Molecular Cell
Review
When Signaling Kinases Meet Histones
and Histone Modifiers in the Nucleus
Sung Hee Baek1,*
1Department of Biological Sciences, Creative Research Initiative Center for Chromatin Dynamics, Seoul National University,
Seoul 151-742, South Korea
*Correspondence: sbaek@snu.ac.kr
DOI 10.1016/j.molcel.2011.03.022
Signaling pathways involve cascades of protein phosphorylation and ultimately affect regulation of transcrip-
tion in the nucleus. However, most of the kinases in these pathways have not been generally considered to
directly modulate transcription thus far. Here, recent significant progress in the field elucidating direct modi-
fications of histones and histone modifiers by upstream kinases is summarized, and future directions are dis-
cussed.
Introduction
Histones are subject to diverse posttranslational modifications
including phosphorylation, acetylation, methylation, ubiquitina-
tion, SUMOylation, ADP ribosylation, deimination, and proline
isomerization (Bhaumik et al., 2007; Campos and Reinberg,
2009; Chi et al., 2010; Jenuwein and Allis, 2001; Kouzarides,
2007; Shilatifard, 2006). These diverse histone modifications
can modulate the activity of transcription factors, nucleosome re-
modelers, histone chaperones, and other histone modifiers by
altering the chromatin state for either activation or repression
(Berger, 2007; Ho and Crabtree, 2010; Kubicek and Jenuwein,
2004; Lee et al., 2010; Ruthenburg et al., 2007; Suganuma and
Workman, 2008; Weake and Workman, 2010). The identification
of the enzymes that directly modify histones has been
intense focus over the last 15 years. Most of these
modifications are dynamic, and the corresponding enzymes in-
clude kinases/phosphatases, acetyltransferases/deacetylases,
methyltransferases/demethylases, ubiquitin ligases/deubiquiti-
nating enzymes, and SUMO ligases/deSUMOylating enzymes
(Berger, 2007; Bhaumik et al., 2007; Klose and Zhang, 2007;
Kouzarides, 2007; Mosammaparast and Shi, 2010; Sims and
Reinberg, 2008; Weake and Workman, 2008; Yang and Seto,
2008). Among the enzymes that modify histones, kinases mainly
depend on the activation of specific upstream signaling path-
ways leading to cascades of protein phosphorylation and regula-
tion of transcription in the nucleus. Recently, significant progress
in understanding the roles of this particular type of modification
has been made through the elucidation of mechanisms by which
gene expression is directly affected through specific kinase-
dependent phosphorylation of histones (Bungard et al., 2010;
Cerutti and Casas-Mollano, 2009; Dawson et al., 2009; Metzger
et al., 2010; Pérez-Cadahı́a et al., 2009). Furthermore, accumu-
lating evidence suggests that some kinases that modify histones
can also modify nonhistone substrates including chromatin
remodeling factors and transcription factors (Cha et al., 2005;
Fischle et al., 2003; Huang and Chen, 2005; Huang et al., 2007;
Lee et al., 2010; Lehtinen et al., 2006).
This review will discuss some of the recent headway that has
been made in understanding the role of upstream kinase-depen-
dent phosphorylation of the histones and nonhistone proteins in
274 Molecular Cell 42, May 6, 2011 ª2011 Elsevier Inc.
mammals. The classical histone H3 mitotic kinases functioning in
cell cycle, recently identified kinases and phosphorylation of
histone variants will be discussed briefly in this review; however,
other excellent reviews on histone H3 (Cheung et al., 2000;
Nowak and Corces, 2004) or histone variants (Elsaesser et al.,
2010; Hake and Allis, 2006; Sarma and Reinberg, 2005; Talbert
and Henikoff, 2010) are available for further details. Considering
the recent rapid progress in this field and its significant influence
in understanding the function of phosphorylation within the
nucleus, it is a great time to summarize current status of the field
and discuss future directions.
Upstream Kinases and Histone Substrates Modified
during Transcription and Apoptosis: H2BS14/S36
and H3T6/S10/T11/S28/Y41/T45
In comparison to other histone modifications, phosphorylation of
histones requires specific activation of upstream signaling path-
ways (Hans and Dimitrov, 2001; Nowak and Corces, 2000, 2004;
Vermeulen et al., 2009; Wyrick and Parra, 2009). Although these
upstream kinases in the signaling pathways regulate transcrip-
tion via activation of phosphorylation-dependent signal trans-
duction cascades, the central kinases are not considered to
directly modify histones or histone modifiers in the nucleus
due to their major cytoplasmic localization. However, accumu-
lating evidence has shown that the upstream kinases have
a more profound effect on gene expression through direct phos-
phorylation of the chromatin. On the basis of a genome-wide
chromatin immunoprecipitation (ChIP)-on-chip analysis of the
yeast mitogen-activated protein kinases (MAPKs), it was pre-
dicted that the activated signal transduction kinases may
frequently occupy target genes by binding to transcription
factors and coregulators in the nucleus (Edmunds and Mahade-
van, 2004; Pokholok et al., 2006). Recently, direct colocalization
of estrogen receptor a (ERa) and extracellular signal-regulated
kinase 2 (ERK2) for regulation of target genes across the genome
has been reported (Madak-Erdogan et al., 2011).
The upstream kinases that directly modify histones are
summarized (Table 1) (Anest et al., 2003; Bungard et al., 2010;
Cerutti and Casas-Mollano, 2009; Cheung et al., 2003; Choi
et al., 2005; Dawson et al., 2009; Dyson et al., 2005; Hurd
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Review

Table 1. Kinases that Modify Histones and/or Histone Modifiers

Substrates Modified
Kinases that Modify Histones Transcription Factors and Functions/Downstream
and/or Histone Modifiers Histones Histone Modifiers Consequences
Haspin H3T3 n/a Chromatin Condensation (H)
VRK1 H3T3, H3S10 CREB-S133, p53-T18 Chromatin Condensation (H)/Txn Activation (TF)
PKCa H3T6 RORa-S35 Txn Activation (H)/Repression (TF)
PKCb H3T6 p65-S276/S536 Txn Activation (H/TF)
PIM1 H3S10 p21-T145 Txn Activation (H)/Apoptosis (TF)
IKKa H3S10 CBP-S1382 Txn Activation (H/TF)
Rsk2 H3S10 IkBa-S32, p53-S15 Txn Activation (H/TF)
PKB/Akt H3S10 EZH2-S21, p300-S1834 Txn Activation (H/TF)
Aurora B H3S10, H3S28 MYBBP1A-S1303 Chromatin Condensation (H/TF)
MSK1/2 H3S10, H3S28 CREB-S133, p65-S276, STAT3-S727 Txn Activation (H/TF)
JNK1 H3S28 SIRT1-S27/S47/T530 Chromatin Condensation (H)/Txn Repression (TF)
MLTKa H3S28 n/a Chromatin Condensation (H)
PRK1 H3T11 HDAC5-T292 Txn Activation (H/TF)
Chk1 H3T11 BLM-S646, p53-S313/S314/T377/S378 Txn Activation (H/TF)
Dlk/ZIP H3T11 Par-T155 Chromatin Condensation (H)/Apoptosis (TF)
PKCd H3T45 TBLR1-S123, p53-S46 Apoptosis (H)/Txn Activation (TF)
MST1 H2BS14 FOXO3-S207 Apoptosis (H/TF)
AMPK H2BS36 ChREBP-S568, p53-S15 Txn Activation (H)/ Txn Repression (TF)
JAK2 H3Y41 STAT5-Y694 Txn Activation (H/TF)
Abl n/a SRC3-Y1357 Txn Activation (TF)
BMK1 n/a PML-S403/T409 Txn Repression (TF)
CaMK n/a HDAC5-S259/S498, CBP-S301 Txn Activation (TF)
S6K1 n/a Mdm2-S163, SREBP1/2 Txn Activation (TF)
SIK1 n/a HDAC5-S259, TORC2-S171 Txn Activation (TF)
PKC, protein kinase C; PIM, proto-oncogene serine/threonine-protein kinase; IKK, IkB kinase; Rsk, p90 ribosomal S6 protein kinase; PKB, protein
kinase B; MSK, mitogen- and stress-activated protein kinase; JNK, c-Jun N-terminal protein kinase; MLTK, mixed lineage kinase-like mitogen-acti-
vated protein triple kinase; PRK, protein-kinase-C-related kinase; Chk, checkpoint kinase; AMPK, AMP-activated protein kinase; MST, mammalian
sterile twenty kinase; JAK, Janus kinase; Txn, transcription; H, histones; TF, transcription factors.

et al., 2009; Metzger et al., 2008, 2010; Pérez-Cadahı́a et al.,
2009; Sassone-Corsi et al., 1999; Shimada et al., 2008; Wang
et al., 2002; Yamamoto et al., 2003; Zhong et al., 2001; Zippo
et al., 2007). Some of these histone kinases also act on transcrip-
tion factors and histone modifiers. Therefore, the unexpected
recent findings shed light on this exciting link between upstream
signaling kinases and direct phosphorylation of histones and/or
histone modifiers.
H2B Ser Phosphorylation
There are two serine phosphorylation sites in the tail of histone
H2B in mammals (Figure 1A). Two of the kinases that can
phosphorylate H2B are MST1 and AMPK, which are responsible
for S14 and S36 phosphorylation, respectively. MST1 is
a member of the sterile 20-like superfamily that is regarded as
upstream regulator of MAPK pathways with functions in cellular
morphogenesis and apoptosis (Dan et al., 2001). Upon cleavage
by caspase-3, MST1 directly phosphorylates H2BS14, suggest-
ing that this phosphorylation might be associated with the onset
of apoptosis (Cheung et al., 2003; Ling et al., 2008). Considering
that both DNA fragmentation and chromatin condensation are
hallmark features of apoptosis, it is worth emphasizing that
H2BS14 phosphorylation is selectively associated with the
apoptotic chromatin condensation. In contrast to the potentially
deadly phosphorylation on S14, the phosphorylation of S36 by
AMPK is connected to a prosurvival pathway. AMPK is activated
by high AMP levels under conditions of energetic stress, and
activated AMPK triggers a program of metabolic adaptation to
conserve ATP and maintain cellular viability (Cantó and Auwerx,
2010). AMPK has been shown to activate transcription through
direct association with chromatin both in stress-responsive
target gene promoters and transcribed regions by direct phos-
phorylation of H2BS36, revealing the first AMPK-dependent
substrate in the nucleus (Bungard et al., 2010). Phosphorylation
of H2BS36 by AMPK may fine-tune specific transcriptional
responses and be essential for survival in response to metabolic
stress.
Given that using ChIP provided evidence for direct association
of AMPK and H2BS36 phosphorylation both in promoters and
transcribed regions, it can be applied in search of other upstream
kinases that target histones and/or nonhistones. Indeed, histone
H2B tail contains a variety of potential phosphorylation sites,
including four serine and two threonine residues, compared to

Molecular Cell 42, May 6, 2011 ª2011 Elsevier Inc. 275


histone H2A which has two serine and one threonine residues or
histone H4 which contains only one serine residue as a targeting
site. Considering the potential diversity of phosphorylation sites
on histone H2B, it seems that future work could yield new
functional connections between this histone and other signaling
pathways.
H3 Ser/Thr Phosphorylation
There are six serine/threonine phosphorylation sites (H3T3/T6/
S10/T11/S28/T45) modified by serine/threonine kinases and
one site (H3Y41) phosphorylated by tyrosine kinase in the tail
of histone H3 in mammals (Figure 1B). H3T6 phosphorylation
was newly identified to be modified by PKCb in prostate cancer
cells (Metzger et al., 2010). During androgen receptor (AR)-acti-
vated gene expression, lysine-specific histone demethylase 1
(LSD1) removes mono- and dimethyl marks from H3K9 for tran-
scriptional activation. LSD1 functions as both corepressor and
coactivator by demethylating mono- and dimethyl marks from
H3K4 and H3K9, respectively, yet it was unclear how this dual
specificity might be regulated. PKCb-dependent H3T6 phos-
phorylation turned out to be the key step in preventing LSD1
from demethylating H3K4 and in promoting demethylation of
H3K9 exclusively during AR-dependent gene activation,
providing an intriguing example of crosstalk between the phos-
phorylation state of H3T6 and methylation of H3K4 and H3K9.
Perhaps this case provides potentially plausible explanation
why the same modification by different kinases or the different
modifications by the same kinase might be associated with
different outcomes. A more systemic approach searching for
the histone and nonhistone substrates of a particular kinase
would be valuable. In parallel, phosphorylation of H3T11 by
PRK1 establishes another novel chromatin mark during
AR-dependent transcriptional activation (Metzger et al., 2008).
Phosphorylation of H3T11 by PRK1 accelerates removal of
repressive methyl marks from H3K9 by the Jumonji C (JmjC)-
276 Molecular Cell 42, May 6, 2011 ª2011 Elsevier Inc.
Molecular Cell
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Figure 1. Kinases that Modify Histones H2B
and H3 and Residues Modified
(A) H2B phosphorylation functions in transcription
and apoptosis. MST1 and AMPK phosphorylate
H2BS14 and H2BS36 in mammals, respectively.
(B) H3 phosphorylation functions both in chro-
mosome condensation and in transcription.
Haspin, VRK1, Aurora B, and Dlk/ZIP are involved
in chromosome condensation in mitosis and
meiosis, whereas other kinases are involved in
transcriptional activation during interphase and
apoptosis in mammalian cells.
transcription or apoptosis occurs on
H3T45 in PKCd-dependent manner. This
modification on H3T45 is detected from
untreated neutrophils at basal level, but
robustly induced in the latter phases of
apoptosis, suggesting a potential link
between H3T45 phosphorylation and
apoptosis (Hurd et al., 2009). Consis-
tently, the induction of H3T45 phosphory-
lation is paralleled with caspase-3
activation, and it is increased in apoptotic
domain containing protein, JMJD2C, leading to the transcrip-
tional activation of AR-dependent target genes. Although both
PKCb-dependent H3T6 phosphorylation and PRK1-dependent
H3T11 phosphorylation are novel histone phosphorylation
marks, an open question remains as to why their mode of action
appears to be limited to AR-dependent transcriptional units.
Thus, it will be a challenging task to conduct ChIP-sequencing
or ChIP-chip analysis using good quality of antibodies to search
for other transcriptional units that are regulated by PKCb and
PRK1 as well as other H3T6 and H3T11 histone kinases depend-
ing on cellular context. On the other hand, it will be of interest to
find other nonhistone substrates phosphorylated by PKCb or
PRK1. For this, mass spectrometry would be a very useful tool
to probe the modified resides.
Chk1 has the same histone H3 substrate specificity as PRK1,
H3T11 phosphorylation, but the upstream activation signal and
mechanism of action do not overlap with PRK1. Under unper-
turbed conditions, Chk1 associates with chromatin and phos-
phorylates H3T11. Phosphorylation of H3T11 results in
enhanced recruitment of GCN5 histone acetyltransferase to
targets, perhaps best exemplified by the cyclin B1 and cdk1
promoters, leading to the acetylation of H3K9 and H3K14
(Shimada et al., 2008). Upon DNA damage, Chk1 releases from
the promoters leading to dephosphorylation of H3T11, and the
decreased H3T11 phosphorylation leads to transcriptional
repression due to the impaired GCN5 recruitment to its target
promoters. Therefore, Chk1 functions as a histone kinase
responsible for the regulation of DNA damage-induced tran-
scriptional repression by inducing diminished histone acetyla-
tion. Future works will further clarify whether there is concomi-
tant recruitment of HDACs for transcriptional repression upon
release of GCN5 from target promoters. H3T11-specific phos-
phatases might exist for counteracting function of Chk1. Finally,
the remaining threonine phosphorylation of histone H3 during
Molecular Cell
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cells around the time of DNA nicking. Given that the position of
H3T45 lies in a structurally important region involved in nucleo-
some structure and stability (Hurd et al., 2009), it is highly
possible that H3T45 phosphorylation facilitates activation of
apoptotic program by inducing structural changes of nucleo-
some. Further biochemical studies will be needed to test this
idea.
Serine phosphorylation on histone H3 includes S10 and S28
residues. H3S10 and H3S28 phosphorylation have a dual role
both in interphase where chromatin needs to be decondensed
for transcriptional activation of genes, and in mitosis where chro-
matin condensation is necessary. As to the upstream kinases
responsible for H3S10 and H3S28 phosphorylation in inter-
phase, a variety of H3S10 kinases including PIM1, IKKa,
MSK1/2, PKB/Akt, and Rsk2, and for H3S28 phosphorylation
kinases including MSK1/2, JNK1, and MLTKa, have been
reported (Anest et al., 2003; Bungard et al., 2010; Cerutti and
Casas-Mollano, 2009; Cheung et al., 2003; Choi et al., 2005;
Dawson et al., 2009; Dyson et al., 2005; Hurd et al., 2009;
Metzger et al., 2008, 2010; Pérez-Cadahı́a et al., 2009;
Sassone-Corsi et al., 1999; Shimada et al., 2008; Wang et al.,
2002; Yamamoto et al., 2003; Zhong et al., 2001; Zippo et al.,
2007). Compared to the mitotic kinases, these kinases are
activated by upstream signals including cytokines, growth
factors, and ultraviolet (UV), and activated kinases directly target
histones in the nucleus. IKKa-dependent H3S10 phosphoryla-
tion is induced by cytokines and is critical for the activation of
NF-kB-directed gene expression (Yamamoto et al., 2003).
Growth factor-dependent stimulation leads to the formation of
a PIM1-MYC complex, and PIM1-dependent phosphorylation
of H3S10 is required for MYC-dependent transcriptional activa-
tion, leading to elevated oncogenic transformation ability (Zippo
et al., 2007). UV irradiation is also responsible for inducing
H3S28 phosphorylation by JNK1 (Zhong et al., 2001). Further-
more, JIL-1 histone H3S10 kinase reported in Drosophila is
interesting due to its distinct functions in interphase, although
there appears no mammalian counterpart. JIL-1 kinase activity
functions to mark euchromatic domains and counteract
heterochromatinization by Su(var)3-9-mediated histone H3K9
dimethylation and heterochromatin protein 1 (HP1) recruitment
(Zhang et al., 2006). Su(var)3-9 turns out to be a substrate phos-
phorylated by JIL-1 kinase, and the kinase activity of JIL-1 is
required for the maintenance of chromatin structure in
Drosophila (Boeke et al., 2010; Wang et al., 2001). Considering
the diverse functions and sites identified for H3 Ser/Thr phos-
phorylation thus far in interphase, it will be of particular interest
to understand the specific mechanism regulating the dynamic
phosphorylation and dephosphorylation of histones in
promoter-, cell- and signal-dependent manner, in addition to
understanding how these phosphorylation events can ultimately
results in different outcomes in different contexts.
Tyr Phosphorylation
The first tyrosine phosphorylation site in the tail of core histone
H3 identifies Y41 and the corresponding kinase turns out to be
JAK2 (Dawson et al., 2009). Given that JAK2 is a nonreceptor
tyrosine kinase that modulates diverse cellular process by
inducing cytoplasmic signaling cascades, it was striking to
discover the phosphorylation of histone H3Y41 by JAK2 in the
nucleus. JAK2 has a previously unrecognized nuclear pool in
hematopoietic cells (Dawson et al., 2009). Upon phosphorylation
of H3Y41 by JAK2, HP1a binding to the chromatin is significantly
decreased, leading to the transcriptional activation of JAK2-
regulated genes, including the hematopoietic oncogene lmo2.
HP1a has been shown to function as a potential tumor
suppressor. Therefore, it is conceivable that the consequences
of constitutive JAK2 activation (JAK2V617F) identified in human
myeloproliferative diseases that are exemplified by the
increased genetic instability are consistent with the reversal of
the HP1a functions. The JAK2-dependent displacement of
HP1a might be tightly regulated in normal cells, whereas in
malignancies, uncontrolled displacement of HP1a may override
its potential tumor suppressive functions. The HP1a recruitment
mechanism has been shown to be widely used in JAK2 signaling.
JAK2 turned out to regulate canonical JAK2-STAT target genes
as well as noncanonical target genes such as lmo2, confirming
the importance of this chromatin pathway in cancer (Rui et al.,
2010). Since JAK2 also phosphorylates STAT5 at Y694 as
a nonhistone substrate (Gouilleux et al., 1994), it will be exciting
to study how JAK2 signals pass through STAT5 and H3Y41 and
converge into the regulation of downstream target genes. Impor-
tantly, JAK2-dependent phosphorylation of STAT5 at Y694 is
necessary for STAT5 DNA binding activity and phosphorylated
STAT5 functions as a transcriptional activator. In parallel,
JAK2-dependent phosphorylation of H3Y41 results in the activa-
tion of JAK2-regulated genes, including lmo2, which lacked
a predicted STAT5 binding site. In both cases, the outcome is
transcriptional activation of target genes.
Williams-Beuren syndrome transcription factor (WSTF) has
been shown to phosphorylate Tyr142 of H2AX, and its intrinsic
tyrosine kinase activity is required for foci formation during the
DNA damage response in mammalian cells (Xiao et al., 2009).
As a counterpart to WSTF, a protein tyrosine phosphatase EYA
has been shown to promote DNA repair rather than apoptosis
by executing a damage signal-dependent dephosphorylation
of Tyr142 of H2AX (Cook et al., 2009). It will be of particular
interest to investigate the functional link between kinases and
phosphatases and their potential interplay for the regulation of
targets for DNA repair and apoptosis in the cellular context.
Upstream Kinases and Histone Substrates Modified
during Chromosome Condensation: H3T3/S10/T11/S28
Although recent studies on the specific upstream kinase-depen-
dent direct phosphorylation of histones allowed significant
progress in this field, phosphorylation of H3 initially gained
considerable interest when this modification was discovered to
be linked to chromosome condensation and segregation during
mitosis and meiosis (Cheung et al., 2000; Nowak and Corces,
2004). Phosphorylation of H3S10 is considered to be a crucial
event for the onset of mitosis, because this modification appears
early in the G2 phase within pericentromeric heterochromatin
and spreads coincident with mitotic chromosome condensation
by metaphase. Aurora B, Haspin, vaccinia-related kinase 1
(VRK1), and death-associated protein-like kinase (Dlk)/zipper-
interacting protein (ZIP) have been shown to play a role in chro-
mosome condensation during mitosis (Crosio et al., 2002; Dai
et al., 2005; Kang et al., 2007; Preuss et al., 2003), whereas
Molecular Cell 42, May 6, 2011 ª2011 Elsevier Inc. 277

most of the other kinases have been shown to regulate transcrip-
tion or apoptosis.
Aurora kinases are a highly conserved family of serine/threo-
nine kinases consisting of Aurora A, B, and C, and Aurora B is
a well-known primary mitotic H3S10 and S28 kinase (Carmena
et al., 2009; Crosio et al., 2002; Keen and Taylor, 2009; Kelly
and Funabiki, 2009; Vader and Lens, 2008). The Haspin
phosphorylates H3T3 during mitosis and meiosis and is required
for normal metaphase chromosome alignment (Dai et al., 2005;
Higgins, 2010). Recently, Haspin was shown to position Aurora
B at centromeres in order to regulate selected targets of Aurora
B during mitosis (Wang et al., 2010). Compared to Aurora B
having specificity on H3S10 and H3S28 phosphorylation, chro-
matin-localized VRK1 has been shown to be responsible for
H3T3 and H3S10 phosphorylation depending on the cell-cycle
phase (Kang et al., 2007). Indeed, VRK1 being a mitotic histone
H3 kinase is differentially expressed during cell cycle progres-
sion and its level peaks in the G2/M phase. Dlk/ZIP phosphory-
lates histone H3T11 rather than H3S10, which is characteristic of
mitotic chromosome, and this H3T11 phosphorylation is
discernable from prophase to early anaphase and is particularly
enriched at centromeres (Preuss et al., 2003). Therefore, Dlk/ZIP
has been shown to function as a centromere-specific histone
kinase for subsequent mitotic processes. Although H3T11 phos-
phorylation shows correlation with the level of chromosome
condensation, the functional consequence of H3T11 phosphor-
ylation during mitosis and meiosis is not clear and requires
further investigation.
The histone H3 family contains three main classes of histone
H3 genes: the canonical, replication-dependent histone H3,
the replication-independent histone variant H3.3, and the
centromeric H3 variant CENP-A (Elsaesser et al., 2010; Hake
and Allis, 2006; Sarma and Reinberg, 2005; Talbert and Henikoff,
2010). In contrast to H3S10 and H3S28, which are phosphory-
lated in prophase, H3.3S31 phosphorylation is observed only
in late prometaphase and metaphase stages of mitosis, suggest-
ing that H3.3S31 phosphorylation is a mitosis-specific event
(Hake et al., 2005). CENP-A substitutes for histone H3 in the
nucleosome core of centromeric chromatin at the inner plate of
the kinetochore, and it implicates an important function in
kinetochore assembly. Prevention of CENP-A phosphorylation
on S7 in prophase leads to chromosome misalignment during
mitosis as a result of a defect in kinetochore attachment to
microtubules (Kunitoku et al., 2003; Sullivan et al., 1994; Yoda
et al., 2000).
It can be postulated that the phosphorylation status of H3 is
dynamically regulated by opposing actions of specific kinases
and phosphatases during mitosis. Compared to the known
mitotic H3 kinases, finding the counteracting phosphatases is
an emerging area of investigation. It has been shown that Aurora
B binds to a type 1 protein phosphatase (PP1) for spatiotemporal
regulation of H3 phosphorylation during mitosis and is activated
by the PP1/PP2A inhibitor okadaic acid (Sugiyama et al., 2002).
In mammals, four isoforms of catalytic subunit of PP1 have
distinct subcellular localizations. For example, during mitosis,
PP1a is localized to the centrosome, while PP1g associates
with the microtubules of the mitotic spindle, and PP1d associ-
ates with chromosome. It will be challenging to search for novel
278 Molecular Cell 42, May 6, 2011 ª2011 Elsevier Inc.
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H3 phosphorylation sites in addition to T3, S10, T11, and S28
residues co-occupied with corresponding new kinases and
phosphatases in the future. Perhaps mass spectrometry and
ChIP sequencing approaches will be useful techniques along
with highly sensitive imaging techniques to address the issues.
Nonhistone Substrates Modified: Transcription Factors
and Histone Modifiers
As touched on above, the enzymes that modify histones can also
modify nonhistone proteins, including chromatin remodeling
factors and transcription factors/coregulators. Like histone
modification, it is important to determine whether there is and
to decipher any potential modification code of nonhistone
substrates in consideration of specific signaling pathways and
cellular contexts. Some kinases exemplified by PKCs, PIM1,
IKKa, Rsk2, PKB/Akt, MSK1/2, JNK1, PRK1, Chk1, Dlk/ZIP,
MST1, AMPK, and JAK2 that modify histones can also modify
nonhistone substrates (Cha et al., 2005; Fischle et al., 2003;
Huang and Chen, 2005; Huang et al., 2007; Lee et al., 2010;
Lehtinen et al., 2006). In these cases, we should be careful in
analyzing as the signal-dependent responses may be going
through unsuspected nonhistone substrates. It has been
suggested that there exist histone-like modification cassettes
or histone mimics as a distinct class of nonhistone targets that
might extend the principles of the proposed histone code to
the regulation of nonhistone proteins (Boosen et al., 2009;
Fischle et al., 2003; Huang et al., 2007; Kaur et al., 2010; Peng
et al., 2010; Takemori et al., 2009).
H3 Ser/Thr Kinases with Nonhistone Substrates
The Ser/Thr PKC family comprises about 2% of the kinases in
human. Although 12 isoforms of PKC exist in mammals,
increasing evidence supports individual and nonredundant roles
within the members of this family (Rosse et al., 2010). It is evident
in the case of different PKC family members targeting different
sites on histone H3: PKCa and PKCb phosphorylate H3T6,
whereas PKCd phosphorylates H3T45. Recently, orphan nuclear
receptor RORa turned out to be a nonhistone substrate phos-
phorylated by PKCa in the nucleus, and the consequence of
RORa phosphorylation is the inhibition of canonical Wnt/b-cate-
nin target genes in colon cancer (Lee et al., 2010). A significant
inverse correlation between reduction of RORa phosphorylation
and PKCa activation in human colorectal tissues provides the
clinical relevance of the findings. Another PKC isoform, PKCd
has a nonhistone substrate transducer b-like-related protein 1
(TBLR1) in addition to histone H3T45. TBLR1 phosphorylation
at S123 by PKCd is required for overcoming C-terminal binding
protein (CtBP)- and nuclear receptor corepressor (NCoR)-
dependent transcriptional repression checkpoint (Perissi et al.,
2008). Considering a large number of PKC family members
and the importance as key transducers in signaling networks, it
is conceivable that many more nonhistone targets as well as
new histone substrates could be uncovered in the future.
IKKa phosphorylates nonhistone substrate such as CREB
binding protein (CBP) at serine 1382 and 1386 as well as histone
H3S10, and phosphorylation of CBP by IKKa is responsible for
accelerating cell growth by switching the binding preference of
CBP from p53 to NF-kB (Huang et al., 2007). CBP is also phos-
phorylated by Ca2+/calmodulin-dependent protein kinase IV
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(CaMKIV) at S301 leading to the CREB/CBP-dependent tran-
scriptional activation in hippocampal neurons (Impey et al.,
2002). Although the transcriptional activation function of CBP
and p300 is overlapping, CaMKIV-mediated phosphorylation
appears to be CBP-specific. PKB/Akt is also responsible for
phosphorylating p300 at S1884, which enhances its enzymatic
activity (Huang and Chen, 2005).
In many cases, the functional consequence of phosphoryla-
tion of histone-modifying enzymes is the modulation of their
enzymatic activity either positively, such as CBP and p300, as
mentioned above, or negatively. In other cases, phosphorylation
of histone modifiers regulates protein stability functioning as
a ‘‘phosphodegron’’ by inducing ubiquitin/26S proteasome-
dependent degradation process. One example is the case of
steroid receptor coactivator-3 (SRC-3) where a phosphodegron
is crucial for both SRC-3 stability and coactivator function
(Li et al., 2008). Further, it is conceivable that phosphorylation
of histone modifiers can regulate protein-protein interaction by
affecting recruitment of binding modules to target proteins
depending on cellular context. PKB/Akt has a histone modifier,
enhancer of zeste homolog 2 (EZH2) as a nonhistone substrate.
PKB/Akt-mediated phosphorylation of EZH2 at serine 21 has
a negative effect on the methyltransferase activity of EZH2 by
impeding binding of EZH2 to histone H3, which results in
a decrease of H3K27 trimethylation and derepression of silenced
target genes (Cha et al., 2005). Although cyclin-dependent
kinase 1 (CDK1) is not categorized into H3 Ser/Thr kinases
discussed here, it is notable that EZH2 is also phosphorylated
by CDK1 at multiple sites and each phosphorylation confers
different functions. CDK1-dependent phosphorylation of EZH2
at T350 leads to the release of EZH2 from the other PRC2
components and thereby inhibits EZH2 enzymatic activity
(Chen et al., 2010), while phosphorylation at T487 is involved in
suppression of methylation of H3K27 and promotion of osteo-
genic differentiation of human mesenchymal stem cells
(Wei et al., 2011). Further, a domain in EZH2 comprising T345
phosphorylated by CDK1 is important for the interaction with
HOTAIR and the 50 end of Xist (Kaneko et al., 2010). In a similar
case in which enzymatic activity is modulated by phosphoryla-
tion, JNK1 leads to the phosphorylation of silent mating type
information regulation 2 homolog 1 (SIRT1) histone deacetylase
at S27, S47, and T530 and the phosphorylated SIRT1 exhibits
increased enzymatic activity (Nasrin et al., 2009). The SIRT1
phosphorylation by oxidative stress-activated JNK1 provides
a mechanistic insight on the regulation of the enzymatic activity
of SIRT1 by oxidative stress. Although detailed molecular mech-
anisms of how phosphorylation can enhance or inhibit enzymatic
activity have not been investigated extensively, we can specu-
late that phosphorylation affects nucleosome accessibility,
protein-protein interactions, and altered nuclear localization.
Kinases without Known Histone Substrates
Although salt-inducible kinase 1 (SIK1), S6K1, Abl, CaMK, and
big mitogen-activated protein kinase 1 (BMK1) have been shown
to phosphorylate histone modifiers such as class II histone
deacetylases (HDACs) and SRC-3 possessing weak histone
acetyltransferase activity, direct histone substrates have not
yet been reported (Berdeaux et al., 2007; Lai et al., 2010; McKin-
sey et al., 2000; Oh et al., 2008; Yang et al., 2010). Considering
that most MAPKs turn out to phosphorylate histone residues
directly in the nucleus, it is worthwhile to determine whether
there are other histone residues that are directly modified by
this family of kinases. SIK1 was reported to be a class II HDAC
kinase that conducts an important role in the SIK1-HDAC
pathway by promoting survival of skeletal myocytes (Berdeaux
et al., 2007). SIK1 regulates activity of myocyte enhancer factor
2 (MEF2) by phosphorylating HDAC5 at S259. Recently, BMK1
was shown to interact with promyelocytic leukemia protein
(PML) and suppress its tumor suppressor function through phos-
phorylation at S403 and T409, emphasizing the importance of
PML phosphorylation in tumor growth (Yang et al., 2010). With
regard to the kinase-dependent tumor-related regulation, S6K1
is a good example for a multifaceted regulator of Mdm2 and
p53. Under genotoxic stress, S6K1 phosphorylates Mdm2 on
S163 and inhibits Mdm2-mediated p53 ubiquitination and
degradation (Lai et al., 2010). The Abl tyrosine kinase phosphor-
ylates SRC-3 at Y1357, which is required for the enhanced tran-
scriptional activity of SRC-3 in cancer cells (Oh et al., 2008).
Tyrosine phosphorylation of SRC-3 allows crosstalk between
hormone and growth factor-related kinase signaling pathways
in cancer. It is tempting to speculate that the Abl tyrosine kinase
might use its Src homology domain 2 (SH2) to confer substrate
specificity which recognize tyrosine phosphorylated residues
as well as autoregulation of kinase activity.
Many of the nonhistone substrates can be modified by the
same histone-modifying enzymes. Thus, it is conceivable that
modification of other substrates by the same enzyme might
provide other unidentified regulatory signaling pathways or
redundant signaling pathways where the biological responses
may be going through or in parallel with those of histones. For
example, JAK2-dependent phosphorylation of H3Y41 results in
the exclusion of HP1a from the promoter and JAK2-dependent
phosphorylation of STAT5 is necessary for STAT5 DNA binding
activity, leading to the activation of JAK2 target genes in both
cases. Furthermore, we should keep in mind that modifications
such as phosphorylation are very dynamic and rapidly changing
within cells, suggesting that not only signaling kinases but also
corresponding phosphatases will conduct a balancing role for
timely regulated programs.
How Histone Phosphorylation Is ‘‘Read’’:
Phosphoprotein Binding Domains
Phosphorylated histone tails may act as an integrating platform
to receive information from upstream signals and transmit it to
the downstream effectors. An important aspect of signaling
through histone phosphorylation is the presence of ‘‘reader’’
molecules that recognize phosphorylated residues by specific
domains and thus create an attractive deciphering mechanism
to respond to alterations within cells. Proteins bind phosphory-
lated serines via specific phosphoprotein binding domain exem-
plified by 14-3-3 domain (Taverna et al., 2007). The mammalian
14-3-3 family of proteins have well-conserved phosphoserine-
binding modules and are involved many signal transduction
pathways, chromosome condensation, and apoptosis (Muslin
et al., 1996; Yaffe et al., 1997). 14-3-3 proteins have been shown
to form homo- and heterodimers, allowing interaction with at
least two phosphoserine sites, but engagement of two different
Molecular Cell 42, May 6, 2011 ª2011 Elsevier Inc. 279

gene activation. An asterisk (*) indicates a variety of kinases responsible for H3S10 phosphorylation leading to increase in H3K4 methylation, decrease in H3K9
methylation, or increase in H3K14 acetylation in mammalian cells. These kinases include PIM1, IKKa, PKB/Akt, Rsk2, and MSK1/2.
histone tails at the same time has not yet been reported. While
14-3-3 proteins target phosphoserine in the core histone
H3S10 context, BRCA1 carboxyl-terminal (BRCT) domains of
DNA damage checkpoint protein 1 (MDC1) target phosphoserine
in histone variant H2AX. What happens when DNA double-
strand breaks are induced and DNA repair machinery needs to
be activated in the context of chromatin? Histone phosphoryla-
tion may assist in the recognition and accessibility of regions
where DNA repair needs to take place. Phosphorylation of
histone variant H2AX at S139 in mammalian cells occurs rapidly
by the phosphatidylinositol 3-kinase (PI3K)-like family of kinases,
which includes ATM and AT-related (ATR) and DNA-dependent
protein kinase (DNA-PK) for DNA repair (Burma et al., 2001;
Rogakou et al., 1998). MDC1 localizes to sites of DNA breaks
and associates with checkpoint kinase 2 (Chk2) after DNA
damage responses (Lou et al., 2006).
Tyrosine phosphorylation is mainly involved in signaling
through transmembrane receptors that regulate cell proliferation
and differentiation. Readers that recognize phosphotyrosine
residues are distinct from those of phosphoserine residues,
and the SH2 and phosphotyrosine binding (PTB) domains are
phosphotyrosine readers (Lim and Pawson, 2010). Given that
certain tyrosine kinases and phosphatases possess phospho-
binding domains exemplified by the case of Abl kinase, it is
plausible that perhaps they use these domains directly to bind
phosphorylated tyrosines in histones and nonhistone proteins
for more elaborate and efficient substrate recognition, binding,
and regulation. Bioinformatics and systems biology as large-
scale studies could be used to explore phospho-binding
domains within kinases and phosphatases.
The binary switch hypothesis predicts that phosphorylated
histones will prevent the binding of repressive complexes from
the methylated lysine residues or rather aggressively block
specific lysine methylation (Fischle et al., 2003). Histone
phosphorylation can potentially regulate the binding of bromo-
domain-containing factors to the acetyl-lysine residues or the
binding of chromodomain-containing factors to the methyl-
lysine residues to establish a combinatorial regulatory code in
many biological processes, including regulation of gene expres-
sion, heterochromatin formation, and developmental pathways.
For example, a ‘‘binary methylation-phosphorylation switch’’
mechanism can explain how HP1 proteins are released from
mitotic chromatin (Fischle et al., 2003). At the onset of mitosis,
Aurora B mediates the phosphorylation of H3S10 right next to
H3K9me3, the HP1 binding site, leading to the disruption of
280 Molecular Cell 42, May 6, 2011 ª2011 Elsevier Inc.
Molecular Cell
Review
Figure 2. Crosstalk between Histone
Phosphorylation and Other Modifications
The negative effect of phosphorylation is depicted
by a dashed line, whereas the positive influence of
phosphorylation over another is depicted by an
arrow. PKCb-dependent phosphorylation of H3T6
increases H3K4 methylation but inhibits H3K9
methylation for transcriptional activation. PRK1-
dependent phosphorylation of H3T11 blocks
H3K9 methylation, and Chk1-dependent phos-
phorylation of H3T11 increases H3K14 acetylation
for transcriptional activation. MSK1/2-mediated
phosphorylation relieves H3K27methylation for
the interaction of the chromodomain of HP1 proteins with the
H3K9me3 target site. At the end of mitosis, dephosphorylation
of H3S10 by phosphatase dynamically establishes HP1 binding
to the stable H3K9me3 mark during the cell cycle. It appears that
this binary switch hypothesis might apply to nonhistone proteins
and future work might yield insight into the potential generality of
this type of mechanism.
Crosstalk between Histone Phosphorylation and Other
Modifications
Histone phosphorylation controls many important cellular
processes, including transcription, apoptosis, DNA repair, and
chromosome condensation. Given that the H3S10 phosphoryla-
tion is critical during interphase because it confers transcrip-
tional activation on a number of genes depending on upstream
signaling pathways, the location of S10 residue in close prox-
imity to other modifiable residues in the H3 tail enables a variety
of crosstalk between phosphorylation and other modifications
that lead to synergistic or parallel effects on transcription. In
addition to S10, S28, T6, and T11 residues are located in close
proximity to other modifiable residues of the histone H3 tail,
which allow various crosstalks between phosphorylation and
other modifications such as methylation and acetylation
(Figure 2). For gene activation, it is well established that H3S10
phosphorylation inhibits H3K9 methylation, facilitates H3K4
methylation, and enhances H3K14 acetylation, which allows
further chromatin decondensation. A variety of H3S10 kinases
including Aurora B, VRK1, MSK1/2, PIM1, Rsk2, PKB/Akt, and
IKKa are involved in this crosstalk. Two cases demonstrating
strong crosstalk between phosphorylation and methylation
during AR-dependent gene activation process in prostate
cancer include the phosphorylation of H3T6 by PKCb and the
phosphorylation of H3T11 by PRK1 (Metzger et al., 2008;
2010). Importantly, PKCb-dependent phosphorylation of H3T6
prevents LSD1 from demethylating H3K4 and accelerates the
demethylation of H3K9 by altering substrate specificity of LSD1.
A more recently identified link between phosphorylation and
acetylation is the crosstalk between MSK1/2 and Chk1. Methyl-
ation of H3K27 by the polycomb repressive complex 2 (PRC2) is
well established as a repression mark during differentiation.
MSK1/2 preferentially phosphorylates H3S28 in the context of
H3K27me3-marked promoters, leading to the displacement of
polycomb group proteins for transcriptional activation, support-
ing the binary switch hypothesis (Gehani et al., 2010). With the
aid of antibodies against the H3K27me3S28ph double mark, it
Molecular Cell
Review
was successful to investigate extensively the coexisting mecha-
nism of the K27me3 and S28ph on the same histone tail H3.
Generation of various antibodies against the double or triple
mark will be very helpful in addressing the interplay and crosstalk
between these modifications. Another example of crosstalk can
be seen from Chk1-mediated phosphorylation of H3T11 regu-
lating DNA damage-induced transcriptional repression of cell-
cycle regulatory genes (Canton and Scott, 2010; Shimada
et al., 2008). Loss of H3T11 phosphorylation correlates with
reduced binding of the GCN5 histone acetyltransferase following
reduced H3K9 and H3K14 acetylation. As to the crosstalk
between histone phosphorylation and other modifications, there
is still a lot to learn.
While crosstalk between histone phosphorylation and other
modifications adds another layer of complexity and a dynamic
view of modifications, it also raises a host of interesting and
important questions. With multiple histone modifications
including phosphorylation present at the same time on the
N-terminal histone tails, what kind of interplay does it exist
between each modification? Could these modifications function-
ally affect each other? Could they work in concert to achieve
a synergy? There is no doubt that phosphorylation can affect
other modifications nearby cooperatively or antagonistically. To
this end, it will be of great interest to monitor temporal and spatial
dynamics in crosstalk between phosphorylation and other modi-
fications using real-time imaging techniques in living cells.
Future Perspectives and New Insights
There is no doubt that signaling to chromatin is an exciting
research area that has already provided and will continue to
provide striking discoveries. In particular, the combinatorial
modification of histones and nonhistone proteins offers
numerous regulatory opportunities at the level of upstream
kinases and downstream signal transduction cascades,
including crosstalk. Histone phosphorylation, acting sequentially
or in concert with other modifications, allows initiation, propaga-
tion, termination, and fine-tuning of signal transduction
cascades. Systems biology approaches will help understand
the interplay of each modification at the network levels. It is of
particular interest that rapid development of genomics, epige-
nomics, and bioinformatics technologies allow us to investigate
these fields at a global level. Moreover, by using sensitive detec-
tion techniques for phosphorylation, it should be possible to
define new histone phosphorylation sites regulated by certain
kinases that have not been considered yet to act directly in the
nucleus and attribute unidentified functions to known histone
phosphorylation. Development of highly sensitive detection
methods for phosphorylation with advanced technologies and
instruments would be central to answer a lot of the unanswered
questions.
Major challenges for the future will be in the establishment of
biological relevance for phosphorylation of histone and nonhis-
tone proteins in vivo. This would entail dissection and decipher-
ing of signaling pathways using in vivo model animals. In doing
this, genetically engineered mice to analyze the physiological
functions of kinases and phosphatases involved in adding or
removing phosphorylation will be valuable. Further, knockin
mice, in which endogenous genes encoding nonhistone proteins
are replaced with the ones lacking a particular phosphorylation
by point mutation, will be extremely valuable in studying the
physiological meaning of a particular phosphorylation. This
approach is applicable to other model organisms including
Drosophila, C. elegans, and zebrafish. Some phosphorylation
sites are well conserved among species, so model organism
studies with genetic approaches allow us to search for in vivo
physiological functions easily and rapidly. Considering that
many histone genes exist in mammalian genome, it is very
difficult to investigate each phosphorylation of histones resulting
in a biological outcome. In this case, another model organism
such as yeast is possible. The chemical-genetic methods for
developing a specific small-molecule inhibitor of single protein
kinase will be particularly promising for addressing the function
of signaling kinase-dependent phosphorylation of histones and
nonhistone proteins.
It is exciting to predict that there might be specific potential
code for modification of nonhistone proteins as in the case of
a proposed histone code for histones. It is possible that there
is exciting unrevealed crosstalk between histone phosphoryla-
tion and nonhistone phosphorylation to decipher. Given that
histone modifications have been implicated in a number of
epigenetic phenomena, it is conceivable that modification of
nonhistone proteins such as transcription factors and histone
modifiers in the nucleus may be involved in a number of biolog-
ically important epigenetic phenomena. Only time will tell us
more exciting stories.
ACKNOWLEDGMENTS
I would like to apologize to all scientists whose work could not be cited due to
space limitation. I would like to thank members of the Chromatin Dynamics
Research Center, Keun Il Kim, and Jongkyeong Chung for critical readings,
discussions, or ideas and Eun Soon Hong for manuscript preparation. This
Creative Research Initiative work was supported by the National Research
Foundation of Korea grant funded by the Korean government (Chromatin
Dynamics Research Center, 2009-0081563) to S.H.B.
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