants from
NIH (GM55360 and RR020839 to S.L.B., GM62268 to E.S.J., and
GM60464 to P.B.M.).
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