MOLECULAR AND CELLULAR BIOLOGY, Nov. 2005, p. 10060–10070
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 22
Insights into the Role of Histone H3 and Histone H4 Core Modifiable
Residues in Saccharomyces cerevisiae†
Edel M. Hyland,1Michael S. Cosgrove,2‡ Henrik Molina,3Dongxia Wang,4§ Akhilesh Pandey,3
Robert J. Cottee,4and Jef D. Boeke1*
High Throughput Biology Center,1Department of Biophysics and Biophysical Chemistry,2Mass Spectrometry Facility,
Department of Biological Chemistry,3and Middle Atlantic Mass Spectrometry Laboratory, Department of
Pharmacology,4Johns Hopkins University School of Medicine, Baltimore, Maryland
Received 5 May 2005/Returned for modification 6 June 2005/Accepted 22 August 2005
The biological significance of recently described modifiable residues in the globular core of the bovine
nucleosome remains elusive. We have mapped these modification sites onto the Saccharomyces cerevisiae
histones and used a genetic approach to probe their potential roles both in heterochromatic regions of the
genome and in the DNA repair response. By mutating these residues to mimic their modified and unmodified
states, we have generated a total of 39 alleles affecting 14 residues in histones H3 and H4. Remarkably, despite
the apparent evolutionary pressure to conserve these near-invariant histone amino acid sequences, the vast
majority of mutant alleles are viable. However, a subset of these variant proteins elicit an effect on transcrip-
tional silencing both at the ribosomal DNA locus and at telomeres, suggesting that posttranslational modifi-
cation(s) at these sites regulates formation and/or maintenance of heterochromatin. Furthermore, we provide
direct mass spectrometry evidence for the existence of histone H3 K56 acetylation in yeast. We also show that
substitutions at histone H4 K91, K59, S47, and R92 and histone H3 K56 and K115 lead to hypersensitivity to
DNA-damaging agents, linking the significance of the chemical identity of these modifiable residues to DNA
metabolism. Finally, we allude to the possible molecular mechanisms underlying the effects of these
The fundamental unit of compaction of eukaryotic DNA is
the nucleosome, consisting of two molecules each of the four
core histones, H2A, H2B, H3, and H4. Not surprisingly, all
four core histone genes are essential. Histones H3 and H4
flank the dyad axis of the structure, bind to the terminal seg-
ments of the DNA that enter and leave the nucleosome, and
are the most highly conserved histones, suggesting that they
play a more prominent regulatory role in chromatin formation.
Furthermore, nucleosomes assembled in vitro from histone H3
and H4 tetramers retain the ability to impede transcription (2).
As chromatin creates a degree of inaccessibility to the genetic
information, proteins involved in DNA-templated reactions
must overcome this. Perturbation to chromatin architecture is
accomplished by the action of ATP-dependent chromatin re-
modeling factors, which reposition nucleosomes relative to the
underlying DNA (1), and the histone modifying enzymes,
which chemically alter histone proteins that comprise the nu-
cleosomes (25, 41). Evidence supports the concerted action of
these proteins in dictating transitions between transcriptionally
active euchromatin and silent heterochromatin (4, 35).
There is a large repertoire of histone modifications, includ-
ing lysine (K) and arginine (R) methylation, serine (S) and
threonine (T) phosphorylation, and lysine acetylation, ubiqui-
tylation, and sumoylation. Particular examples of these modi-
fications underlie dynamic alterations in histone structure,
mostly in the N-terminal “tail” domains of the histones, and
are known to act epigenetically to regulate gene expression
(19). The histone code hypothesis proposes that numerous
effector proteins, which regulate chromatin-based processes,
recognize particular patterns of posttranslational modifications
in the histone tails (20, 48). Indeed, bromodomain (7, 17, 23)
and chromodomain (13, 22, 29) proteins that recognize acety-
lated and methylated histone residues, respectively, have been
identified and are implicated in the regulation of transcription.
Most publications to date have focused on modifications
within the amino-terminal tails of the histones. These highly
basic stretches of amino acids protrude from the nucleosome
core, are flexible and unstructured, and are believed to act as
a platform accommodating the binding of chromatin-associ-
ated proteins to the modified residues. Although these modi-
fications are quite biologically significant, deletion of the tail
domains from the genes encoding individual core histones sup-
ports viability (24) and, in fact, “tailless” H3, H2A, and H2B
proteins retain the ability to organize into nucleosomes (11),
suggesting that the essential function of histone proteins re-
sides in their globular core domain. Indeed, deletions of cer-
tain regions of the core domain of histone H4 are lethal (24).
Residues that lie in the globular core of the nucleosome have
also been shown to be a substrate for modifying enzymes.
Histone H3 K79, for example, is methylated by the Dot1 pro-
tein in Saccharomyces cerevisiae or the homologous protein in
humans, Dot1L (27, 49). Methylation of H3 K79 appears to be
* Corresponding author. Mailing address: 339 Broadway Research
Building, 733 North Broadway, Baltimore MD 21205. Phone: (410)
955-0398. Fax: (410) 502-1872. E-mail: email@example.com.
† Supplemental material for this article may be found at http://mcb
‡ Present address: Department of Biology, Syracuse University,
§ Present address: Biotechnology Core Facility, National Center
for Infectious Diseases, Centers for Disease Control and Prevention,
Atlanta, GA 30333.
cell cycle regulated (14), and its methylation status is important
for defining transcriptionally active regions of the genome (36).
However, core modifications are not limited to this residue. A
report by Zhang et al. (54) provided evidence for the presence
of an extensive network of covalent modifications on the glob-
ular histone fold domains of Bos taurus histones. They proved
that numerous histone core residues have the potential to be
acetylated, methylated, and phosphorylated, similar to that of
the tail residues. Mapping these residues to the three-dimen-
sional structure of the nucleosome core particle (28) illustrates
that these modifiable residues form a pattern of chemical
groups along the lateral surface of the nucleosome in addition
to mapping to its solvent-exposed surface (4). It should be
noted that the Zhang et al. study (54) did not use purified
nucleosomes as the starting material, and so it is not necessar-
ily the case that all of these modifications occur on nucleo-
somes per se; some may occur only on free histones. Impor-
tantly, the biological significance of most of these modifications
has not been investigated.
In our analysis, we systematically generated a library of his-
tone core domain mutations in the yeast S. cerevisiae at resi-
dues corresponding to those identified by Zhang et al. The
mutations were designed to structurally mimic either constitu-
tively modified or unmodified versions of potential modifica-
tion sites. Using this genetic tool, we then explored the re-
quirement of these core modifications in chromatin structure
by analyzing the effects of the altered histones on heterochro-
matin structure and function at transcriptionally silent regions
of the genome. We also explored the possibility that modifiable
residues within the histone core play roles in the DNA damage
response pathway, as has been documented for amino-terminal
tail modifications (3, 33, 42). Here, we provide direct physical
mass spectrophotometric evidence that one of the residues
tested, histone H3 K56, can be acetylated in S. cerevisiae. This
result corroborates the genetic data obtained and is in accord
with recent results from at least two other groups (30, 51). Our
findings are the first to illustrate in vivo that several modifiable
residues within histone H3 and H4 cores are vital for hetero-
chromatin integrity. We also demonstrate that several of these
residues are required to elicit a normal cellular response to
DNA damage, which we propose is likely to be controlled by
their modification status.
MATERIALS AND METHODS
Strains and media. Standard laboratory methods and techniques for budding
yeast manipulations were used. The previously described yeast strain JPY12 (38)
was employed for all investigations. All media were supplemented with 0.64 mM
adenine, except for those monitoring silencing of ADE2. Lead plates were pre-
pared as described elsewhere (5). Synthetic complete (SC) medium was used to
prepare plates containing DNA-damaging agents.
Plasmids. Plasmids pDM18 (43) and pJP11 (38), both carrying HHT2 (encod-
ing histone H3) and HHF2 (encoding histone H4), were utilized in this study. A
derivative of pDM18, pEMH7, was constructed in which the 3? untranslated
region of histone H4 was replaced by the ADH1 3? untranslated region by
inserting a 230-bp fragment amplified using primers JB8727 and JB8728 at
EcoRI and KpnI restriction sites. Each gene was mutagenized using Stratagene’s
QuikChange protocol. Initially, pEMH7 was amplified with a distinct set of
primers specific for the desired mutation, followed by Dpn1 digestion to elimi-
nate unmutagenized parental strands. All plasmids were sequenced with oligo-
nucleotides JB6505 and JB6504 subsequent to recovery from bacteria to verify
the presence of the mutation. A random selection of 10 positively identified
mutants was subcloned back into pEMH7 to verify that their associated pheno-
types were not dependent upon erroneous mutations incorporated into the
backbone of the plasmid during the PCR.
Silencing assays. JPY12 (38) was transformed with pEMH7 harboring the
mutation of interest. Transformants were streaked on yeast extract-peptone-
dextrose to allow loss of the wild-type pJP11 (LYS2 HHT1 HHF1) plasmid and
replica plated separately to SC-Trp, SC-Lys, and SC-His plates. His?, Trp?, and
Lys?colonies were selected and studied further, except in those rare cases in
which the mutations required the presence of wild-type histones for viability and
were therefore Lys?. Silencing of MET15 at the ribosomal DNA locus was
assayed on lead plates as described elsewhere (5). For photography, colonies
were incubated at 30°C for 7 days. The strength of ribosomal DNA silencing of
the mURA3 marker was monitored by plating fivefold serial dilutions with a
starting optical density at 600 nm (OD600) of 0.5 on SC-Trp-His plus 5-fluoroo-
rotic acid (5-FOA) plates. Plates were incubated at 30°C for 4 days and photo-
graphed. The silencing of telomeric ADE2 reporter (44) was assayed on SC-Trp
plates. Cells were spotted on plates and incubated at 30°C for 3 days and
subsequently at room temperature for a further 3 days to allow for color devel-
opment. All data are representative of at least three independent phenotypic
assays undertaken on at least three independent isolates of each mutation in
DNA damage sensitivity assays. Serial dilutions prepared as described above
were plated on SC medium for a growth control and on SC medium containing
either hydroxyurea (HU), methyl methane sulfonate (MMS), or camptothecin
(CPT) at the indicated concentrations. Concentrated stock solutions of each drug
were prepared in dimethyl sulfoxide so as to have a final concentration of 1%
dimethyl sulfoxide in each plate. Plates were incubated for 3 days at 30°C and
photographed, except those that contained HU, which were incubated for a week.
Southern blot analysis. Genomic DNA was prepared for each strain harboring
individual mutations on pEMH7. Genomic DNA was digested for 2 h with KpnI
and run on a 0.8% agarose gel. Following transfer of DNA to nylon membrane,
the membrane was probed with two 500-bp PCR products internally labeled with
[?-32P]dATP. The pEMH7-specific probe was generated using primers JB8455
and JB8456, and the probe specific to ACT1 was created from a yeast genomic
DNA templated PCR using primers JB6761 and JB6762. Hybridization of the
probes was detected using a phosphorimager screen and quantified using Image-
Real-time PCR. Strains were grown in rich medium supplemented with
0.64 mM adenine and harvested at log phase. RNA was prepared from these cells
and treated with DNase, and 450 ng of this RNA was reverse transcribed using
Superscript III (Invitrogen). Following RNase H digestion, cDNA equivalent to
25 ng of input RNA was added to a real-time PCR, using the Applied Biosystems
SYBR green RT-PCR system. Reactions were run in a 96-well plate using the
Applied Biosystems Prism 7900HT fast real-time PCR system. The Ctvalues for
ADE2 expression were compared with that of an internal ACT1 control. The
primers used for ACT1 amplification were JB9639 and JB9640, and those for
ADE2 amplification were JB9638 and JB9641.
Mass spectrometry analysis (quadrupole time of flight). For liquid chroma-
tography-tandem mass spectrometry (LC-MS/MS) analysis, excised gel slices
from sodium dodecyl sulfate-polyacrylamide gel electrophoresis were digested
with trypsin as follows. The gel bands were washed twice in 0.1 M NH4HCO3
(Fluka, Buch, Switzerland) and twice in 50% acetonitrile and subsequently cut
into 2- by 2-mm pieces. The gel pieces were shrunk using 100% acetonitrile and
then allowed to swell in 10 ng/?l trypsin–0.1 M NH4HCO3(Promega U.S.,
Madison, WI) on ice for 20 min. After the rehydration, excess trypsin solution
was removed and samples were incubated overnight at 37°C. The digestion was
stopped by adding 10 ?l of glacial acetic acid, and the supernatant containing the
tryptic peptides was harvested. An extraction step was carried out to recover the
peptides from the gel slices by adding 50% acetonitrile and incubating at room
temperature for 30 min. The supernatant was harvested again and pooled. The
pooled peptide extracts were dried down to approximately 5 ?l and subjected to
LC-MS/MS analysis as follows. Samples were injected onto a 10-cm C18column
(inner diameter, 75 ?m) packed with Vydac MS218 5-?m beads (Vydac, Co-
lumbia, MD) and eluted using a gradient increasing from 7% solvent B–93%
solvent A (solvent A, 0.4% acetic acid, 0.005% heptafluorobutyric acid; solvent
B, 90% acetonitrile, 0.4% acetic acid, 0.005% heptafluorobutyric acid) to 45%
solvent B–55% solvent A in 30 min (1100 CapLC; Agilent, Palo Alto, CA).
Eluting peptides were analyzed using a quadrupole time-of-flight mass spectrom-
eter (QSTAR Pulsar; Sciex, Toronto, Canada). The LC system was connected to
the mass spectrometer using a nanoelectrospray source from Proxeon (Odense,
Denmark). MS/MS data were searched against a yeast-only database (NCBInr)
using Mascot (Matrix Sciences Ltd., London, England). Two missed cleavage
sites and the relevant variable modification were allowed, and the mass accuracy
was set to 0.2 Da for both precursor and fragment ions.
VOL. 25, 2005 ROLES FOR NUCLEOSOMAL CORE MODIFIABLE RESIDUES10061
Mass spectrometry analysis (MALDI). The mixture of histone proteins was
digested with trypsin (20:1 [wt/wt]) in 25 mM ammonia bicarbonate at 37°C for
18 h. Digested peptide mixtures were separated by reversed-phase high-perfor-
mance LC on a C18column with an acetonitrile–water–0.1% trifluoroacetic acid
solvent system. The collected fractions were dried completely and resuspended
in 3 ?l of water. The derivatization reaction was carried out by mixing 3 ?l of
4-sulfophenyl isothiocyanate (10 ?g/?l in 20 mM NaHCO3, pH 9.5) with 1 ?l of
the fraction at 55°C for 30 min. The reaction was terminated by adding 1 ?l
of 5% trifluoroacetic acid, and the sample was cleaned by using a micropipette
tip (C18 ZipTip; Millipore, Bedford, MA). For mass spectrometer measure-
ments, 0.5 ?l of the sample was spotted on a matrix-assisted laser desorption
ionization (MALDI) target followed by the addition of 0.5 ?l of ?-cyano-4-
hydroxycinnamic acid matrix and was allowed to dry at room temperature. All
MS and MS/MS spectra were acquired in the positive ion mode using a Kratos
Analytical (Manchester, United Kingdom) AXIMA-CFR MALDI-TOF mass
spectrometer equipped with a pulsed extraction source, a 337-nm pulsed nitro-
gen laser, and a curved-field reflectron. The acceleration voltage was 20 kV. The
MS/MS spectra were interpreted manually or by computer-assisted database
searches using the Mascot program (www.matrixscience.com) against the NCBI
Nucleosome views. Representations of nucleosomes were generated using
Mapping modifiable histone residues on the S. cerevisiae
nucleosome structure. The positions of modified residues in
the globular core of the bovine histones were mapped onto the
amino acid sequence of S. cerevisiae histone H3 and H4 pro-
teins. As depicted in Fig. 1A, all of the modifiable residues
identified from bovine histones have unambiguous counter-
parts in the yeast sequence. In fact, residues H4 S47, H3 K79,
and T118 were invariant over a wide representation of eukary-
otic species, and residues H3 R52, K56, K115, K122, H4 K31,
K59, K79 K91, and R92 showed amino acid variation only
among deeply rooted eukaryotes, such as Giardia lamblia (see
Fig. S1 in the supplemental material). The preservation of the
FIG. 1. (A) Sequence alignment of histones H3 and H4 from Saccharomyces cerevisiae and Bos taurus, generated using CLUSTALW. Shaded
residues represent the N-terminal tails of the histone proteins. Modified residues identified in bovine histones are labeled and highlighted in both
sequences according to the type of modification. (B) Mapping of modifiable residues on the surface of the yeast nucleosome crystal structure (50).
A surface representation of the nucleosome, without histone tails, is shown and is viewed down the DNA superhelical axis. Potentially acetylated
residues are green, methylated residues are blue, and phosphorylated residues are orange. The DNA helix is represented in bright green.
(C) Rotation of view in panel B 90° around the horizontal axis. This is defined as the lateral surface of the nucleosome.
10062 HYLAND ET AL.MOL. CELL. BIOL.
exact positioning of these modifiable residues in the yeast
nucleosome was verified by comparing the structural context of
each amino acid in the Xenopus laevis (28) and S. cerevisiae
(50) nucleosome crystal structures, the former of which shares
more sequence identity with the bovine histones. The location
of each modifiable residue is highlighted on the yeast nucleo-
some crystal structure in Fig. 1B.
Generation of S. cerevisiae histone mutations. The muta-
genic strategy undertaken generated alleles at each potentially
modifiable residue so as to mimic both modified and unmod-
ified versions of the amino acid. Acetylated lysines, for exam-
ple, were mutated to both arginine and glutamine, which are
the natural amino acids that most closely mimic deacetylated
and acetylated lysine, respectively. All lysine residues were
treated in this way, even those known only to be methylated in
bovine, because there are instances of lysine residues that can
be either acetylated or methylated. Additionally, all modified
residues tested were systematically replaced with alanine. This
generated a library of 39 histone alleles at 14 core residues in
both histone H3 and histone H4 (Table 1). We anticipated that
many of these substitutions would be lethal, given the unusu-
ally high conservation of histone sequences from yeast to mam-
mals. To probe this, a previously developed plasmid shuffle
strategy was used (38). In this strategy, histone function per-
mits growth on medium containing ?-amino-adipate, which
selects against the wild-type histone gene present on the LYS2
“shuffle” plasmid pJP11 (38). As shown in Fig. 2A, all substi-
tutions in histone H4, and the majority of altered histone H3
proteins, supported viability. Notably, only changes at two his-
tone H3 residues, R52 and T118, were lethal. A few mutant
strains had a slow growth phenotype (see Table S1 in the
supplemental material). These include those strains harboring
histone H4 S47E, histone H3 K122A, and histone H3 K122Q
mutations. To determine to what extent strains containing vi-
able substitutions compensated for the presence of the aber-
rant histones by increasing the copy number of these histone
genes, Southern blotting analysis was employed. Data showed
that the ratio between the abundance of the plasmid and a
single-copy gene, ACT1, was not significantly altered in the
mutated strains compared with wild type, indicating that, sur-
prisingly, the majority of these substitutions of invariant amino
acids were very well tolerated in yeast cells (Fig. 2B).
High-throughput phenotypic analysis of histone mutations.
All of the engineered histone mutations were examined for si-
lencing properties in the ribosomal DNA and in telomeres and
also for sensitivity to three distinct DNA-damaging treatments, as
described in Materials and Methods. The majority of the histone
mutants showed one or more phenotypes in these assays. For
by 23 of the 39 alleles, 50% of the mutants induced an lrs (loss-
of-ribosomal DNA silencing) phenotype, and one-third were
more sensitive to HU. These data are summarized in Table 3,
below, and in Table S1 in the supplemental material. In the
following sections we examine specific cases where evidence was
obtained which is consistent with control of heterochromatin or
DNA damage sensitivity by the covalent modification state.
FIG. 2. (A) Plasmid shuffle experiment of mutant histones to de-
termine their viability. Strains expressing aberrant histones in the pres-
ence of wild-type proteins were plated with an initial OD600of 0.5 and
serially diluted fivefold on both SC-Trp-Lys and on plates containing
?-amino-adipate, which selects against the wild-type histone gene
present on the LYS2 “shuffle” plasmid. As a negative control, the
JPY12 yeast strain was transformed with a plasmid from which either
histone H3 or H4 was completely deleted. (B) Southern blot analysis to
determine whether cells expressing the mutated histones were com-
pensating for their presence by increasing the copy number of the
plasmid harboring the altered histone. Genomic DNA was extracted
from each strain and probed for ACT1, a single-copy gene, and the
plasmid backbone in a Southern blot analysis. The ratio of these band
intensities was then compared with that of cells containing wild-type
TABLE 1. Library of histone alleles engineered through
Histone H4 Lys 31
K31A, K31R, K31Q
K59A, K59R, K59Q
K77A, K77R, K77Q
K79A, K79R, K79Q
K91A, K91R, K91Q
R52A, R52K, R52Q
R53A, R53K, R53Q
K56A, K56R, K56Q
K79A, K79R, K79Q
K115A, K115R, K115Q
K122A, K122R, K122Q
VOL. 25, 2005ROLES FOR NUCLEOSOMAL CORE MODIFIABLE RESIDUES 10063
Modifiable nucleosome core residues are important for
transcriptional silencing at telomeres and ribosomal DNA.
Using a genetic approach, we sought indirect evidence for the
existence of nucleosome core modifications in S. cerevisiae and
to infer the potential modification status of each residue at
three transcriptionally silent regions, a strategy previously em-
ployed to study tail modifications (21, 31). The intrinsic logic is
that mutations that represent different modified states of a
residue will display contrasting phenotypes, enabling inference
of the potential modification state. To assess the effect of
altered histones on heterochromatin, we studied transcrip-
tional silencing, a genetically tractable yeast system that al-
lowed us to monitor the existence of various states of hetero-
Histone H4. Peptide mass fingerprinting of bovine histone
H4 indicated that K77 and K79 are acetylated in vivo in that
species (54). To study the effects that mutation of these resi-
dues has on telomeric silencing in yeast, we employed strains
that utilized an ADE2 reporter at the telomeres of chromo-
some V. Substitutions at H4 K79 displayed a pattern of phe-
notypes for telomeric silencing similar to that of a well-char-
acterized acetylated residue, histone H4 K16. We found that
the K79Q substitution leads to a loss of telomeric silencing,
whereas K79R has no effect on silencing, suggesting that the
positive charge is required for telomere silencing. These results
are consistent with K79 being maintained in a deacetylated
state at silent telomeres (Fig. 3A). The substitutions at histone
H4 K77, however, gave the opposite result: K77Q appears
normal for telomeric silencing, whereas K77R causes silencing
to increase. These results would be consistent with the acety-
lation of K79 at silent telomeres (Fig. 3A). Consistent with the
notion that modification of these residues is involved in the
regulation of telomeric silencing, both H4 K77A and H4 K79A
aberrant histone proteins cause telomeric silencing to be lost.
Quantitative real-time PCR was undertaken to verify that the
color changes reported in the silencing assay accurately repre-
sent differences in levels of ADE2 transcript. Values obtained
did indeed corroborate the genetic data, as we saw that the
trend in ADE2 mRNA levels for the mutants mirrored their
effects on silencing (Table 2).
The ribosomal DNA locus on chromosome XII is also main-
tained within a transcriptionally repressed chromatin structure
(15, 45). A previously described strain was utilized that con-
tains both MET15 and mURA3 insertions in the ribosomal
DNA locus (38). Silencing could therefore be analyzed using
two independent reporter systems. Consistent with the telo-
meric silencing results, K79R maintained wild-type levels of
ribosomal DNA silencing, whereas K79Q induced an lrs phe-
notype as indicated by its white coloring on lead plates and its
increased sensitivity to 5-FOA (Fig. 3B).
The support for acetylation of residue H4 K77 in the
ribosomal DNA is less apparent. The K77R substitution has
no effect on ribosomal DNA silencing, consistent with it
being deacetylated at the ribosomal DNA locus. However,
neither the K77Q nor the K77A mutation leads to the lrs
phenotype but, rather, confers an irs (increased ribosomal
DNA silencing) phenotype (Fig. 3B), suggesting a more
repressed chromatin structure. This suggests that either (i)
the positive charge of lysine 77 limits the amount of ribo-
somal DNA silencing, or (ii) perhaps K77 modification does
not play a role in silencing at this locus.
Additionally, our genetic analysis of K59 in histone H4 sup-
ports a role for its modification at ribosomal DNA. An earlier
study suggested that ribosomal DNA silencing was not affected
by mutations in this residue (54), but we saw clear silencing
defects in the H4 K59A and K59Q mutants (Fig. 4B). How-
ever, as K59 has been shown to be a substrate for methylation
in B. taurus, we are unable to draw conclusions from the K59Q
mutation. Nonetheless, we can conclude from this analysis that
the positive charge on an unmethylated K59 is necessary for
effective ribosomal DNA silencing. The discrepancy with the
earlier study is likely due to the fact that the strength of ribo-
FIG. 3. (A) Telomeric silencing assay of histone H4 K16, K77, and
K79 substitutions. Strains containing an ADE2 reporter inserted into
the telomeric regions of chromosome V were transformed with plas-
mids containing the substituted histones and plated onto SC-Trp
plates. After incubation at 30°C for 2 days, plates were put at 4°C for
a further 7 days to facilitate the development of the red color. WT and
loss-of-telomeric-silencing (LTS) strains are shown for comparisons.
(B) ribosomal DNA (denoted rDNA in the figure) silencing assay of
histone H4 K77 and K79 substitutions utilizing a strain containing
mURA3 and a MET15 marker inserted into ribosomal DNA repeats.
Cells were plated with an initial OD600of 0.5 and serially diluted
fivefold on SC-Trp for a growth control and SC-Trp-His plus 5-FOA to
analyze silencing of the mURA3 reporter. Additionally, colonies were
streaked onto complete medium containing PbNO3and incubated at
30°C for 5 days. Brown color formation was analyzed after 1 week at
4°C. WT, loss-of-ribosomal DNA-silencing (LRS), and met15 null
strains are shown as controls.
10064 HYLAND ET AL.MOL. CELL. BIOL.
somal DNA silencing varies between strain backgrounds, and
our silencing studies are done in a strain background in which
differences are easily detected (38, 45).
Histone H3. In an earlier study, the peptide fragment con-
taining residues 52 to 62 of histone H3 showed an increase of
mass of 14 Da by mass spectrometry that was consistent with
methylation of a residue contained within this sequence (54).
The possible candidates for this modification were R52, R53,
and K56. We analyzed mutations at all three residues and
noted that the genetics were consistent with K56 acetylation
leading to a loss of silencing in the ribosomal DNA and to a
lesser extent in the telomeres (Fig. 5). The K56Q mutant led to
mild derepression of the reporter gene at telomeres, whereas
the aberrant histone K56R retained wild-type levels of silenc-
ing (Fig. 5A). Substitution of K56 with glutamine additionally
interfered with heterochromatin formation at ribosomal DNA
and led to an lrs phenotype, whereas replacement of lysine with
an arginine residue had no effect (Fig. 5B). Assuming that the
presence of arginine mimics a persistent deacetylated state,
these data suggest that the lack of modification at K56 sites is
required for efficient silencing at both ribosomal DNA and
telomeres. Consistent with this hypothesis, K56A caused a loss
of ribosomal DNA silencing. Although the initial data gener-
ated from B. taurus suggested that K56 was monomethylated,
the genetic analysis supported its acetylation in S. cerevisiae.
We therefore undertook mass spectrometry analysis to recon-
cile these conflicting data and determine the exact state of K56
in yeast. We showed by both MALDI post-source decay (Fig.
5D) and quadrupole time-of-flight mass spectrometry analysis
(Fig. 5C) that K56 is acetylated in bulk yeast histones (Fig. 4C).
(K56 acetylation was first reported to us by Hiroshi Masumoto
and Alain Verreault at London Research Institute, who have
obtained extensive independent evidence for this modification
[personal communication].) Additionally, during the prepara-
tion of the manuscript H3 K56 acetylation was reported by
another independent group (51).
A second possible site of modification within the core of
histone H3 is K115 acetylation. In support of this, the substi-
tutions of this residue show the predicted pattern of transcrip-
tional silencing phenotypes for a deacetylated residue both at
telomeres and at ribosomal DNA (see Table S1 in the supple-
mental material). Similarly, it appears that the previously ob-
served acetylation of histone H3 K122 might affect ribosomal
DNA silencing. Mutations that incorporate either a glutamine
or an arginine residue at these sites result in lrs and irs phe-
notypes, respectively, which suggests that the unmodified ver-
sion of K122 is important for ribosomal DNA silencing but also
FIG. 4. Transcriptional silencing assays for reporter strains expressing
substitutions at histone H4 K59 and K31. (A) Cells were plated and
analyzed as for Fig. 3. (B) ribosomal DNA silencing was monitored by
plating and analysis as for Fig. 3.
TABLE 2. Real-time PCR analysis of effects of histone alleles
on expression of telomeric ADE2a
aRNA was prepared from the indicated substitution strains and reverse tran-
scribed into cDNA. Quantitative PCR was undertaken to amplify both ADE2
and ACT1 cDNA in the presence of SYBR green. ??Ctwas calculated using the
formula (CtADE2 ? CtACT1)mutant? (CtADE2 ? CtACT1)wild type. Values
were also compared with those of an RT-negative control for each sample.
Results are representative of RNA that was prepared from two individual col-
onies of each sample, both of which were assayed in quadruplicate.
VOL. 25, 2005 ROLES FOR NUCLEOSOMAL CORE MODIFIABLE RESIDUES10065
indicates that a permanently deacetylated state is undesirable.
Therefore, our genetic results could be interpreted as follows:
tight regulation of the modified state of K122 is required at
ribosomal DNA (see Table S1 in the supplemental material).
Predicted modifiable core histone residues that play a mod-
ification-independent role in heterochromatin formation. Cer-
tain predicted modifiable residues, however, did not show phe-
notypic patterns consistent with modification state (Table 3).
For example, all substitutions made at histone H4 K31 and H4
S47 resulted in increased telomeric and ribosomal DNA silenc-
ing, suggesting that the mutations generate a form of chroma-
tin that is presumably less accessible to the transcriptional
machinery. Conversely, mutations at H4 K59 and R92 and H3
R52, T118, and K122 facilitated the complete expression of the
ADE2 reporter at telomeres, regardless of the replacement.
This suggests that telomeric heterochromatin depends upon
the identity of the particular amino acid side chain at each of
these sites (see Fig. 4A for representative data). However, we
cannot assume that these amino acids are not sites of modifi-
cation in S. cerevisiae, as indeed we have previously discussed
that the mutation of residues H4 K59 and H3 K122 produced
phenotypes at rRNA that supported their acetylation. Perhaps
the modifications of H4 K31, S47, and R92 and H3 R52 and
T118 might be critical for other unexplored cellular processes,
or perhaps the substitutions do not faithfully mimic the effects
of the modifications in these instances.
The DNA damage response is impaired by histone H3 and
H4 core mutations. To determine whether mechanisms under-
lying DNA-templated cellular processes utilize modification of
the nucleosome core, we initially explored the sensitivity of
each of the 39 histone alleles to various DNA-damaging re-
agents, including MMS, HU, and CPT. None of the single
amino acid substitutions conferred resistance or hypersensitiv-
ity to MMS. However, as depicted in Fig. 6, certain mutant
FIG. 5. Acetylation of H3 K56 in S. cerevisiae. (A) Silencing reporter strains expressing alanine, glutamine, and arginine substitutions at histone
H3 K56 were plated and analyzed as for Fig. 3. (B) ribosomal DNA silencing strains were plated and analyzed as for Fig. 3. WT, loss-of-ribosomal
DNA-silencing (LRS), and met15 null strains are shown as controls. (C) MS/MS spectrum of the doubly charged lysine acetylated peptide ion,
FQKacetylSTELLIR. The complete sequence including the acetylated lysine residue was deduced from the y-ion series as shown. Diagnostic
fragment ions (53) for an acetylated lysine residue are observed at m/z 84.1 and m/z 126.1. (D) The MALDI postsource decay spectrum of a histone
H3 peptide isolated from a trypsin-digested total yeast histone preparation. The peptide was N-terminally sulfonated prior to mass spectrometric
10066 HYLAND ET AL.MOL. CELL. BIOL.
histones led to an impaired response to HU treatment. Histone
H4 variant proteins S47E, K59A, K59Q, R92A, and R92K
displayed growth defects on medium containing 200 mM HU
in comparison to wild-type H4 strains. Residues S47, K59, and
K91 therefore showed contrasting phenotypes for the different
substitutions, suggesting that it is the modification at these sites
that may control accessibility to DNA damaging agents or the
efficacy of DNA repair pathways. The exact structural at-
tributes of residue H4 R92 on the other hand appear necessary
for recovery from DNA damage, as all substitutions made at
this residue confer increased HU sensitivity. Histone H3
K115A, K115Q, T118A, T118E, K56A, and K56Q mutants also
were hypersensitive to HU, suggesting that the modification of
these amino acids is influencing DNA repair. Of the above
mutants, only H3 K56A and H3 K56Q were sensitive to CPT
(Fig. 6B). The differential sensitivity to these two drugs, both of
which are proposed ultimately to produce double-stand breaks,
may be explained by their different modes of action. HU in-
hibits ribonucleotide reductase, leading to replication defect
and subsequent fork collapse, whereas CPT is a topoisomerase
I inhibitor. Presumably, the detailed structure, positioning, or
timing of these two types of double-strand breaks is different
enough that they end up packaged differently in the different
types of mutant nucleosomes.
Histones have been mutagenized and subjected to a wide
variety of genetic screens (47). Numerous investigators have
mutagenized individual histone residues to test specific hy-
potheses for their roles. More general screens have been per-
formed for histone mutants that perturb interactions with
chromatin remodelers (12, 18, 26, 40) or that decrease or
increase silencing (10, 38) and histone variants important in
centromere function (16). In each case, multiple alleles with
the desired phenotypes were isolated, although the screens
have probably not been done to saturation, suggesting there is
much more to learn through such an approach.
In this study we have taken a more structural approach,
probing systematically the residues known to be modifiable and
testing their biological roles. A conclusion that may seem sur-
prising is that the identity of these modifiable residues, which
are very highly phylogenetically conserved, can be changed
without killing the cell—we infer that drastically different side
chains do not compromise the basic function of these histones
to form nucleosomes. On the other hand, since these residues
are on the nucleosome surface at a solvent interface and can
clearly function in one or more chemically modified states, it is
perhaps not so surprising that the side chains can be modified.
On the other hand, the percentage of these mutants that dis-
play obvious phenotypes relating to altered silencing (92%) or
sensitivity to DNA-damaging agents (33%) is astonishingly
high and suggests possible active roles for the nucleosome
surfaces associated with these residues.
At least eight residues within the histone fold domains of
bovine histones H3 and H4 are subject to posttranslational
modification; however, the functional significance of these
modifications is unknown. The genetic data presented here
FIG. 6. The DNA repair response is affected by substitutions of
core nucleosome residues. (A) Strains expressing mutated histones
were plated with an initial OD600of 0.5 and serially diluted fivefold on
SC as a growth control and SC plus 200 mM HU to analyze the ability
of these cells to recover from DNA damage-induced stress. A rad52
null strain was used as a positive control. (B) Cells harboring substi-
tutions at H3 K56 were examined on plates containing CPT at the
indicated concentrations. The initial density of cells corresponded to
an OD600of 0.5, and cells were serially diluted fivefold.
TABLE 3. Summary of phenotypes observed for
indicated histone allelesa
aThe mutants were analyzed and their phenotypes were scored arbitrarily with
respect to wild type. For a given process, wild type was denoted ???, and those
mutants with a negative effect were scored as ?? or ?, depending on the
severity of the mutation. Alleles that induced an increase in silencing are indi-
cated with ???? or ?????, the latter of which corresponds to almost-
complete repression of the reporter gene.
VOL. 25, 2005ROLES FOR NUCLEOSOMAL CORE MODIFIABLE RESIDUES10067
indicate that several of these residues are critical for epigenetic
regulation of gene expression in S. cerevisiae. Moreover, dif-
ferent amino acid substitutions confer distinct and indeed op-
posite phenotypes on gene silencing, strongly suggesting that
these core residues are also subject to posttranslational mod-
ification in S. cerevisiae. The mechanistic pathways underlying
the consequences of these modifications may involve direct
effects on nucleosomal structure or packing, or they may im-
plicate the interaction with a nonhistone protein, or both. By
considering the observed phenotypes of mutations engineered
at these sites with respect to the precise location of the specific
residue on the nucleosome structure, we generated testable
hypotheses to address this.
Lateral surface residues. Histone H4 residues K77 and K79
are both located on the lateral surface of the nucleosome
within the DNA-protein interface (Fig. 1B). H4 K77 does not
directly contact the DNA but is positioned close enough that
an interaction could be facilitated through a water molecule
(6). H4 K79, however, directly interacts with the DNA. Both
residues are located in a region of histone H4 called the L2
loop, which has been shown in the nucleosome crystal structure
to interact with the H3 L1 loop, generating a DNA binding
surface known as L1L2 (28). The lack of an observable silenc-
ing phenotype for the K79R substitution indicates that the
presence of a positive charge at this location is sufficient for
normal histone H4 functions at heterochromatin. As the K79Q
mutation leads to a destabilization of heterochromatin, we can
hypothesize that acetylated K79 will interfere with DNA-pro-
tein interactions and render the nucleosome more mobile,
suggesting that its acetylation increases the accessibility of
DNA. This is consistent with the recently proposed “regulated
nucleosome mobility” model (4), which suggests that modifi-
cations that alter histone-DNA interactions regulate the equi-
librium between mobile and relatively stationary nucleosomes.
In contrast, the H4 K77 mutations are less easily interpreted by
this model. Mutations in residue K77 are consistent with acet-
ylation of this residue being essential for telomeric silencing
but inhibitory to silencing at the ribosomal DNA locus. This
paradox can be reconciled by the fact that different proteins
are required in the establishment of silencing at each of these
loci, and perhaps K77 modification is important for this dis-
tinction. This observed effect at telomeres is consistent with
findings that knockout of class I histone deacetylases, which
presumably leads to increased acetylation of histones (and
perhaps other proteins), paradoxically enhances silencing in
several organisms (9, 37, 46).
Other modifiable residues that lie on the lateral surface of
the nucleosome include H3 K115, K122, H4 K31, and S47.
Similar to H4 K77, they all have the potential to participate
indirectly in DNA binding. K115 and K122 specifically lie in
the L2 loop region of H3, which interacts with H4 L1 loop,
forming another L1L2 DNA binding surface in the dyad axis of
the nucleosome. Both residues were shown to be acetylated in
bulk histones from B. taurus. The phenotypes we observed for
K115 mutations are consistent with K115 acetylation in the
regulation of DNA-protein interactions, as the K115Q sub-
stitution causes derepression at both ribosomal DNA and telo-
meres, whereas the K115R mutation has no effect. Similar
observations were made for H3 K122 mutant phenotypes at
the ribosomal DNA locus. However, at telomeric regions, the
contribution of K122 appears to be dependent on side chain
identity, as arginine cannot substitute for its function, and both
K122A and K122Q also result in the loss of silencing. There-
fore, as for H4 K77, it appears that H3 K122 behaves differ-
ently at distinct silent regions of the genome.
The decrease in accessibility of DNA to the transcription
machinery observed at telomeres for all substitutions at H4
K31 and S47 is intriguing. In spite of their location directly
beneath the DNA on the lateral surface, alterations to their
chemical make-up, mimicking a persistently modified state,
actually lead to an increase in silencing. The molecular mech-
anisms of this remain to be elucidated, but hypotheses pre-
dicted by these data are that (i) these residues play a role in
recognition of a silencing inhibitor in the unmodified state, or
(ii) they have a direct effect on nucleosome compaction.
H3 T118I has previously been identified as a SWI/SNF-
independent or SIN allele (26). Sin?mutations are thought to
suppress mutations in the ATP-dependent nucleosome remod-
eling complex by causing increased nucleosome mobility. In
the crystal structure, H3 T118 directly interacts with DNA and
H4 R45, which protrudes into the DNA minor groove, and this
latter interaction is presumed to ensure a nonspecific interac-
tion between R45 and DNA. A nucleosome containing H3
T118I has been crystallized (34) and indeed exhibits weakened
DNA contacts in this region. Furthermore, H3 T118I sup-
presses a SUC2 UAS deletion mutation, thereby allowing tran-
scription of this gene solely from its basal promoters (39). This
additionally supports its effect on DNA accessibility through
increased nucleosome mobility. Phosphorylation of H3 T118 is
predicted to similarly weaken histone-DNA interactions; how-
ever, this modification is presumably tightly regulated and/or
highly localized within the cell, since replacing H3 T118 with
glutamic acid (or alanine) is lethal to yeast and both H3 T118E
and T118A exert a dominant-negative effect at silent loci. In
addition, T118A showed a slight increase in sensitivity to HU.
Undoubtedly, further analysis is required to uncover the exact
biological significance of phosphorylated T118. It should be
noted that both potentially phosphorylated residues, H4 S47
and H3 T118, lie in close proximity on the nucleosome surface,
therefore having the potential to generate an acidic patch.
H3 K56 is located near a terminal DNA segment that enters
(or leaves) the nucleosome. Its side chain is surface exposed,
and it is positioned close to the edge of the lateral surface of
the nucleosome. Our mass spectrometry results verify acetyla-
tion of this residue in S. cerevisiae, and it is evident from our
genetic data that this modification plays a role in heterochro-
matin formation. K56 mutants also show an increase in sensi-
tivity to numerous DNA damage-causing agents. Given its
location on the three-dimensional structure of the nucleosome,
we hypothesize that acetylation of K56 might induce detach-
ment of DNA from the nucleosome, increasing DNA accessi-
bility to both transcription machinery and repair proteins. Con-
sistent with this theory, we show that the unmodifiable K56R
variant protein, which presumably would not facilitate such
unraveling, is the most sensitive to DNA-damaging drugs. As
many ATP-dependent nucleosome remodeling complexes act
through this mechanism, it will be interesting to probe for
interactions between enzymes that modulate K56 modification
and such remodeling complexes. Indeed, the recent indepen-
dent report of H3 K56 acetylation (51) showed that this mod-
10068 HYLAND ET AL.MOL. CELL. BIOL.
ification is required for recruitment of Snf5, a SWI/SNF nu-
cleosome remodeler to both the HTA1 and SUC2 genes.
Residues on the nucleosome face. Certain modifiable resi-
dues map to the face of the nucleosome. One such residue is
histone H4 K59, clearly shown to be methylated in bovine
histones. Replacement of this lysine with alanine, arginine, or
glutamine inhibited the silencing mechanisms at telomeres.
The exposure of this side chain on the nucleosome surface
suggests that it can act as part of a binding surface that regu-
lates internucleosomal interactions or is recognized by a silenc-
ing factor (e.g., COMPASS). In the latter case, as no amino
acid substitution accurately mimics the methylated state, we
speculate that such a factor could interact with methyl lysine.
At the ribosomal DNA, however, we see that silencing is main-
tained in the presence of H4 K59R, consistent with hetero-
chromatin formation at ribosomal DNA relying on the unmod-
Finally, there are two modifiable residues in histone H4,
namely K91 and R92, that are not surface exposed but buried
in the nucleosome core. They are located close to the interface
between the H3/H4 tetramer and H2A/H2B dimer, and K91
participates in a salt bridge with H2B E63. During the prepa-
ration of the manuscript, it was revealed by Ye et al. (52) that
the acetylation of K91 is important for regulating chromatin
assembly. Our data are consistent with theirs in that the un-
modifiable K91R variant protein can substitute for the native
protein and no phenotypes were observed for this mutation in
the assays under examination. K91Q, however, showed slight
loss of ribosomal DNA silencing as well as increased sensitivity
to hydroxyurea, indicating that perturbing this interaction may
cause generalized destabilization of chromatin structure. Ei-
ther substitution of R92 to alanine or lysine eliminated silenc-
ing at telomeres and rendered the cell more sensitive to DNA
damage, indicating an important role for this residue in the
The genetic analysis presented here provides insights into
the role of previously uncharacterized nucleosome core mod-
ifications. Our systematic mutational analysis has permitted us
to probe for the influence of these modifications on silencing as
well as on the DNA repair response pathway. While our ob-
servations are consistent with the modification of several of
these residues in S. cerevisiae, we acknowledge that the results
could reflect the major charge changes introduced at these
locations. However, earlier insights into tail modifications
gained using this strategy (31) and the recent independent
identification of both H3 K56 and H3 K91 acetylation in
S. cerevisiae lend credence to our claim and suggest that direct
physical evidence supporting the remaining sites of modifica-
tion may be uncovered in the future. Although at this point we
can only speculate as to the detailed mechanisms that imple-
ment the functions of these modifications, it is evident that
they are biologically significant and warrant further investiga-
tion. Several studies have shown that combining mutations in
the N-terminal modifiable residues can lead to complex and
sometimes synergistic phenotypes (10, 31, 32). Considering the
potentially complex network defined by the modifiable resi-
dues on the nucleosome core, it would be interesting for future
work to include an analysis of different combinations of these
This work was supported by the Technology Center for Networks
and Pathways of Lysine Metabolism (NIH RR020839).
We are grateful to Alain Verreault and Hiroshi Masumoto for
sharing unpublished data and Pamela Meluh for critical reading of the
1. Becker, P. B., and W. Horz. 2002. ATP-dependent nucleosome remodeling.
Annu. Rev. Biochem. 71:247–273.
2. Chang, C. H., and D. S. Luse. 1997. The H3/H4 tetramer blocks transcript
elongation by RNA polymerase II in vitro. J. Biol. Chem. 272:23427–23434.
3. Cheung, W. L., F. B. Turner, T. Krishnamoorthy, B. Wolner, S. H. Ahn, M.
Foley, J. A. Dorsey, C. L. Peterson, S. L. Berger, and C. D. Allis. 2005.
Phosphorylation of histone H4 serine 1 during DNA damage requires casein
kinase II in S. cerevisiae. Curr. Biol. 15:656–660.
4. Cosgrove, M. S., J. D. Boeke, and C. Wolberger. 2004. Regulated nucleosome
mobility and the histone code. Nat. Struct. Mol. Biol. 11:1037–1043.
5. Cost, G. J., and J. D. Boeke. 1996. A useful colony colour phenotype asso-
ciated with the yeast selectable/counter-selectable marker MET15. Yeast
6. Davey, C. A., D. F. Sargent, K. Luger, A. W. Maeder, and T. J. Richmond.
2002. Solvent mediated interactions in the structure of the nucleosome core
particle at 1.9 Å resolution. J. Mol. Biol. 319:1097–1113.
7. de la Cruz, X., S. Lois, S. Sanchez-Molina, and M. A. Martinez-Balbas. 2005.
Do protein motifs read the histone code? Bioessays 27:164–175.
8. Delano, W. L. 2002. The PyMOL molecular graphics system. Delano Scien-
tific, San Carlos, Calif.
9. De Rubertis, F., D. Kadosh, S. Henchoz, D. Pauli, G. Reuter, K. Struhl, and
P. Spierer. 1996. The histone deacetylase RPD3 counteracts genomic silenc-
ing in Drosophila and yeast. Nature 384:589–591.
10. Dion, M. F., S. J. Altschuler, L. F. Wu, and O. J. Rando. 2005. From the
cover: genomic characterization reveals a simple histone H4 acetylation
code. Proc. Natl. Acad. Sci. USA 102:5501–5506.
11. Dorigo, B., T. Schalch, K. Bystricky, and T. J. Richmond. 2003. Chromatin
fiber folding: requirement for the histone H4 N-terminal tail. J. Mol. Biol.
12. Duina, A. A., and F. Winston. 2004. Analysis of a mutant histone H3 that
perturbs the association of Swi/Snf with chromatin. Mol. Cell. Biol. 24:
13. Eissenberg, J. C. 2001. Molecular biology of the chromo domain: an ancient
chromatin module comes of age. Gene 275:19–29.
14. Feng, Q., H. Wang, H. H. Ng, H. Erdjument-Bromage, P. Tempst, K. Struhl,
and Y. Zhang. 2002. Methylation of H3-lysine 79 is mediated by a new family
of HMTases without a SET domain. Curr. Biol. 12:1052–1058.
15. Fritze, C. E., K. Verschueren, R. Strich, and R. Easton Esposito. 1997.
Direct evidence for SIR2 modulation of chromatin structure in yeast rDNA.
EMBO J. 16:6495–6509.
16. Glowczewski, L., P. Yang, T. Kalashnikova, M. S. Santisteban, and M. M.
Smith. 2000. Histone-histone interactions and centromere function. Mol.
Cell. Biol. 20:5700–5711.
17. Hassan, A. H., P. Prochasson, K. E. Neely, S. C. Galasinski, M. Chandy,
M. J. Carrozza, and J. L. Workman. 2002. Function and selectivity of bro-
modomains in anchoring chromatin-modifying complexes to promoter nu-
cleosomes. Cell 111:369–379.
18. Hirschhorn, J. N., A. L. Bortvin, S. L. Ricupero-Hovasse, and F. Winston.
1995. A new class of histone H2A mutations in Saccharomyces cerevisiae
causes specific transcriptional defects in vivo. Mol. Cell. Biol. 15:1999–2009.
19. Iizuka, M., and M. M. Smith. 2003. Functional consequences of histone
modifications. Curr. Opin. Genet. Dev. 13:154–160.
20. Jenuwein, T., and C. D. Allis. 2001. Translating the histone code. Science
21. Johnson, L. M., P. S. Kayne, E. S. Kahn, and M. Grunstein. 1990. Genetic
evidence for an interaction between SIR3 and histone H4 in the repression
of the silent mating loci in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci.
22. Jones, D. O., I. G. Cowell, and P. B. Singh. 2000. Mammalian chromodomain
proteins: their role in genome organisation and expression. Bioessays 22:
23. Kasten, M., H. Szerlong, H. Erdjument-Bromage, P. Tempst, M. Werner,
and B. R. Cairns. 2004. Tandem bromodomains in the chromatin remodeler
RSC recognize acetylated histone H3 Lys14. EMBO J. 23:1348–1359.
24. Kayne, P. S., U. J. Kim, M. Han, J. R. Mullen, F. Yoshizaki, and M.
Grunstein. 1988. Extremely conserved histone H4 N terminus is dispensable
for growth but essential for repressing the silent mating loci in yeast. Cell
25. Kouzarides, T. 2002. Histone methylation in transcriptional control. Curr.
Opin. Genet. Dev. 12:198–209.
26. Kruger, W., C. L. Peterson, A. Sil, C. Coburn, G. Arents, E. N. Moudri-
anakis, and I. Herskowitz. 1995. Amino acid substitutions in the structured
VOL. 25, 2005ROLES FOR NUCLEOSOMAL CORE MODIFIABLE RESIDUES10069
domains of histones H3 and H4 partially relieve the requirement of the yeast Download full-text
SWI/SNF complex for transcription. Genes Dev. 9:2770–2779.
27. Lacoste, N., R. T. Utley, J. M. Hunter, G. G. Poirier, and J. Cote. 2002.
Disruptor of telomeric silencing-1 is a chromatin-specific histone H3 meth-
yltransferase. J. Biol. Chem. 277:30421–30424.
28. Luger, K., A. W. Mader, R. K. Richmond, D. F. Sargent, and T. J. Richmond.
1997. Crystal structure of the nucleosome core particle at 2.8 Å resolution.
29. Lusser, A., D. L. Urwin, and J. T. Kadonaga. 2005. Distinct activities of
CHD1 and ACF in ATP-dependent chromatin assembly. Nat. Struct. Mol.
30. Masumoto, H., D. Hawke, R. Kobayashi, and A. Verreault. 2005. A role for
cell-cycle-regulated histone H3 lysine 56 acetylation in the DNA damage
response. Nature 436:294–298.
31. Megee, P. C., B. A. Morgan, B. A. Mittman, and M. M. Smith. 1990. Genetic
analysis of histone H4: essential role of lysines subject to reversible acetyla-
tion. Science 247:841–845.
32. Megee, P. C., B. A. Morgan, and M. M. Smith. 1995. Histone H4 and the
maintenance of genome integrity. Genes Dev. 9:1716–1727.
33. Moore, J. D., and J. E. Krebs. 2004. Histone modifications and DNA double-
strand break repair. Biochem. Cell Biol. 82:446–452.
34. Muthurajan, U. M., Y. Bao, L. J. Forsberg, R. S. Edayathumangalam, P. N.
Dyer, C. L. White, and K. Luger. 2004. Crystal structures of histone Sin mutant
nucleosomes reveal altered protein-DNA interactions. EMBO J. 23:260–271.
35. Narlikar, G. J., H. Y. Fan, and R. E. Kingston. 2002. Cooperation between
complexes that regulate chromatin structure and transcription. Cell 108:
36. Ng, H. H., R. M. Xu, Y. Zhang, and K. Struhl. 2002. Ubiquitination of
histone H2B by Rad6 is required for efficient Dot1-mediated methylation of
histone H3 lysine 79. J. Biol. Chem. 277:34655–34657.
37. Olsson, T. G., K. Ekwall, R. C. Allshire, P. Sunnerhagen, J. F. Partridge, and
W. A. Richardson. 1998. Genetic characterisation of hda1?, a putative fission
yeast histone deacetylase gene. Nucleic Acids Res. 26:3247–3254.
38. Park, J. H., M. S. Cosgrove, E. Youngman, C. Wolberger, and J. D. Boeke.
2002. A core nucleosome surface crucial for transcriptional silencing. Nat.
39. Prelich, G., and F. Winston. 1993. Mutations that suppress the deletion of an
upstream activating sequence in yeast: involvement of a protein kinase and
histone H3 in repressing transcription in vivo. Genetics 135:665–676.
40. Recht, J., and M. A. Osley. 1999. Mutations in both the structured domain
and N-terminus of histone H2B bypass the requirement for Swi-Snf in yeast.
EMBO J. 18:229–240.
41. Roth, S. Y., J. M. Denu, and C. D. Allis. 2001. Histone acetyltransferases.
Annu. Rev. Biochem. 70:81–120.
42. Sanders, S. L., M. Portoso, J. Mata, J. Bahler, R. C. Allshire, and T.
Kouzarides. 2004. Methylation of histone H4 lysine 20 controls recruitment
of Crb2 to sites of DNA damage. Cell 119:603–614.
43. Sikorski, R. S., and P. Hieter. 1989. A system of shuttle vectors and yeast
host strains designed for efficient manipulation of DNA in Saccharomyces
cerevisiae. Genetics 122:19–27.
44. Singer, M. S., and D. E. Gottschling. 1994. TLC1: template RNA component
of Saccharomyces cerevisiae telomerase. Science 266:404–409.
45. Smith, J. S., and J. D. Boeke. 1997. An unusual form of transcriptional
silencing in yeast ribosomal DNA. Genes Dev. 11:241–254.
46. Smith, J. S., E. Caputo, and J. D. Boeke. 1999. A genetic screen for ribo-
somal DNA silencing defects identifies multiple DNA replication and chro-
matin-modulating factors. Mol. Cell. Biol. 19:3184–3197.
47. Smith, M. M., and M. S. Santisteban. 1998. Genetic dissection of histone
function. Methods 15:269–281.
48. Strahl, B. D., and C. D. Allis. 2000. The language of covalent histone
modifications. Nature 403:41–45.
49. van Leeuwen, F., P. R. Gafken, and D. E. Gottschling. 2002. Dot1p modu-
lates silencing in yeast by methylation of the nucleosome core. Cell 109:
50. White, C. L., R. K. Suto, and K. Luger. 2001. Structure of the yeast nucleo-
some core particle reveals fundamental changes in internucleosome interac-
tions. EMBO J. 20:5207–5218.
51. Xu, F., K. Zhang, and M. Grunstein. 2005. Acetylation in histone H3 glob-
ular domain regulates gene expression in yeast. Cell 121:375–385.
52. Ye, J., X. Ai, E. E. Eugeni, L. Zhang, L. R. Carpenter, M. A. Jelinek, M. A.
Freitas, and M. R. Parthun. 2005. Histone H4 lysine 91 acetylation A core
domain modification associated with chromatin assembly. Mol. Cell 18:
53. Zhang, K., P. M. Yau, B. Chandrasekhar, R. New, R. Kondrat, B. S. Imai,
and M. E. Bradbury. 2004. Differentiation between peptides containing
acetylated or tri-methylated lysines by mass spectrometry: an application for
determining lysine 9 acetylation and methylation of histone H3. Proteomics
54. Zhang, L., E. E. Eugeni, M. R. Parthun, and M. A. Freitas. 2003. Identifi-
cation of novel histone posttranslational modifications by peptide mass fin-
gerprinting. Chromosoma 112:77–86.
10070 HYLAND ET AL.MOL. CELL. BIOL.