Identification of Lysine 37 of Histone H2B as a Novel Site
Kathryn E. Gardner, Li Zhou¤, Michael A. Parra, Xian Chen, Brian D. Strahl*
Department of Biochemistry and Biophysics, School of Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina,
United States of America
Recent technological advancements have allowed for highly-sophisticated mass spectrometry-based studies of the histone
code, which predicts that combinations of post-translational modifications (PTMs) on histone proteins result in defined
biological outcomes mediated by effector proteins that recognize such marks. While significant progress has been made in
the identification and characterization of histone PTMs, a full appreciation of the complexity of the histone code will require
a complete understanding of all the modifications that putatively contribute to it. Here, using the top-down mass
spectrometry approach for identifying PTMs on full-length histones, we report that lysine 37 of histone H2B is dimethylated
in the budding yeast Saccharomyces cerevisiae. By generating a modification-specific antibody and yeast strains that harbor
mutations in the putative site of methylation, we provide evidence that this mark exist in vivo. Importantly, we show that
this lysine residue is highly conserved through evolution, and provide evidence that this methylation event also occurs in
higher eukaryotes. By identifying a novel site of histone methylation, this study adds to our overall understanding of the
complex number of histone modifications that contribute to chromatin function.
Citation: Gardner KE, Zhou L, Parra MA, Chen X, Strahl BD (2011) Identification of Lysine 37 of Histone H2B as a Novel Site of Methylation. PLoS ONE 6(1): e16244.
Editor: Mary Bryk, Texas A&M University, United States of America
Received September 29, 2010; Accepted December 8, 2010; Published January 13, 2011
Copyright: ? 2011 Gardner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Institute of Diabetes, Digestive and Kidney Diseases grant 5 R21 DK082706-02 (B.D.S. and X.C.). The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
¤ Current address: Jiangsu Skyray Instrument Co., Ltd. Kunshan, Jiangsu, China
In eukaryotic cells, DNA is packaged in the form of chromatin.
Approximately 147 base pairs of DNA wrap around an octomer
composed of two H2A–H2B dimers and one H3–H4 tetramer to
form nucleosomes, the fundamental repeating unit of chromatin
[1,2]. Because nucleosomes are organized into progressively
higher-ordered structures, significant chromatin remodeling is
necessary for the numerous DNA-templated processes that must
occur for normal cellular function, such as transcription, DNA
replication, DNA repair, and chromosome segregation.
One means by which alterations to chromatin structure is
accomplished is through post-translational modifications (PTMs) of
the histone proteins. The core histones are largely globular, with the
exception of unstructured N-terminal tails that protrude from the
surface of the core particle. Although numerous PTMs have been
shown to occur on residues located on the histone tails , it is
becoming increasingly evident that residues within the globular
domain are also subject to modifications [4,5,6]. The type of PTMs
demonstrated to occur on histone proteins include acetylation,
methylation, phosphorylation, ubiquitylation, sumoylation, ADP
ribosylation, proline isomerization, citrullination, butyrylation,
propionylation and glycosylation [3,7,8]. While the functional
significance of some of the aforementioned modifications remainsto
be elucidated, it is well established that other histone PTMs function
by at least one of the following mechanisms: (1) disruption of
nucleosomalcontacts between histonesandtheirassociated DNAor
between histones in contiguous nucleosomes, or (2) recruitment of
non-histone proteins [3,4]. Acetylation of lysine residues is the best-
characterized modification shown to affect higher-order chromatin
on which it occurs, thereby inhibiting histone-histone or histone-
DNA interaction and thus chromatin compaction [9,10,11]. With
regard to the other means by which histone PTMs can function, the
recruitment of non-histone proteins is facilitated by the ability of
specialized domains to recognize and bind to defined marks .
For example, methylation of specific lysine residues in a defined
state (mono-, di-, or trimethyl) can serve as a binding platform for
effector proteins containing one of the following types of methyl-
binding domains: chromodomain, tudor domain, PHD finger,
MBT, Ankyrin repeat, PWWP domain and WD40 repeats
The complexity of the number and diverse types of PTMs has led
to the hypothesis of a ‘‘histone code’’ [15,16], which posits that
combinatorial patterns of histone PTMs lead to defined biological
outcomes brought about by the recruitment of effector proteins
necessary for function in DNA-templated processes. For example,
TAF1(thelargestsubunit of the TFIIDcomplexwhichisinvolvedin
initiating the assembly of transcriptional machinery) contains a
histone H4 , and itself canfunction as a histone acetyltransferase
. There are numerous other examples of how defined
combinations of histone modifications positively or negatively affect
recruitment of specific proteins [19,20,21,22,23,24]. Despite the
PLoS ONE | www.plosone.org1January 2011 | Volume 6 | Issue 1 | e16244
identification of numerous histone PTMs to date, it is likely that
other modifications still await discovery. Thus, of immediate
importance in deciphering the histone code is the need for
identifying all the PTMs that are present on histones, so that
subsequentstudiescan be completed todetermine the combinatorial
and what the functional outcomes of such combinations are.
In recent history, mass spectrometry (MS) has widely been used
as the primary method to identify histone PTMs. MS studies have
commonly employed the bottom-up approach, in which short
peptides derived from proteolytic cleavage of reverse-phase HPLC
(RP-HPLC)-purified histones are analyzed by MS with peptide
mass fingerprinting (PMF) or a combination of liquid chromatog-
raphy (LC) and tandem MS (MS/MS) using electron transfer or
collision-induced dissociation methods (ETD and CID, respec-
tively) . While this technique is a highly effective means by
which to determine the molecular mass (by MS-PMF) or the
sequence of a protein (by LC-MS/MS), it is limited in that
incomplete sequence coverage of the protein of interest often
occurs, and proteins with multiple cleavage sites (including the
histone core proteins, which are rich in lysine and arginine
residues) result in peptide segments that are too small for effective
retention and/or detection [26,27,28,29,30]. More recently,
advances in MS have led to the development of the top-down
approach as a complementary method to bottom-up analysis as a
highly useful means by which to identify PTMs on histones
[31,32,33,34,35,36,37]. Full-length proteins are analyzed with top-
down MS, as samples are infused into the mass spectrometer by
electrospray ionization (ESI), allowing for MS/MS fragmentation
via ETD or electron capture dissociation (ECD) of intact proteins
. A major advantage of top-down MS is that combinatorial
patterns of modifications that exist on a single histone molecule
can be identified , which is particularly valuable in outlining
the global landscape of PTMs on histone proteins.
In this study, we sought to use top-down MS to analyze the
global landscape of PTMs on histone H2B. From this analysis, we
identified lysine 37 of histone H2B (H2BK37) as a novel site of
methylation in the budding yeast Saccharomyces cerevisiae, and that
this modification exists in the dimethyl state. We generated an
antibody specific for dimethylated H2BK37 (H2BK37me2), with
which we were able to confirm that this mark does in fact occur in
vivo. Though our candidate approach to identify the methyltrans-
ferase responsible for placing this mark and phenotypic analysis to
reveal a biological function did not offer conclusive results, we
provide evidence that this modification is evolutionarily conserved
supporting its overall importance as a novel histone modification.
Furthermore, these results demonstrate that despite the numerous
rounds of previous MS analysis, additional series of MS analyses
employing recent technological advancements are necessary for
continued identification of novel sites of modifications to generate
a more complete atlas of the factors that putatively function in the
context of the histone code.
H2B is dimethylated at lysine 37
To date, only three lysine residues have been well-characterized
as sites of methylation in budding yeast (namely lysines 4, 36, and
79 of histone H3) . In higher eukaryotes, methylation is known
to also occur on histone H3 at lysine residues 9 and 27 and histone
H4 at lysine 20 . To begin to address whether histone
methylation occurs on other sites in budding yeast, as well as to
acquire a more comprehensive atlas of histone PTMs, we sought
to use MS analysis to identify novel histone modifications. Given
recent advancements in MS technology, it is now possible to use
the top-down MS approach to analyze intact histone proteins,
thereby allowing for more precise delineation and quantification of
the complex modified forms in which the histones exist . We
initially performed our top-down MS studies on histone H2B, as
this histone has more recently been shown to be monomethylated
at lysine 5 in humans [41,42], and we were interested in
determining whether this modification is conserved or if
alternative sites of methylation exist in budding yeast.
According to its amino acid sequence, the theoretical mono-
isotopic mass ([M+H]2) of yeast histone H2B is 14113.6056 Da.
Using a 12 Tesla Bruker Daltonics mESI-FTICR-MS with
ultrahigh mass accuracy and resolution, exact mass measurement
of the protein was performed to validate sample preparation of
histone H2B following isolation from yeast nuclei and RP-HPLC
purification. The experimental monoisotopic mass of one of the
major peaks (peak 2) was at 14113.6028 Da, extremely close to the
theoretical value (mass error,1 ppm) (Figure 1A). Patterns of
PTMs of yeast histone H2B were also mapped by exact mass
measurement. The PTM site(s) on each form was further identified
and characterized based on exact masses and sequence informa-
tion from MS and MS/MS experiments. Relative abundances of
modified forms were obtained by integrating the four most
abundant isotopic peaks in three different charge states of MS
spectra and taking their sum (Table 1).
With a mass of 14141.6352 Da, the second strongest peak (peak
4) exactly matched the theoretical monoisotopic mass of yeast
histone H2B with two methyl marks (mass error,1 ppm). To
identify the modification site(s), the precursor ion corresponding to
the modified protein (m/z 1415.9 Da, 10+ charge state) was
isolated for top-down experiments using mESI-FTICR-MS with
ECD (Figure 1B, upper panel). Inspection of the c and z fragment
ions derived from the ECD MS/MS spectrum revealed +28 Da
mass shifts of c37 to c49 ions, indicating that lysine 37 is
dimethylated (Figure 1B, lower panel). As indicated in Table 1,
the relative abundance of dimethylated lysine 37 on histone H2B is
over 25.7% in all yeast protein isoforms. Other PTMs (e.g., sites of
acetylation and methylation) could be identified based on ECD
MS/MS experiments. However, with the exception of N-terminal
acetylation at serine 1 (data not shown), which has previously been
identified [29,43,44], additional PTMs could not be conclusively
The finding that lysine 37 of histone H2B is dimethylated is in
agreement with recently published MS results from a study
surveying for sites of lysine propionylation and butylyration .
However, very little is known about this lysine residue. Physically,
lysine 37 of histone H2B is located between the DNA gyres of the
nucleosome structure (Figure 1C). A previous study surveying the
role of the N-terminal domain of histone H2B in transcription on a
genome-wide level demonstrated that residues 30–37 of histone
H2B are necessary and sufficient for the repression of a subset of
genes in the budding yeast genome, and subsequently termed this
region the H2B repression (HBR) domain . This study posited
a model by which the changes in gene expression that are observed
upon deletion of the HBR could be due to elimination of yet to be
identified PTMs that function in repression, and specifically
suggest lysine 37 as a potential site of methylation .
To validate the finding that H2B is dimethylated on lysine 37 in
budding yeast, we first raised an antibody specific for this modified
state in rabbit. Western blot analysis of acid-extracted wild-type
histones using crude serum compared to pre-immune serum
demonstrated that this mark exists in vivo (data not shown). To further
corroborate this finding and characterize this novel mark, a-
H2BK37me2 antibody was affinity purified from crude serum and
Histone H2B Lysine 37 Is Dimethylated
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peptide competition analysis was completed using acid-extracted
wild-type H2B, H2B K37A, and H2B K123R mutant histone
samples. Where affinity purified a-H2BK37me2 antibody shows a
clear signal in histone samples containing wild-type H2B, mutation
of lysine 37 to a non-modifiable alanine (K37A) abrogates this
signal (Figure 2A, No peptide controls: left column, upper panels).
Mutant H2B harboring a K123R mutation was used as a control
to demonstrate specificity of this antibody for lysine 37. As a
further measure of control, we showed that H2BK37me2 was not
detectable in Western blot analysis using IgG purified from pre-
immune serum (Figure 2A, lower panels). The affinity purified
antibody is specific for dimethylation of lysine 37, as pre-
incubation of the a-H2BK37me2 antibody with a dimethylated
H2BK37 peptide resulted in a loss of signal in all three histone
samples, but preincubation with an unmodified H2BK37 peptide
did not alter reactivity (Figure 2A, middle and right columns,
upper panels). Altogether, these data support the in vivo existence
of dimethylation of histone H2B on lysine 37 and the generation of
an antibody that is capable of specifically recognizing this
Given that mutation of lysine 123 of histone H2B results in a
loss of H2B monoubiquitylation at this site as well as a loss of
methylation of histone H3 on lysines 4 and 79 [47,48,49,50,51],
we sought to determine whether crosstalk existed between histone
H2B lysine 37 methylation and other known sites of histone
methylation in budding yeast. Western blot analysis, using acid-
Table 1. Yeast histone H2B patterns of PTMs.
Yeast H2B PTM Relative Abundance* (%)
**PTM sites cannot be assigned.
Figure 1. Top-down mass spectrometry (MS) analysis reveals
histone H2B is dimethylated at lysine 37. (A) Top-down mESI-
FTICR-MS analysis of yeast histone H2B. Shown is a mass spectrum of
H2B revealing multiply modified forms of this histone, as indicated by
peaks numbered 1–9. Each peak was analyzed by top-down mESI-FTICR-
MS/MS analysis and modifications identified are denoted in the legend.
Asterisks indicate PTMs that were not assigned. 100 scans per spectrum
were acquired in the ICR cell with a resolution of 580,000 at m/z 400 Da.
(B) Top-down mESI-FTICR-MS/MS analysis of peak 4. ECD MS/MS
spectrum of histone H2B with two methyl marks (precursor: m/z
1415.9 Da, 10+ charge state) reveals lysine 37 is dimethylated. N-
terminal (c ions) and C-terminal (z ions) fragment ions are assigned and
shown in the upper panel. Lower panel denotes the ions in the
sequence. Unassigned ions are either internal fragment ions or
electronic noise. 100 scans per spectrum were acquired in the ICR cell
with a resolution of 580,000 at m/z 400 Da. (C) Lysine 37 of H2B is
located within the DNA gyres in the nucleosomal structure. Histones
H2A, H2B, H3 and H4 are shaded green, yellow, red, and blue,
respectively. The DNA backbone is colored gray. The yellow arrow
points to the location of lysine 37 of histone H2B. The nucleosomal
representation was generated using open-source PyMOL software
(PyMOL 0.99rev10, DeLan Scientific LCC) with structural data taken from
 (PDB file 1kx5).
Histone H2B Lysine 37 Is Dimethylated
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extracted histones from wild-type H2B and H2B K37A mutant
strains, showed that the loss of H3K37 methylation did not disrupt
H3K4, H3K36 or H3K79 methylation (Figure 2B). In contrast,
and as a control, the H2B K123R mutant resulted in a loss of both
H3K4 and H3K79 methylation, in agreement with previously
published results (Figure 2B, and ). Finally, the H2B K123R
mutation does not disrupt H2BK37 methylation (Figure 2B).
Together, these results suggest that dimethylation of H2BK37 is
neither affected by H2B K123 ubiquitylation nor affects the ability
of additional lysine residues to be methylated.
Elucidating the enzymes that place and remove H2BK37
We next sought to identify the putative histone methyltransfer-
ase responsible for placing this mark. To this end, a candidate
screen in which acid-extracted histones from individual deletion
strains from the Yeast Knockout Collection (Open Biosystems)
were analyzed by Western blot analysis using our a-H2BK37me2
antibody (Figure 3A). Included in the list of candidates were: the
budding yeast SET-domain containing proteins; the histone lysine
methyltransferase Dot1; known non-histone lysine methyltransfer-
ases; known yeast arginine methyltransferases (specific for both
histone and non-histone substrates); and putative methyltransfer-
ases (Table 2). The SET domain is the catalytic domain of all
identified histone lysine methyltransferases to date, with the
exception of Dot1 . To date, there are 12 proteins in budding
yeast that harbor a SET domain (including Set1 through Set7,
Rkm1 through Rkm3, and Ctm1) . Of these proteins, only
Set1 and Set2 have been demonstrated to function as histone
lysine methyltransferases, and are specific for histone H3 lysine
residues 4 and 36, respectively [54,55,56,57]. Methylation of
histone H3 at lysine 79 is catalyzed by Dot1, which is structurally
unrelated to the other identified methyltransferases, as it lacks a
SET domain altogether [58,59]. In addition to histone lysine
methyltransferases, budding yeast enzymes from the SET domain
family that are capable of methylating non-histone substrates on
lysine residues (namely, Ctm1, Rkm1, Rkm2, and Rkm3;
[60,61,62,63]) were also tested in this screen. As arginine
methylation is also known to occur in budding yeast, it is possible
that enzymes responsible for such modification on arginine
residues could demonstrate substrate promiscuity, and thus the
known arginine methyltransferases Hmt1, Rmt2, and Hsl7
[64,65,66] were also included in this screen. Finally, a number
of annotated proteins (of both known and unknown function)
predicted to function as methyltransferases based on structural
predictions were also screened for activity toward histone H2B
lysine 37, including the following: Trm12, Mtq1, Ylr137w,
Ynl092w, Mni1, Ybr271w, Tae1, Ymr209c, Ylr063w, Ybr141c,
Crg1, Yjr129c, and See1 [65,67,68,69,70,71].
We predicted that deletion of the responsible histone methyl-
transferase would result in a loss of signal in Western blot analysis
using the a-H2BK37me2 antibody, as is observed in a parallel
manner with Western blot analysis of samples derived from strains
harboring individual deletions of the other known histone
methyltransferases and the antibodies specific for their respective
substrates. Unfortunately, all candidates screened to date (Table 2)
did not give insight into the identity of the responsible methyltrans-
ferase. A loss of H2BK37me2 signal by Western blot analysis was
not detected upon deletion of the individual candidates, as was
observed for the control H2B K37R and H2B K37A mutants
compared to their isogenic strain expressing wild-type H2B
(Figure 3A, bottom). This could be due functional redundancy
amongst methyltransferases, which would be masked by single gene
deletions. This, however, seems unlikely, as histone methyltransfer-
Figure 2. a-H2BK37me2 antibody is specific for dimethylated lysine 37 on histone H2B. (A) A polyclonal antibody was purified from
antiserum raised by immunizing rabbits with the peptide SKARKme2ETYS-C, where me2 is dimethyl lysine. Peptide competition assay demonstrates
specificity of purified a-H2BK37me2 antibody for dimethyl lysine 37 of histone H2B. Western blot analysis was completed using acid-extracted
histones from strains harboring wild-type Flag-H2B (YKG001), Flag-H2B K37A (YKG007), and Flag-H2B K123R (YKG002), demonstrating that
dimethylation of lysine 37 on histone H2B occurs in vivo, as the antibody is able to recognize this modification in wild-type and H2B K123R-derived
histone samples, but not histones extracted from the Flag-H2B strain harboring a K37A mutation (No peptide controls: left column, upper panels).
Preincubation of the purified antibody with H2K37me2 peptide resulted in a loss of the ,15 kDa band in all three histone samples, whereas
preincubation with unmodified H2BK37 peptide did not alter the reactivity (middle and right columns, upper panels). H2BK37me2 signal was not
detectable in Western blot analysis using IgG purified from pre-immune serum (lower panels). All blots were stripped and reprobed with an a-H2B
antibody to demonstrate equal loading. (B) Western blot analysis using modification specific antibodies indicates that mutation of lysine 37 on
histone H2B does not affect methylation at other known sites of methylation in budding yeast, including histone H3 lysines 4, 36, and 79. A H2B
K123R mutation abrogates methylation at H3K4 and H3K79, in agreement with previously published results , but does not affect H2BK37
Histone H2B Lysine 37 Is Dimethylated
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ases are typically highly specific for both the lysine residue that they
target as well as the degree to which they can methylate their
respective substrate [72,73]. Alternatively, another class of yet to be
identified histone methyltransferases or a methyltransferase that is
essential for viability could facilitate placement of this mark, in
which case a candidate screen of non-essential ORFs would fail to
reveal the responsible enzyme and rather an unbiased approach
would have to be employed to identify the catalytic enzyme.
Recently, the JmjC domain has been identified as the catalytic
domain of a family of histone demethylases [74,75]. There are five
JmjC-domain-containing proteins in budding yeast: Jhd1, Rph1,
Gis1, Jhd2, and Ecm5 . Jhd1, Rph1, and Jhd2 have all been
demonstrated to possess histone demethylase activity, with
specificity for H3K36me2/1, H3K36me3/2, and H3K4me3/2,
respectively [75,77,78,79,80,81]. We also tried a candidate
approach using deletion analysis of the five JmjC-domain-
containing proteins to identify a putative demethylase for this
mark. Again, acid-extracted histones were analyzed by Western
blot analysis using the a-H2BK37me2 antibody, with wild-type
H2B and H2B K37A mutant histones serving as controls
(Figure 3B). We anticipated that deletion of the putative
demethylase would result in an increase in the total H2BK37me2,
Figure 3. Candidate approach by Western blot analysis does not reveal the methyltransferase and demethylase responsible for
H2B lysine 37 methylation. (A) Following validation of correct deletion of the ORF of interest and replacement with kanMX by genomic PCR (data
not shown), histones were acid-extracted from candidates from the Yeast Knockout Collection (Open Biosystems), and putative histone
methyltransferase activity was tested by Western blot analysis using the purified a-H2BK37me2 antibody. A Coomassie-stained gel illustrating a
representative purification of histones is shown in upper panel, and representative Western blots results from the candidate screen are shown below.
The blots were first probed with the a-H2BK37me2 antibody (upper) and then striped and reprobed with an a-H2B antibody (lower) to demonstrate
equal loading. Histones derived from strains harboring wild-type Flag-H2B (YKG001) and Flag-H2B K37R (YKG006) or K37A (YKG007) were loaded on
all gels to demonstrate loss-of-signal upon mutation of lysine 37, thereby serving as a control for antibody specificity. The presence of a Flag-tag on
histone H2B results in the slight shift in electrophoretic mobility observed in the control strains, as compared to untagged H2B species in the
candidate deletion strains. Deletion of candidate genes did not reveal a putative H2BK37me2 histone methyltransferase by Western blot analysis. (B)
Histones were acid-extracted from the five JmjC-domain-containing protein deletions in Saccharomyces cerevisiae, and putative histone demethylase
activity was analyzed by Western blot analysis using the purified a-H2BK37me2 antibody. Shown are Western blot results from the candidate screen,
in which the blots were first probed with the a-H2BK37me2 antibody (upper) and then striped and reprobed with an a-H2B antibody (lower) to
demonstrate equal loading. Again, histones derived from strains harboring wild-type Flag-H2B (YKG001) and Flag-H2B K37A (YKG007) were used as a
control for antibody specificity, and the presence of a Flag-tag on histone H2B results in the slight shift in electrophoretic mobility observed in the
control strains, as compared to untagged H2B species in the candidate deletion strains. Deletion of each individual candidate did not result in an
enhanced signal, suggesting that none of these candidates function as the histone demethylase for H2BK37me2.
Histone H2B Lysine 37 Is Dimethylated
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but deletion of the individual JmjC-domain-containing proteins
did not show global changes in the level of H2BK37me2. This was
not entirely surprising, as individual deletion of demethylases such
as Jhd1 or Rph1 fails to show global changes in the levels of their
target substrates [77,79]. Collectively, both the methyltransferase
and demethylase enzymes specifically responsible for placing and
removing dimethyl marks on H2BK37 remain to be identified.
Mutation of H2BK37 leads to no overt cellular phenotype
In parallel to identifying enzymes that catalyzetheplacementand
removal of this methylation event, we sought to definethe biological
function of this mark. To this end, a number of phenotypic assays
were completed using a series of strains harboring wild-type H2B,
H2B K37A, H2B K37R, or H2B K123R mutant histones (in most
cases, except where specifically noted, the H2B K123R mutant
strain was included as a positive control). General growth at various
temperatures and on various types of complete media was assessed,
but both the H2B K37R and H2B K37A strains failed to show
differential growth as compared to the isogenic wild-type strain.
This was in contrast to the H2B K123R strain, which exhibited a
slow growth phenotype at all of the temperatures and various
medias assessed (data not shown). Examination of growth under
Table 2. Candidates screened for putative H2BK37me2 histone methyltransferase activity.
Candidate Annotated SGD description(s)
CRG1 Putative S-adenosylmethionine-dependent methyltransferase; mediates cantharidin resistance
CTM1 Cytochrome c lysine methyltransferase; trimethylates residue 72 of apo-cytochrome c (Cyc1p) in the cytosol; not required for normal respiratory
DOT1Nucleosomal histone H3-Lys79 methylase; methylation is required for telomeric silencing, meiotic checkpoint control, and DNA damage response
HMT1 Nuclear SAM-dependent mono- and asymmetric arginine dimethylating methyltransferase that modifies hnRNPs, including Npl3p and Hrp1p,
affecting their activity and nuclear export; methylates U1 snRNP protein Snp1p and ribosomal protein Rps2p
HSL7Protein arginine N-methyltransferase that exhibits septin and Hsl1p-dependent bud neck localization and periodic Hsl1p-dependent
phosphorylation; required along with Hsl1p for bud neck recruitment, phosphorylation, and degradation of Swe1p
MNI1 AdoMet-dependent methyltransferase involved in a novel 3-methylhistidine modification of ribosomal protein Rpl3p; seven beta-strand MTase
family member; null mutant exhibits a weak vacuolar protein sorting defect and caspofungin resistance
MTQ1 S-adenosylmethionine-dependent methyltransferase; methylates translational release factor Mrf1p
RKM1 SET-domain lysine-N-methyltransferase, catalyzes the formation of dimethyllysine residues on the large ribsomal subunit protein L23a (RPL23A and
RKM2 Ribosomal protein lysine methyltransferase, responsible for trimethylation of the lysine residue at position 3 of Rpl12Ap and Rpl12Bp
RKM3Ribosomal lysine methyltransferase specific for monomethylation of Rpl42ap and Rpl42bp (lysine 40); nuclear SET domain containing protein
RMT2 Arginine N5 methyltransferase; methylates ribosomal protein Rpl12 (L12) on Arg67
SEE1 Probable lysine methyltransferase involved in the dimethylation of eEF1A (Tef1p/Tef2p); sequence similarity to S-adenosylmethionine-dependent
methyltransferases of the seven beta-strand family; role in vesicular transport
SET1Histone methyltransferase, subunit of the COMPASS (Set1C) complex which methylates histone H3 on lysine 4; required in transcriptional silencing
near telomeres and at the silent mating type loci; contains a SET domain
SET2Histone methyltransferase with a role in transcriptional elongation, methylates a lysine residue of histone H3; associates with the C-terminal domain
of Rpo21p; histone methylation activity is regulated by phosphorylation status of Rpo21p
SET3Defining member of the SET3 histone deacetylase complex which is a meiosis-specific repressor of sporulation genes; necessary for efficient
transcription by RNAPII; one of two yeast proteins that contains both SET and PHD domains
SET4 Protein of unknown function, contains a SET domain
SET5Zinc-finger protein of unknown function, contains one canonical and two unusual fingers in unusual arrangements; deletion enhances replication of
positive-strand RNA virus
SET6SET domain protein of unknown function; deletion heterozygote is sensitive to compounds that target ergosterol biosynthesis, may be involved in
SET7/RKM4Ribosomal lysine methyltransferase specific for monomethylation of Rpl42ap and Rpl42bp (lysine 55); nuclear SET-domain containing protein
TAE1AdoMet-dependent proline methyltransferase; catalyzes the dimethylation of ribosomal proteins Rpl12 and Rps25 at N-terminal proline residues;
has a role in protein synthesis; fusion protein localizes to the cytoplasm
TRM12S-adenosylmethionine-dependent methyltransferase of the seven beta-strand family; required for wybutosine formation in phenylalanine-accepting
YBR141C Putative S-adenosylmethionine-dependent methyltransferase; GFP-fusion protein localizes to the nucleolus
YBR271WPutative S-adenosylmethionine-dependent methyltransferase of the seven beta-strand family; GFP-fusion protein localizes to the cytoplasm;
predicted to be involved in ribosome biogenesis
YJR129CPutative protein of unknown function; predicted S-adenosylmethionine-dependent methyltransferase of the seven beta-strand family; GFP-fusion
protein localizes to the cytoplasm
YLR063WPutative S-adenosylmethionine-dependent methyltransferase; GFP-fusion protein localizes to the cytoplasm
YLR137W Putative S-adenosylmethionine-dependent methyltransferase
YMR209C Putative S-adenosylmethionine-dependent methyltransferase
YNL092W Putative S-adenosylmethionine-dependent methyltransferase of the seven beta-strand family
Histone H2B Lysine 37 Is Dimethylated
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anaerobic conditions, as well as following release from stationary
phase, also failed to show a difference between the K37 mutant and
wild-type histone strains (data not shown). Mutation of lysine 37 to
either arginine or alanine also did not affect the ability of yeast cells
to properly sporulate as compared to an isogenic strain expressing
wild-type H2B (data not shown). We next posited that H2BK37me2
might be cell-cycle regulated, and therefore synchronized wild-type
cells in G2/M with nocodazole and harvested cells at defined points
along the cell cycle following nocodazole release. Western blot
analysisofthesecellsatvarious stagesofthe cell cyclefailedtoreveal
an enrichment and/or depletion of H2BK37me2 at any defined cell
cycle stage (as compared to known cell-cycle regulated marks such
as phosphorylation of histone H3 on serine 10 and threonine 45,
which occur during mitosis and S-phase, respectively [82,83]) (data
We also performed assays to screen for phenotypes related to
DNA replication and repair. To that end, wild-type H2B and the
H2B K37 mutant strains were spotted on media containing the
agents hydroxyurea (HU, an agent which blocks replication
leading to replication fork collapse) or methyl methanesulfonate
(MMS, an alkylating agent that causes DNA lesions and ultimately
DNA strand breaks). However lysine 37 mutations in histone H2B
did not alter cellular growth compared to an isogenic wild-type
parent on media containing 0.05% MMS (data not shown) or
100 mM HU (Figure 4A), where cells bearing a H2B K123R
mutation were sensitive to both. Moreover, to assess the ability of
lysine 37 mutant strains to carry out replication, plasmid
maintenance assays were completed, where the ability of a cell
to replicate a reporter plasmid containing a single origin of
replication and a selectable marker is measured . Mutation of
lysine 37 on histone H2B to either arginine or alanine did not
affect the ability of yeast strains to faithfully replicate the reporter
plasmid as compared to isogenic wild-type cells (data not shown).
Taken together, the results from these screening assays suggest that
Figure 4. Phenotypic analysis of strains harboring H2B K37R/A mutations. (A) Phenotypic spotting assays indicate that cells harboring
mutations at lysine 37 in histone H2B to arginine (YKG006) or alanine (YKG007) do not show sensitivity to YPD media containing 100 mM
hydroxyurea (HU; a DNA damaging agent that leads to replication fork collapse), as is observed in an H2B K123R mutant strain (YKG002) , but
rather grow similarly to yeast containing wild-type H2B (YKG001). (B) Telomeric silencing assay demonstrates that reporter strains harboring H2B
K37R and H2B K37A mutations (YKG028 and YKG029, respectively) exhibit normal silencing like that observed for reporter strains expressing wild-
type H2B (YKG027), but not strains that express an H2B K123R mutation (YZS274) or are deleted for SIR2 (YZS275), which have known defects in
telomeric silencing . Growth on SC-HIS serves as a plating control, as all strains express H2B-containing plasmids carrying a HIS3 auxotrophic
marker. (C) Introduction of H2B K37R or K37A mutations (YKG033 and YKG034, respectively) into strains containing a temperature-sensitive allele of
SPT16 (spt16–197) does not affect cellular growth at the semi- and non-permissive temperatures (32uC and 34uC, respectively), as cells grow at a
similar rate to those harboring wild-type H2B (YKG031). Introduction of an H2B K123R mutation (YKG032) exacerbates growth in the spt16–197
background at the semi-permissive temperature, in agreement with previously published results . The isogenic parental strain Y131 expressing
wild-type SPT16 grows phenotypically normal at the non-permissive temperature for the spt16–197 strain.
Histone H2B Lysine 37 Is Dimethylated
PLoS ONE | www.plosone.org7 January 2011 | Volume 6 | Issue 1 | e16244
histone H2B lysine 37 does not have a significant role in DNA
replication or repair.
As methylation of both lysine 4 and 79 of histone H3 have been
previously demonstrated to be necessary for proper telomeric
silencing [48,51,58,85], we next sought to determine if mutation of
lysine 37 would also result in loss of telomeric silencing. To that
end, H2B K37R and H2B K37A mutations were introduced into
a histone H2A-H2B shuffle strain engineered to assay for defects in
telomeric silencing, where expression of URA3, located at the left-
end telomere of chromosome VII (URA3-TEL), is used as a
readout for proper silencing . If telomeric silencing properly
occurs, the URA3 gene is silenced, and cells grow normally on
media containing 5-fluoroortic (5-FOA), an agent that is toxic only
to cells that express URA3. Introduction of H2B K37R and H2B
K37A mutations in URA3-TEL strains results in comparable
growth on 5-FOA-containing media to the isogenic URA3-TEL
strain expressing wild-type H2B (Figure 4B). This is in direct
contrast to cells expressing H2B K123R or cells deleted of SIR2,
which both fail to grow on media containing 5-FOA due to
improper silencing of the URA3 gene (Figure 4B), in agreement
with previously published results . Together, these data suggest
that lysine 37 of histone H2B is not essential for gene silencing in
Several assays to test for transcriptional defects were also
employed. Spotting assays on media containing 6-azauracil (6-AU)
or mycophenolic acid (MPA), which both deplete intracellular
levels of nucleotides leading to altered cellular viability when
combined with mutations that affect transcriptional elongation,
were completed. In both cases, strains with mutant H2B K37R or
K37A grew comparably to cells with wild-type H2B, where an
H2B K123R mutation resulted in a slow growth phenotype (data
not shown). Transcription induction was also assessed by measuring
the induction of GAL1 and GAL10 transcripts in wild-type H2B
and H2B K37 mutant strains. However, gene expression analysis
by reverse-transcription quantitative PCR (RT-qPCR) revealed
that mutation of lysine 37 on histone H2B does not alter induction
of either GAL1 or GAL10, as compared to wild-type cells,
supporting that this residue does not significantly contribute to
transcriptional induction of these genes. Finally, we were curious
to see how mutation in H2B K37 would behave in combination
with mutant SPT16, a member of the FACT histone chaperone
complex that promotes transcription elongation [86,87,88,89].
Previous results have shown that the growth phenotype observed
upon inactivation of SPT16 is enhanced and suppressed by
mutations in lysine residues 4 and 36 of histone H3, respectively,
suggesting that FACT function is dependent upon H3K4
methylation and is opposed by H3K36 methylation . We
therefore introduced lysine 37 mutations into a histone H2A-H2B
shuffle-strain containing a temperature-sensitive allele of SPT16
(spt16-197), and cellular growth was assessed at range of
temperatures. However, this analysis failed to reveal a combina-
torial effect between mutation of lysine 37 on histone H2B and
inactivation of SPT16, as H2B K37R/A spt16-197 double mutant
strains grew comparably to isogenic spt16-197 containing wild-
type H2B (Figure 4C). This is in direct opposition to a H2B
K123R spt16-197 double mutant strain, which demonstrated a
synthetic effect upon inactivation of the FACT allele. These data
together substantiate that methylation of lysine 37 does not appear
to play a major role in transcription, as mutation of this histone
residue results in no overt phenotype in all transcription-based
assays completed to date.
Finally, given that Parra et al presented a model by which gene
expression changes observed upon deletion of the HBR domain
could be a consequence of eliminating a modified form of this
domain , we sought to address whether methylation lysine 37
of H2B in particular functions in transcriptional regulation on a
genomic level. To this end, gene expression changes upon
mutation of lysine 37 were assessed by microarray analysis.
Comparison of gene expression changes in cells expressing wild-
type H2B versus a H2B K37A mutant revealed that lysine 37 does
not appear to function significantly in genome-wide transcription
regulation, as only 20 genes showed differential gene expression
using a cutoff of a two-fold difference in expression (where two
genes were upregulated (Table S1) and 18 genes were downreg-
ulated (Table S2) in a H2B K37A mutant relative to the isogenic
wild-type strain). RT-qPCR analysis was able to recapitulate the
microarray results of genes shown to be up- or downregulated in a
H2B K37A mutant strain relative to the isogenic parent strain
(Figure S1 and data not shown), thus validating the microarray
results. However, the lack of a significant number of genes showing
differential expression between wild-type and H2B K37A mutant
strains indicates overall that H2BK37me2 alone does not play a
major role in regulation of transcription on a genome-wide level in
Methylation of H2BK37 is conserved in higher eukaryotes
Sequence alignment of histone H2B from Saccharomyces cerevisiae
against multiple species reveals that lysine 37 is conserved along
evolution, despite lower sequence similarity of surrounding amino
acid residues (Figure 5A). To determine if we could detect the
presence of methylated lysine 37 in higher eukaryotes, we
performed Western blot analysis comparing oligonucleosomes
isolated from chicken erythrocyte nuclei and core histones from
HeLa cell nuclei to yeast histones. Western blot analysis using the
a-H2BK37me2 antibody revealed that this mark is indeed
conserved in higher eukaryotes (Figure 5B), as a comparable
species is observed in both the chicken and human histone samples
as to histones extracted from yeast harboring wild-type, but K37A
mutant, H2B. The presence of a discernable signal in samples
derived from higher eukaryotic species suggests that, despite the
lack of an obvious cellular phenotype in yeast to date, this mark is
likely to be biologically important since it was retained during
To date, only six lysines residues have been identified and
characterized as sites of histone methylation (namely, lysines 4, 9,
27, 36 and 79 of histone H3, and lysine 20 of histone H4) .
Recently, a comprehensive study employing LC-ESI MS/MS to
identify PTMs of histones associated with each phase of the yeast
cell cycle revealed that lysine 111 of histone H2B is also a site of
histone methylation , in agreement with additional previously
publishes results . Phenotypic analyses have revealed that
mutation of this lysine residue confers sensitivity to the DNA-
damaging agent MMS and renders telomeric silencing defective
, supporting the importance of this lysine residue and its
methylation in chromatin function. Trimethylation of lysine 64 on
histone H3 has been shown to be enriched at pericentric
heterochromatin in human and mice samples, and is dynamically
regulated during early development, supporting a function for this
modification in the reprogramming process involved in germ cell
development . Additionally, methylation of histone H3 at
lysine 122 has recently been reported in mice , and genome-
wide localization patterns of methylation of lysine 5 on histone
H2B have been reported in humans [41,42]. However, the latter
two sites of histone methylation are largely uncharacterized at
present. It is likely that additional sites of histone lysine
Histone H2B Lysine 37 Is Dimethylated
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methylation remain to be identified, and that much remains to be
discovered with regard to the complexity of histone methylation
and how this PTM in particular contributes to the histone code
and cellular function. That additional sites of modifications critical
for normal cellular function remain to be identified thereby
necessitates further investigations directed toward elucidating a
complete atlas of histone PTMs.
In this manuscript, we reveal the utility of top-down MS analysis
of histone H2B is dimethylated in budding yeast. We also provide
evidence that this modification is evolutionarily conserved. Much
remains to be determined with respect to the placement and
removal, regulation and biological function(s) of this mark. For
example, a candidate screen employing all known lysine methyl-
transferases in budding yeast (both specific for histone and non-
histone substrates) has revealed that the methyltransferase respon-
sible for placement of this mark doesnot fall into the category of one
of the previously identified methyltransferases. This suggests that
either multiple methyltransferases function redundantly to methyl-
ate H2BK37, or that a novel class of methyltransferases capable of
placing this mark exists. Using a similar candidate approach to
screen known histone demethylases for specificity for this mark also
failed to expose a demethylase specific for this mark. Given that
deletion of known JmjC-domain-containing demethylases does not
result in global changes in the levels of histone modifications that
they have been shown to target [77,79], it is likely that identification
of the demethylase responsible for removal of lysine 37 methylation
cannot be revealed by deletion analysis. Alternatively, multiple
demethylases could be functionally redundant in the removal of this
mark, thus making deletion analysis a less ideal assay for
identification of the enzyme responsible for erasing methylation at
H2BK37. It is also possible that a family of enzymes other than
JmjC-domain-containing histone demethylases exists that is respon-
sible for removal of this mark, as well as others (for example, a
demethylase specific for H3K79 remains to be identified), or that
there simply is not a demethylase for this mark.
Saccharomyces cerevisiae provides an advantageous genetic system
for studying the functional consequence of loss of a specific amino
acid residue (a feat that cannot be readily accomplished in higher
eukaryotes ), thus prompting us to carry out phenotypic analysis
in budding yeast. As MS analysis has revealed that H2BK37
dimethylation is a relatively abundant modification, we reasoned
that mutation of lysine 37 would likely cause pleiotropic effects.
However, all assays screened to date have failed to reveal a
functional phenotype when lysine 37 is changed to either arginine
or alanine. It is possible that this modification could function
redundantly with another histone modification, in which case
combinatorial mutations would be necessary to reveal the
functional significance of these marks. Thus, further studies will
have to be completed to determine the biological significance of
this mark in chromatin.
Materials and Methods
Yeast strains and DNA constructs
A list of yeast strains used for these studies can be found in
Table 3. Plasmids harboring wild-type or mutant histone H2B
were introduced into yeast H2A-H2B shuffle strains using
standard transformation  and shuffling  protocols.
The plasmids pZS145 (HTA1-Flag-HTB1 CEN HIS3) and
pZS146 (HTA1-Flag-htb1 (K123R) CEN HIS3) were isolated from
the strains YZS276 and YZS277, respectively, obtained from Z.W.
Sun . The plasmids pKG1 (HTA1-Flag-htb1 (K37R) CEN HIS3)
and pKG2 (HTA1-Flag-htb1 (K37A) CEN HIS3) were derived from
site-directed mutagenesis of pZS145  using the QuikChange II
Site-Directed Mutagenesis kit (Stratagene). The accuracy of all
constructs was verified by DNA sequencing.
Histone acid extraction
Histones were extracted from yeast nuclei using a standard acid
extraction method . Briefly, 250 mL cultures were grown at
30uC to an OD600approximately equal to 1.5. Cells were collected
Figure 5. Methylation of lysine 37 of histone H2B is conserved. (A) Multiple sequence alignment of histone H2B from different species
reveals that budding yeast histone H2B lysine 37 is conserved from yeast to humans. Sequence alignment was completed using ClustalX . NCBI
accession numbers are as follows: Saccharomyces cerevisiae: NP_010510.1; Schizosaccharomyces pombe: NP_588181.1; Drosophila melanogaster:
NP_724342.1; Caenorhabditis elegans: NP_507031.1; Xenopus laevis: NP_001086753.1; Mus musculus: NP_783594.1; Gallus gallus: CAA40537.1; Bos
taurus: DAA31692.1; Homo sapiens: NP_733759.1. Asterisk (*) denotes position of conserved lysine residue. (B) Increasing amounts of
oligonucleosomes purified from chicken erythrocyte nuclei and mononucleosomes isolated from HeLa cell nuclei were run against histones
extracted from yeast strains harboring wild-type Flag-H2B (YKG001), Flag-H2B K37A (YKG007), and wild-type H2B (untagged) (BY4742), as shown by
Coomassie brilliant blue (CBB) staining of histone proteins electrophoresed on 15% SDS-polyacrylamide gels (lower panel). An equivalent loading of
histone substrate was used for Western blot analysis using purified a-H2BK37me2 antibody (upper panel). Similar signals are detected for chicken-
and human-derived histone substrates to that observed for yeast harboring wild-type H2B (either tagged or untagged), but not yeast H2B with an
K37A mutation, thus demonstrating that dimethylation of histone H2B lysine 37 is conserved among species.
Histone H2B Lysine 37 Is Dimethylated
PLoS ONE | www.plosone.org9 January 2011 | Volume 6 | Issue 1 | e16244
Table 3. Yeast Strains.
Strain Genotype Reference/Source
FY406MATa (hta1-htb1)D::LEU2 (hta2-htb2)D::TRP1 leu2D1 ura3-52 lys2D1
lys2-128d his3D200 trp1D63 ,pSAB6 (HTA1-HTB1 URA3).
YKG001MATa (hta1-htb1)D::LEU2 (hta2-htb2)D::TRP1 leu2D1 ura3-52 lys2D1
lys2-128d his3D200 trp1D63 ,pZS145 (HTA1-Flag-HTB1 CEN HIS3).
YKG002MATa (hta1-htb1)D::LEU2 (hta2-htb2)D::TRP1 leu2D1 ura3-52 lys2D1
lys2-128d his3D200 trp1D63 ,pZS146 (HTA1-Flag-htb1 (K123R) CEN HIS3).
YKG006 MATa (hta1-htb1)D::LEU2 (hta2-htb2)D::TRP1 leu2D1 ura3-52 lys2D1
lys2-128d his3D200 trp1D63 ,pKG1 (HTA1-Flag-htb1 (K37R) CEN HIS3).
YKG007 MATa (hta1-htb1)D::LEU2 (hta2-htb2)D::TRP1 leu2D1 ura3-52 lys2D1
lys2-128d his3D200 trp1D63 ,pKG2 (HTA1-Flag-htb1 (K37A) CEN HIS3).
YZS272MATa ura3-1 leu2-3,112 ade2-1 trp1-1 his3-11,15 can1-100 (hta1-htb1)D::LEU2
(hta2-htb2)D ,pZS144 (HTA1-Flag-HTB1 CEN TRP1). URA3-TEL
YKG027MATa ura3-1 leu2-3,112 ade2-1 trp1-1 his3-11,15 can1-100 (hta1-htb1)D::LEU2
(hta2-htb2)D ,pZS145 (HTA1-Flag-HTB1 CEN HIS3). URA3-TEL
YKG028 MATa ura3-1 leu2-3,112 ade2-1 trp1-1 his3-11,15 can1-100 (hta1-htb1)D::LEU2
(hta2-htb2)D ,pKG1 (HTA1-Flag-htb1 (K37R) CEN HIS3). URA3-TEL
YKG029MATa ura3-1 leu2-3,112 ade2-1 trp1-1 his3-11,15 can1-100 (hta1-htb1)D::LEU2
(hta2-htb2)D ,pKG2 (HTA1-Flag-htb1 (K37A) CEN HIS3). URA3-TEL
YZS274MATa ura3-1 leu2-3,112 ade2-1 trp1-1 his3-11,15 can1-100 (hta1-htb1)D::LEU2
(hta2-htb2)D ,pZS146 (HTA1-Flag-htb1 (K123R) CEN HIS3). URA3-TEL
YZS275 MATa ura3-1 leu2-3,112 ade2-1 trp1-1 his3-11,15 can1-100 (hta1-htb1)D::LEU2
(hta2-htb2)D ,pZS145 (HTA1-Flag-HTB1 CEN HIS3). URA3-TEL sir2D::TRP1
YZS276 MATa (hta1-htb1)D::LEU2 (hta2-htb2)D leu2-3,112 his3-11,15 trp1-1 ura3-1
ade2-1 can 1-100 ,pZS145 (HTA1-Flag-HTB1 CEN HIS3).
YZS277MATa (hta1-htb1)D::LEU2 (hta2-htb2)D leu2-3,112 his3-11,15 trp1-1 ura3-1
ade2-1 can 1-100 ,pZS146 (HTA1-Flag-htb1 (K123R) CEN HIS3).
Y131MATa (hta1-htb1)D::LEU2 (hta2-htb2)D leu2-3,112 trp1-1 ura3-1
ade2-1 can1-100 his3-11,15 ,pRS426 (HTA1-HTB1 URA3 2 mm).
YCH278MATa (hta1-htb1)D::LEU2 (hta2-htb2)D leu2-3,112 trp1-1 ura3-1 ade2-1 can1-100
his3-11,15 spt16::kanMX ,pRS426 (HTA-HTB URA3 2 mm). ,pBM46-spt16-197.
YKG031MATa (hta1-htb1)D::LEU2 (hta2-htb2)D leu2-3,112 trp1-1 ura3-1 ade2-1 can1-100
his3-11,15 spt16::kanMX ,pZS145 (HTA1-Flag-HTB1 CEN HIS3). ,pBM46-spt16-197.
YKG032MATa (hta1-htb1)D::LEU2 (hta2-htb2)D leu2-3,112 trp1-1 ura3-1 ade2-1 can1-100
his3-11,15 spt16::kanMX ,pZS146 (HTA1-Flag-htb1 (K123R) CEN HIS3). ,pBM46-spt16-197.
YKG033 MATa (hta1-htb1)D::LEU2 (hta2-htb2)D leu2-3,112 trp1-1 ura3-1 ade2-1 can1-100
his3-11,15 spt16::kanMX ,pKG1 (HTA1-Flag-htb1 (K37R) CEN HIS3). ,pBM46-spt16-197.
YKG034MATa (hta1-htb1)D::LEU2 (hta2-htb2)D leu2-3,112 trp1-1 ura3-1 ade2-1 can1-100
his3-11,15 spt16::kanMX ,pKG2 (HTA1-Flag-htb1 (K37A) CEN HIS3). ,pBM46-spt16-197.
YMP001MATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 rad5-535 HTZ1::myc/7xHisThis study
YBC63MATa lys2-128 leu2D ura3-52 trp1D63 his3D200
YBC1236MATa lys2-128 leu2D ura3-52 trp1D63 his3D200 set1D::HIS3MX6
DY2390 (W303)MATa ade2 can1 his3 leu2 lys2 trp1 ura3
YAR005MATa ade2 can1 his3 leu2 lys2 trp1 ura3 rph1D::kanMXThis study
YAR007MATa ade2 can1 his3 leu2 lys2 trp1 ura3 jhd1D::kanMXThis study
YAR009MATa ade2 can1 his3 leu2 lys2 trp1 ura3 gis1D::kanMXThis study
YAR011MATa ade2 can1 his3 leu2 lys2 trp1 ura3 jhd2D::kanMXThis study
YAR013MATa ade2 can1 his3 leu2 lys2 trp1 ura3 ecm5D::kanMXThis study
YNL037MATa ade2 can1 his3 leu2 lys2 trp1 ura3 dot1D::kanMXThis study
BY4741 MATa his3D1 leu2D0 met15D0 ura3D0Open Biosystems
BY4742MATa his3D1 leu2D0 lys2D0 ura3D0Open Biosystems
The following deletion strains used for candidate screening are from the Yeast Knockout Collection in the BY4741 background (Open Biosystems): crg1D::kanMX,
ctm1D::kanMX, htm1D::kanMX, mni1D::kanMX, mtq1D::kanMX, rkm1D::kanMX, rkm2D::kanMX, rkm3D::kanMX, rmt2D::kanMX, see1D::kanMX, set2D::kanMX, set3D::kanMX,
set4D::kanMX, set5D::kanMX, set6D::kanMX, set7D::kanMX, tae1D::kanMX, trm12D::kanMX, ybr141cD::kanMX, ybr271wD::kanMX, yjr129cD::kanMX, ylr063wD::kanMX,
ylr137wD::kanMX, ymr209cD::kanMX, ynl092wD::kanMX. The following deletion strain used for candidate screening is from the Yeast Knockout Collection in the BY4742
background (Open Biosystems): hsl7D::kanMX.
Histone H2B Lysine 37 Is Dimethylated
PLoS ONE | www.plosone.org 10January 2011 | Volume 6 | Issue 1 | e16244
by centrifugation at 27006g for 5 minutes, washed once with
sterile water, and collected again by centrifugation. Cells were
resuspended in 7.5 mL Solution 1 [0.1 mM Tris-Cl (pH 9.4),
10 mM DTT], and then incubated at 30uC for 15 minutes with
shaking at 100 rpm. Cells were collected by centrifugation at
27006g for 5 minutes, washed in 15 mL Solution 2 [1.2 M
sorbitol, 20 mM HEPES-OH (pH 7.4)], and pelleted again. Cells
were resuspended in 15 mL Solution 2 containing Zymolyase 20T
at a final concentration of 0.2 mg/mL, and were then incubated at
30uC with shaking at 100 rpm until spheroplasting was greater
than 90% (as determined by measuring the OD600 of 10 mL
sample in 1 mL 1% SDS; typically 45–50 minutes), at which point
15 mL ice-cold Solution 3 [1.2 M sorbitol, 20 mM PIPES-OH
(pH 6.8), 1 mM MgCl2] was added. Cells were pelleted again at
13006g for 5 minutes 4uC. Pellets were resuspended in 7.5 mL
ice-cold Solution 4 [250 mM sucrose, 60 mM KCl, 14 mM NaCl,
5 mM MgCl2, 1 mM CaCl2, 15 mM MES (pH 6.6), 1 mM
PMSF, 0.8% TritonX-100], incubated on ice for 20 minutes, and
spun at 17006g for 5 minutes at 4uC. Nuclei isolation in Solution
4 was completed a total of three times. Nuclei were washed three
times in 12.5 mL Wash 1 [10 mM Tris-Cl (pH 8.0), 0.5% NP-40,
75 mM NaCl, 1 mM PMSF] for 15 minutes on ice for the first two
washes, and 5 minutes on ice for the third wash, followed by two
washes in 12.5 mL Wash 2 [10 mM Tris-Cl (pH 8.0), 400 mM
NaCl, 1 mM PMSF] for 10 minutes on ice for the first wash, and
centrifuged immediately following the second resuspension.
Histones were extracted in 1.5 mL 0.4 N H2SO4with incubation
on ice for 30 minutes, with occasional vortexing. Debris was
pelleted by centrifugation at 10,0006g. Histone proteins were
precipitated from the supernatent by addition of 100% TCA to a
final concentration of 20% with incubation on ice for 30 minutes.
Histone proteins were pelleted at 15,0006g. Pellets were washed
once with acetone containing 1% HCl, and once with acetone.
After being air-dried, histone proteins were resuspended in 300 mL
10 mM Tris-Cl (pH 8.0).
Reverse-phase HPLC purification of histone proteins
Following sulfuric acid extraction, histones derived from the
strain YMP001 were subject to RP-HPLC isolation. Gradient
conditions used for histone isolation were adapted from conditions
previously described . Briefly, proteins from sulfuric acid
extracts were injected onto a Zorbex C-18 column with a pore size
of 3.5 mm using an Agilent 1100 series RP-HPLC (Agilent, Santa
Clara CA). The column was washed and prepared using the
following method: 5–35% Acetonitrile (CH3CN) with 0.1%
Trifluoroacetic acid (TFA) for 5 minutes followed by 35%
CH3CN/0.1% TFA for 10 minutes. Histones were separated
using the following gradient: 35%–60% CH3CN/0.1%TFA for 30
minutes . Protein elution was monitored by UV absorption at
220 nm. Fractions containing histone H2B were determined by
Western blot analysis using an a-H2B antibody (Active Motif, Cat.
using a hybrid Qe-Fourier Transform Ion Cyclotron Resonance -
Mass Spectrometer, equipped with a 12.0 Tesla actively shielded
magnet (Apex Qe-FTICR-MS, 12.0 T AS, Bruker Daltonics,
Billerica, MA, USA), and an Apollo II microelectrospray (mESI)
source. The voltages on mESI spray capillary, spray shield, capillary
exit, deflector, ion funnel and skimmer were set at +4.2 kV,
+3.6 kV, +340 V, +310 V, +185 V and +25 V, respectively. The
temperature of the mESI source was maintained at 120uC.
Desolvation was carried out using a nebulization gas flow (2.0
Acquisition of MS spectra was performed
bar) and a countercurrent drying gas flow (4.0 L/s). Histone H2B
samples were prepared by resuspending lyophilized RP-HPLC
fractions containing H2B in a mixture of acetonitrile/water/acetic
acid (49.0:49.0:2.0 v/v/v) at a concentration of 0.1–0.2 mg/mL,
directlyinfused with asyringe pump (Harvard Apparatus,Holliston,
MA, USA) and a 100-mL syringe (Hamilton, Reno, NV, USA), and
electrosprayed at an infusion flow rate of 90 mL/hr. Before transfer,
ion packets were accumulated inside the collision cell for a duration
of 0.5–1.0 seconds. 100 MS scans per spectrum were acquired in
the ICR cell with a resolution of 580,000 at m/z 400 Da.
FTICR-ECD MS/MS method was
employed to fragment histone H2B. Precursor ions were isolated
with a quadrupole (Q1) and subjected to ICR cell directly. The
isolation window width was 2.0 Da. Low energy electrons were
generated by the heated hollow dispenser cathode with a bias
voltage of 22.5 V. ECD lens voltage was set at +15.0 V. The
electrons, produced by the hollow dispenser cathode (operated at
1.7 A), were pulsed into the ICR cell with a length of 3.0 ms,
which led to fragmentation of the ions that were already trapped in
the ICR cell. To maximize the ion population before irradiation,
the ICR cell was filled with 1–5 iterations of ion accumulation
from the external collision cell . 100 MS/MS scans per
spectrum were acquired with a resolution of 580,000 at m/z
a-H2BK37me2 antibody production and antibody affinity
A synthetic peptide containing H2B sequence from 33 to 41, in
which lysine 37 was dimethylated, was conjugated to keyhole limpet
hemocyanin via a C-terminal cysteine in the peptide and was used
to immunize rabbits (Pocono Rabbit Farm and Laboratory Inc.).
The a-H2BK37me2 antibody was affinity purified from serum.
Briefly, equilibrated Affigel-10 (BIORAD) was incubated with the
peptide SKARKme2ETYS-C (where me2 is dimethyl lysine) in
PBS for 2 hr at 4uC. Unbound peptide was removed, and the
peptide-boundresinwasblocked with 0.2 Methanolamine(pH 8.0)
for 2 hr at 4uC. After washingwith1 M NaCl and PBS, the blocked
peptide-bound resin was incubated with serum for 3 hr at room
temperature with rotation. The flow-through was collected, and the
resin was washed with 0.5 M NaCl followed by PBS. Antibody was
eluted with 0.1 M glycine (pH 3.0) at one-half column volume/
fraction, and 1/10 (v/v) 1 M Tris-Cl (pH 8.0) was added to
neutralize the pH. Purity of antibody fractions were analyzed on
12% SDS-polyacrylamide gels followed by Coomassie-staining,
allowing for pooling of peak antibody fractions.
IgG was purified from pre-immune serum. Briefly, Protein A
beads (GE Healthcare) pre-equilibrated with Tris-salt buffer
[100 mM Tris-Cl (pH 7.95), 135 mM NaCl] were incubated with
pre-immune serum for 2 hr at room temperature with rotation.
The flow-through was collected, and the column was washed with
Tris-salt buffer, followed by 10 mM Tris-Cl (pH 7.95). IgG was
eluted with 0.1 M glycine (pH 3.0) at one-half column volume/
fraction, and 1/10 (v/v) 1 M Tris-Cl (pH 8.0) was added to
neutralize the pH. Purity of IgG fractions were analyzed on 12%
SDS-polyacrylamide gels followed by Coomassie-staining, allow-
ing for pooling of peak IgG fractions.
Western blot analysis and peptide competition assay
Histone samples were run on 15% SDS-polyacrylamide gels,
which were transferred to PVDF membranes (Pall Corporation)
using a semi-dry apparatus (Hoefer) and Towbin buffer.
Membranes were blotted using standard techniques, and probed
with the antibodies at the following dilutions: a-H3 (Active Motif,
Cat. No. 39163; 1:5000), a-H2BK37me2 (PRF&L, generated in
Histone H2B Lysine 37 Is Dimethylated
PLoS ONE | www.plosone.org 11January 2011 | Volume 6 | Issue 1 | e16244
this study; 1:2000), a-H3K4me3 (Active Motif, Cat. No. 39159;
1:10,000), a-H3K36me3 (Abcam, Cat. No. ab9050; 1:2000), a-
H3K79me3 (Abcam, Cat. No. ab2621; 1:2000), or a-H2B (Active
Motif, Cat. No. 39237; 1:10,000).
For peptide competition assays to demonstrate the specificity of
purified a-H2BK37me2 antibody for H2BK37me2, purified IgG
or a-H2BK37me2 antibody was pre-incubated with no peptide, a
H2K37 peptide (SKARKETYS-C) or a H2K37me2 peptide
(SKARKme2ETYS-C, where me2 is dimethyl lysine) at a final
peptide concentration of 0.1 mg/mL for 1.5 hr at room temper-
ature prior to incubation of PVDF membranes with primary
antibody followed by standard Western blot analysis.
RNA isolation, microarray and RT-qPCR mRNA analyses
Yeast cultures were grown at 30uC in YPD simultaneously in
triplicate to an OD600of approximately 1.0. Ten OD600units of
cells were collected, washed once with water, and pellets were flash
frozen in liquid nitrogen. Total RNA was isolated using the hot
acidic phenol-chloroform method . Briefly, cell pellets were
resuspended in 400 mL TES solution [10 mM Tris-Cl (pH 7.5),
10 mM EDTA, 0.5% SDS], to which 400 mL acidic phenol-
chloroform (Ambion) was added. Samples were vortexed vigor-
ously, incubated at 65uC for 1 hour with occasional vortexing, and
then placed on ice for 5 min. The aqueous layer was back-
extracted once with acidic phenol-chloroform and once with
chloroform. Following back-extraction with chloroform, RNA was
precipitated using a standard ethanol precipitation protocol, and
resuspended in RNase-free water. RNA was cleaned up using an
RNeasy Mini Kit (QIAGEN), and RNA quality was determined
using an Agilent Bioanalyzer.
Biotinylated-cRNA was generated using the MessageAmpTMII-
Biotin Enhanced Kit (Ambion) and was hybridized to Yeast
Genome 2.0 arrays (Affymetrix), following manufacturer’s proto-
col. Briefly, hybridizations were completed for 16 hr at 45uC at
60 rpm in a GeneChip Hybridization Oven 640. Arrays were
washed and stained using the GeneChip Fluidics Station 450, and
were scanned with the GeneChip Scanner 3000 7G Plus Scanner
with Autoloader. Microarray hybridization and analysis was
completed at the University of North Carolina at Chapel Hill
Functional Genomics Core Facility.
For real-time quantitative PCR (qPCR) gene expression
analysis, following treatment of isolated RNA with DNA-free
(Ambion) and RNA clean-up using an RNeasy Mini Kit
(QIAGEN), first-strand cDNA was generated from total RNA
using the Improm-II Reverse Transcription System (Promega).
PCR reactions using 1/20 of total cDNA as template were
completed using primers specific to the indicated genes. Primers
used are as follows: ACT1 Forward: GAGGTTGCTGCTTTG
GTTATTGA, Reverse: ACCGGCTTTACACATACCAGAAC.
AQR1 59 Forward: GCTTTGAGGCAGTTGGAAAA, 59 Re-
verse: CACCGCTAACTGTGGGAGAT; AQR1 39 Forward:
TGGGTTCCTTCTTCACAGGT, 39 Reverse: CTCTGCGT
CTTGTGGAATCA. FMP43 59 Forward: ATTAGCGACGG-
CACTGATTT, 59 Reverse: CAGTGCAACCCAGGAAAAA;
FMP43 39 Forward: GGATACGGAACGGTGATTCT, 39 Re-
verse: TCATCGATGTGGATGCAGTT. PCR reactions were
carried out in triplicate for qPCR analysis using SYBR GreenER
qPCR master mix (Invitrogen) and the Applied Biosystems
7900HT Fast Real-Time PCR system.
All microarray data is MIAME compliant. Raw data generated
from these studies have been deposited into the MIAME
compliant database Gene Expression Omnibus (NCBI, http://
www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO
series accession number GSE24380.
Phenotypic spotting assays
To assay for growth in phenotypic spotting assays, five-fold
serial dilutions of saturated overnight yeast cultures grown in YPD
medium, or in synthetic complete medium supplemented as
appropriate for plasmid selection, were plated onto appropriate
media at a starting OD600of 0.5. Growth on plates was imaged
after 2–4 days of incubation at 30uC, unless temperature is
results of gene expression changes upon mutation of
H2B lysine 37. Yeast cells harboring wild-type H2B (YKG001)
or a H2B K37A mutation (YKG007) were grown to mid-log
phase, and RNA samples were isolated. The expression of genes
identified as up- or downregulated upon mutation of lysine 37 by
microarray analysis was verified by RT-quantitative real time
PCR analysis (RT-qPCR). Representative RT-qPCR analysis is
shown for AQR1 and FMP43, which were up- and downregulated,
respectively, in yeast cells harboring the H2B K37A mutation
relative to wild-type H2B according to microarray analysis. Gene
expression was normalized against actin (ACT1).
RT-qPCR analysis recapitulates microarray
H2B K37A mutant cells.
Genes that are upregulated at least two-fold in
fold in H2B K37A mutant cells.
Genes that are downregulated at least two-
We are grateful for the generous sharing of yeast strains by Zu-Wen Sun
(Vanderbilt University), Mary Ann Osley (University of New Mexico),
David Stillman (University of Utah), Bradley Cairns (University of Utah),
and Ashley Rivenbark and Nick Laribee (University of North Carolina at
Chapel Hill). We are thankful to Krzysztof Krajewski (University of North
Carolina at Chapel Hill) for assistance with peptide analysis, to Yi Zhang
(University of North Carolina at Chapel Hill) for generously providing
chicken oligonucleosomes and HeLa mononucleosome substrates, and to
Mike Vernon (University of North Carolina at Chapel Hill) for assistance
with microarray hybridization. We acknowledge members of the labs of
Jean Cook (University of North Carolina at Chapel Hill) and Scott Briggs
(Purdue University) for assisting with DNA plasmid maintenance and
anaerobic growth phenotypic assays, respectively.
Conceived and designed the experiments: KEG BDS. Performed the
experiments: KEG LZ MAP. Analyzed the data: KEG LZ MAP XC BDS.
Contributed reagents/materials/analysis tools: KEG LZ MAP XC BDS.
Wrote the paper: KEG BDS.
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Histone H2B Lysine 37 Is Dimethylated
PLoS ONE | www.plosone.org14 January 2011 | Volume 6 | Issue 1 | e16244