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Review
A pervasive role of histone
acetyltransferases and deacetylases in
an NF-kB-signaling code
Miriam Calao, Arsène Burny, Vincent Quivy, Ann Dekoninck and Carine Van Lint
Laboratory of Molecular Virology, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, 12 Rue des Profs
Jeener et Brachet, 6041 Gosselies, Belgium
Most nuclear factor-kB (NF-kB) inducers converge to
activate the IkB kinase (IKK) complex, leading to NF-kB
nuclear accumulation. However, depending on the inducer and the cell line, the subset of NF-kB-induced genes is
different, underlining a complex regulation network.
Recent findings have begun to delineate that histone
and non-histone protein acetylation is involved, directly
and indirectly, in controlling the duration, strength and
specificity of the NF-kB-activating signaling pathway at
multiple levels. Acetylation and deacetylation events, in
combination with other post-translational protein modifications, generate an ‘NF-kB-signaling code’ and regulate
NF-kB-dependent gene transcription in an inducer- and
promoter-dependent manner. Indeed, the intricate involvement of histone acetyltransferases and histone deacetylases modulates both the NF-kB-signaling pathway and
the transcriptional transactivation of NF-kB-dependent
genes.
The interplay between protein acetylation and
NF-kB activation
The nuclear factor-kB (NF-kB) family of transcription
factors comprises several evolutionarily conserved, structurally related interacting proteins that bind to DNA [1,2].
NF-kB plays a crucial part in the transcriptional regulation of genes involved in controlling cell proliferation,
differentiation, apoptosis, inflammation and stress
responses, in addition to other biological processes. Consistent with its role in regulating the immune response,
abnormal NF-kB regulation has been linked to cancer,
inflammatory and autoimmune diseases, septic shock,
viral infections and improper immune development. To
date, three NF-kB-activating signaling pathways have
been reported (Box 1). Under most basal conditions, NFkB, typically a p50–p65 heterodimer, is sequestered in the
cytoplasm in an inactive state through its interaction with
an inhibitory protein of the inhibitor of NF-kB (IkB) family.
After cell stimulation by a wide variety of inducers, the IkB
kinase (IKK) complex is activated, leading to transcriptional transactivation of NF-kB-dependent genes (Box 1).
Reversible acetylation is a post-translational protein
modification that regulates many cellular processes, including chromatin assembly and gene transcription (Box
2). Specific acetylation marks, together with other covalent
Corresponding author: Van Lint, C. ([email protected]).
post-translational histone modifications (including
phosphorylation and methylation), function sequentially
or in combination to form the ‘histone code’, which is read
by effector proteins to produce distinct biological outcomes
[3]. Histone acetylation by histone acetyltransferases
(HATs) is crucial for chromatin-compaction status and gene
transcription (Box 2). Whereas increased histone acetylation promotes chromatin decompaction and increased
DNA accessibility to transcription factors, usually leading
to gene activation [4,5] (Box 2), histone deacetylase (HDAC)mediated deacetylation of lysine residues renders nucleosomal DNA less accessible to the transcriptional machinery,
thereby usually favoring transcriptional silencing [5] (Box
2). Histone acetylation marks enable the recruitment of
bromodomain-containing proteins (including chromatinremodeling enzymes, transcription factors and HATs),
which in turn regulate gene expression. In addition to
histones, HATs and HDACs have a wide range of nonhistone protein substrates (including transcription factors),
functions of which are regulated by acetylation and deacetylation.
It is now clearly established that reversible acetylation
of histone and non-histone proteins plays a crucial part in
regulating the entire NF-kB pathway. Several members of
the NF-kB pathway regulate NF-kB-dependent and/or independent transcription through the recruitment of
HATs and/or HDACs to gene promoters [6–12]. Moreover,
direct acetylation of NF-kB family members and of proteins
involved in the NF-kB-signaling pathway (in addition to
other signaling pathways) regulates NF-kB activation and
transcriptional activity [13–25]. The newly discovered
post-translational modifications, serine and threonine
protein acetylation, also can regulate the NF-kB-activating
cascade [13,26]. Indeed, YopJ, a bacterial effector from
Yersinia spp., acetylates IKKa at Thr179 and IKKb at
Thr180, located in their respective activation loops, thus
preventing their phosphorylation and subsequent activation [13]. Therefore, by inhibiting the NF-kB pathway,
Yersinia cripples the host-cell defense system. No YopJ
eukaryotic ortholog has been identified to date.
In this review we propose that multi-level acetylation
and deacetylation events in the NF-kB pathway, in concert
with other post-translational protein modifications, generate an ‘NF-kB-signaling code’, which could, by
analogy with the ‘histone code’, shape the distinct,
and sometimes opposing, biological responses elicited by
0968-0004/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2008.04.015
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Trends in Biochemical Sciences Vol.33 No.7
Box 1. Overview of the NF-kB-signaling pathway
Box 2. HATs and HDACs
The NF-kB family of transcription factors is present in most
vertebrate cell types as homo- and heterodimers of five structurally
related Rel and NF-kB proteins, namely p65 (also called RelA), RelB,
c-Rel, NF-kB1 (p50 and its precursor p105) and NF-kB2 (p52 and its
precursor p100) [1,2]. The transactivation domain (TAD) that is
necessary for NF-kB-dependent transcriptional activity is present
only in p65, c-Rel and RelB. However, RelB–p65 heterodimeric
complexes are inhibitory because they cannot bind to DNA. Because
they lack a TAD, DNA-bound p50 or p52 homodimers function as
transcriptional repressors, but they can stimulate transcription when
associated with the IkB-like nuclear protein Bcl-3 (B-cell lymphoma
3; which contains a TAD) or when heterodimerized with a
transactivating Rel subunit. In unstimulated cells, NF-kB is sequestered in the cytoplasm in an inactive form via interaction with the
inhibitors of NF-kB (IkBs). IkBa, IkBb and IkBe bind the Rel
homology domain of NF-kB dimers, masking their nuclear-localization signals (NLSs). p105 and p100 also function as IkBs, tethering
NF-kB subunits in the cytoplasm of unstimulated cells.
The so-called classical NF-kB pathway is triggered by proinflammatory cytokines such as tumor necrosis factor a (TNFa) and
promotes the recruitment and activation of the classical IkB-kinase
(IKK) complex, which includes two catalytic subunits, IKKa and
IKKb, and a scaffold protein, NF-kB essential modulator (IKKg; also
known as NEMO). After activation, the IKK complex phosphorylates
IkBa, which is subsequently ubiquitylated and degraded by the
proteasome. IkBa masks only the p65 NLS, whereas the p50 NLS
remains exposed and is responsible, together with the IkBa nuclearexport sequence, for shuttling IkBa–NF-kB complexes between the
nucleus and the cytoplasm. IkBa degradation unmasks the p65 NLS,
thereby promoting rapid NF-kB translocation into the nucleus.
Nuclear NF-kB binds DNA and activates transcription of several
genes, including those encoding proinflammatory chemokines and
cytokines and several antiapoptotic genes. After NF-kB-dependent
resynthesis, IkBa enters the nucleus, removes NF-kB from DNA and
enhances NF-kB transit to the cytoplasm, thus restoring the pool of
inducible NF-kB. A second pathway, the ‘alternative’ pathway, is
engaged after lymphotoxin-b- or B-cell-activating-factor induction
and activates an IKKa homodimer, which phosphorylates p100.
After phosphorylation, p100 is ubiquitylated and cleaved to
generate p52, which translocates as a heterodimer with RelB into
the nucleus, where it stimulates transcription of genes that are
important for secondary lymphoid organ development, B-cell
homeostasis and adaptive immunity. A third NF-kB-activating
pathway is classified as atypical because it involves IKK-independent IkBa phosphorylation and proteasome degradation [1,2].
Reversible acetylation is a post-translational protein modification
involved in multiple biological processes, including nucleosomal
assembly and gene transcription [5,27]. The packaging of eukaryotic
DNA with histones into chromatin plays an active part in transcriptional regulation by modulating transcription-factor accessibility.
Acetylation of lysine residues in histone N-terminal tails results in
chromatin decompaction and increased transcription-factor access
to DNA and, thus, usually correlates with gene activation, whereas
histone deacetylation mediates transcriptional repression. Moreover, histone lysine acetylation, in concert with other post-translational epigenetic histone modifications, forms the basis for the
‘histone code’. The combination of different modifications provides
unique interaction surfaces for effector proteins, including chromatin-remodeling enzymes and transcription factors, thus further
regulating gene expression [3]. The histone-acetylation state is a
product of competition between two families of enzymes: histone
acetyltransferases (HATs) and histone deacetylases (HDACs). On the
one hand, enzymes with HAT activity catalyze the transfer of an
acetyl group from acetyl coenzyme A to the e-amino group of a
specific internal lysine residue, thereby resulting in neutralization of
one positive charge on the histone protein. As a result, the
electrostatic properties of the protein are strongly modified. On
the other hand, enzymes possessing HDAC activity remove acetyl
groups from acetylated proteins.
Approximately 30 proteins (including hGCN5, CBP, p300, PCAF
and steroid-receptor coactivator 1, SRC-1) are known to possess
acetyltransferase activity. Each HAT has particular histone substrate
specificity. Moreover, HATs present high specificity related to which
histone lysine they can acetylate. Mammals express at least 18
HDACs (HDAC-1 to HDAC-11 and the sirtuins, SIRT1 to SIRT7), which
are grouped into three classes based on their similarity with yeast
genes. Like HATs, HDACs also possess substrate specificity.
In addition to histones, HATs and HDACs have a wide range of
non-histone protein substrates, of which regulated acetylation and
deacetylation determines cellular fate and survival. These substrates
include general and specific transcription factors, non-histone
structural chromosomal proteins, HATs themselves, viral proteins,
non-nuclear proteins (e.g. a-tubulin) and nuclear import factors
(such as human importin-a). Depending on the functional domain
that is modified, acetylation regulates different functions of these
non-histone proteins: for example, DNA recognition, protein
stability, protein–protein interactions and subcellular localization.
different classes of NF-kB-inducing stimuli in a
cell-line- and promoter-dependent manner. We describe
and discuss the complex involvement of protein acetylation
and deacetylation events regulating the entire NF-kBsignaling pathway.
NF-kB recruits antagonistic coregulatory proteins:
acetyltransferases and deacetylases
NF-kB-dependent gene expression requires the involvement of transcriptional coactivators, which bridge specific
transcription factors to the basal transcriptional machinery and also alter chromatin structure (Table 1). Consistent with their role in modifying chromatin structure,
many NF-kB coactivators, including p300, CREB-binding
protein (CBP), p300/CBP-associated factor (PCAF), members of the p160 family of steroid-receptor coactivators and
tat-interactive protein of 60 kDa (Tip60) [27,28], possess a
HAT domain, which catalyzes the acetylation of specific
internal lysine residues in the core-histone N-terminal
tails (Table 1). Moreover, a growing list of proteins can
340
interact with NF-kB dimers and affect NF-kB–DNA
binding and NF-kB-dependent transcription, in some cases
in concert with HATs. The arginine methyltransferase
CARM1 (coactivator-associated arginine methyltransferase 1) interacts directly with p65 at specific promoters,
where it functions as a p300/CBP-dependent coactivator
for NF-kB-mediated transcriptional activation [29]. Similarly, PRMT1 (protein arginine methyltransferase 1)
coactivates NF-kB-dependent gene expression at the
macrophage inflammatory protein 2 (MIP2) and HIV-1
promoters synergistically with p300, CARM1 and poly
(ADP-ribose) polymerase 1 (PARP1; another NF-kB coactivator [30]) [31]. Similarly to CARM1, which methylates
p300 and CBP thereby altering their function [32], PRMT1
also might methylate – and influence the function of – NFkB transcriptional coactivators [31]. It will be interesting
to determine whether these two methyltranferases directly
modify NF-kB, thereby altering its transcriptional activity.
The promoter-specific involvement of different NF-kB
coactivators indicates that these coactivators combinatorially assemble at different NF-kB-regulated promoters to
achieve distinct functional specificity. However, whereas
the kB-site nucleotide sequence governs cofactor binding
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Table 1. p50–p65-interacting HATs and HDACsa
NF-kB subunit
Interacting HAT
p65
p300
NF-kB subunit
acetylated lysine b
Lys314, Lys315
p300
Lys310
Lys218, Lys221
Lys221
–
CBP
–
CBP and PCAF
–
p300 and PCAF
Lys122, Lys123
PCAF and SRC-1
–
p300
Lys431, Lys440, Lys441
SRC-1
–
p50 via Bcl-3
Tip60
–
NF-kB subunit
Interacting HDAC
p65
SIRT1
NF-kB subunit
deacetylated lysine b
Lys310
p50
p65 and p50
p50
HDAC-1 and HDAC-2
–
HDAC-3
Lys122, Lys123
(Lys218, Lys221, Lys310) c
HDAC-4 and HDAC-5
–
HDAC-6
HDAC-1
–
–
HDAC-3 d
–
Functional consequences
Refs
Both gene-specific transcriptional activation and repression,
although NF-kB–DNA binding and shuttling are not affected
Enhanced NF-kB transcriptional activity
Inhibition of IkBa binding
Enhanced DNA binding
Histone H3 and H4 acetylation at the E-selectin and VCAM
promoters after TNFa induction
Histone H3 and H4 acetylation at the HIV-1 promoter after
cell cycle arrest in G2 or PMA induction
Histone H3 and H4 acetylation at the TNFa and COX-2
promoter after TNFa or high glucose inductions
Reduced NF-kB–DNA binding and postinduction repression
of NF-kB-mediated transcription
Enhanced p65-dependent transcriptional activation at the
E-selectin promoter
Enhanced p50–DNA binding in vitro
Enhanced p50 recruitment to the COX-2 and iNOS promoters
Stimulation of NF-kB-mediated transactivation of an IL-2- (kB)4luciferase reporter gene
After IL-1b induction, H3 and H4 KAI1/CD82 promoter acetylation
and transcriptional activation
[53]
[23]
[23]
[23]
[80,81]
[82,83]
[84]
[20]
[12]
[16]
[17,18]
[85]
[41]
Functional consequences
Refs
Apoptosis induction in response to TNFa via SIRT1-mediated
inhibition of p65 transactivation potential
Inhibition of proinflammatory mediator release in response to
cigarette smoke via SIRT1-mediated inhibition of p65
transactivation potential
Inhibition of NF-kB-regulated gene expression, including IL-8,
by deacetylating histones within the surrounding chromatin
Enhanced NF-kB–DNA binding
Enhanced NF-kB–IkBa binding and postinduction repression
of the NF-kB transcriptional response
Repression of the MIS promoter, which does not contain kB
sites. The NF-kB–HDAC complex is recruited by the orphan
nuclear receptor SF-1, which binds to its responsive element
located within MIS promoter
Repression of the HKa2 promoter
Local histone deacetylation at the HIV-1 and IL-6 promoters
and repression of NF-kB-mediated transcription
HDAC-3 is part of corepressor complexes recruited by p50 to
the KAI1, cIAP-2 and IL-8 promoters in unstimulated cells to
repress basal transcription
[21]
[22]
[35,36]
[20]
[19]
[38]
[37]
[36,43]
[39,41]
a
Abbreviations: HKa 2, H+-K+-ATPasea2; MIS, mullerian inhibiting substance; SRC-1, steroid-receptor coactivator 1; SF-1, steroidogenic factor 1; VCAM, vascular cell adhesion
molecule.
b
–, functional effects mentioned have not been demonstrated to directly depend on lysine acetylation of the NF-kB subunit considered.
c
Not directly established.
d
HDAC3–p50 interaction has not been demonstrated by coimmunoprecipitation assays.
and NF-kB–DNA binding affinity in some cases, the
specificity of the NF-kB response cannot be reduced to
kB sites alone. Recent studies have identified proteins that
serve as ‘specifiers’ that select particular genomic kB sites
to be activated under certain conditions. In this regard,
ribosomal protein S3 (RPS3) is a functional subunit of
specific NF-kB–DNA-binding complexes that can confer
gene target specificity by binding to DNA with some
sequence specificity [33]. Another nuclear protein, Akirin2,
fine-tunes NF-kB transcriptional activity by modulating
the expression of a subset of lipopolysaccharide (LPS)- and
interleukin (IL)-1b-inducible NF-kB-dependent genes
through its interaction with components of the chromatin
machinery [34]. However, how Akirin2 controls and specifies gene expression requires further investigation.
In addition to coactivators, NF-kB also directly or
indirectly recruits corepressor complexes, which possess
HDAC activity and repress both basal and induced
NF-kB-dependent or -independent transcription (i.e. transcription from gene promoters containing or lacking kB
sites, respectively) [19,20,35–38] by deacetylating histone
N-terminal tails in addition to the p50 and p65 NF-kB
subunits (Table 1). Specific HDAC isoforms are recruited
via distinct NF-kB complexes, depending on the promoter
context and on other coregulatory molecules.
In unstimulated cells, promoters containing NF-kBbinding sites are repressed by transcriptionally inactive
p50 or p52 homodimers that directly or indirectly bind
DNA and recruit HDACs and/or other corepressor proteins
[39–43]. HDACs usually are components of large corepressor complexes containing proteins such as mouse SWIindependent 3A (mSin3A), nuclear receptor corepressor
(N-CoR) and/or silencing mediator of retinoic acid and
thyroid hormone receptor (SMRT). For transcription
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Figure 1. Derepression of NF-kB-regulated genes repressed by p50 homodimers. p50 (red) homodimers recruit repressor complexes composed of SMRT (dark gray) and
HDAC-3 (orange) (a) or Bcl-3 (purple), N-CoR (light blue) and HDAC-3 (b). (a) (i) In the unstimulated state, the SMRT–HDAC-3 complex, tethered by p50 homodimers,
regulates the basal repression of classical NF-kB-regulated genes, including cIAP-2, also known as BIRC3. (ii) Upon cellular stimulation, IKKa (light green) promotes the
removal of the SMRT–HDAC-3 complex. Indeed, IKKa-mediated SMRT phosphorylation induces its nuclear export and proteasomal degradation, thereby releasing HDAC-3
from the promoter (iii). This initial derepression enables transcriptionally active p50–p65 heterodimers to bind the promoter; concomitantly, the SMRT corepressor returns
to the chromatin-bound NF-kB complex (iv). In the context of the appropriate stimuli, IKKa remains chromatin associated and phosphorylates both p65 (dark green) and
SMRT, thereby preventing HDAC-3 recruitment. p300 (light pink) then binds p65–p50 heterodimers, thereby facilitating subsequent p65 acetylation, which is required for
full NF-kB-mediated transcription [24]. (b) In the small subset of NF-kB-regulated promoters that tether Bcl-3 (i), such as the Kangai 1 (KAI1; also known as CD82) promoter,
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to proceed, these corepressors must be replaced by
coactivator proteins, a mechanism termed derepression.
Derepression requires phosphorylation of the repressor
protein for two genes that are basally repressed by p50
homodimers: the classical NF-kB-regulated gene cellular
inhibitor of apoptosis 2 (cIAP2), also called baculoviral IAP
repeat-containing 3 (BIRC3) [24,39] (Figure 1a), and Kangai 1 (KAI1), also termed CD82 (Figure 1b), which belongs
to the small subset of NF-kB-regulated genes that tether Bcell CLL/lymphoma 3 (Bcl-3) [24,39,41].
The Perkins group [44] reported that the NF-kB p52
subunit switches from recruiting activator (Bcl-3) to
repressor (HDAC-1) proteins to target promoters in
response to UV light and subsequent p53 induction. Moreover, although p52 is required for HDAC-1 recruitment at
some promoters, resulting in transcriptional repression, it
contributes to p300 coactivator recruitment and transcriptional stimulation at other promoters [42].
Although the NF-kB p65 subunit possesses a transactivation domain, it can function either as a transcriptional activator or as a repressor by interacting with HATs
or HDACs, respectively [45]. This dual functionality of
p65, indicating that several distinct stimuli can produce
repressive forms of NF-kB, is regulated on at least two
levels: inducible p65 post-translational modifications and
the action of coregulatory proteins. Indeed, PKAc (the
catalytic subunit of the cAMP-dependent protein
kinase)-mediated phosphorylation of p65 at Ser276
(within its Rel homology domain) enhances p300 and
CBP recruitment and simultaneously decreases
HDAC-1 affinity for p65 [10,36]. Moreover, Mayo and
colleagues [24] demonstrated that IKKa-mediated phosphorylation of p65 at Ser536 (within its transactivation
domain) displaces the SMRT–HDAC-3 corepressor complex, enabling p300 to interact with p65. Among the NFkB coregulatory proteins, ARF (alternative reading
frame), a tumor suppressor, expression of which is induced
upon oncogene activation, and LZAP (LxxLL/leucine-zipper-containing ARF-binding protein) inhibit NF-kBinduced antiapoptotic gene expression by increasing the
interaction between HDACs and p65, without altering
NF-kB–DNA binding [46,47]. ARF- and LZAP-dependent
inhibition of p65-mediated antiapoptotic gene expression
reflects differential p65 phosphorylation status: ARF activation induces p65 phosphorylation at Thr505, whereas
LZAP expression decreases phosphorylation of p65 at
Ser536 [47,48]. Importantly, increased NF-kB association
with HDAC-containing corepressor complexes is a common mechanism through which many tumor-suppressor
proteins inhibit the antiapoptotic function of NF-kB,
which contributes to tumorigenesis.
Therefore,
stimulus-dependent
phosphorylation
events modulate switches between HDAC-containing
NF-kB repressor complexes and HAT-containing NFkB-activator complexes. The mechanisms through which
HATs and other coactivators enhance NF-kB transcriptional activity (whereas it is inhibited by HDACs and
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Vol.33 No.7
other corepressors) are multifactorial and involve direct
effects on NF-kB, other transcription factors and chromatin structure.
Regulation of NF-kB biological functions by direct
acetylation
In addition to acetylating the N-terminal tails of core
histones that surround NF-kB-regulated genes, HATs also
directly acetylate several NF-kB subunits: p52 and its
precursor p100, p50 and p65. Hu and Colburn [14] have
reported that p100 acetylation enhances processing of
p100 to p52, whereas Wu and colleagues [15] demonstrated
that p300-dependent p52 acetylation enhances its DNAbinding activity. These differences could reflect acetylation
at different lysine residues; however, the acetylation sites
in p52 and its precursor p100 have not been mapped.
The p50 subunit of the classical NF-kB p50–p65 heterodimer is acetylated in vitro and in vivo (Figure 2; Table 1).
In vitro, p300-mediated acetylation of p50 occurs only in
the presence of the HIV-1 viral protein Tat [16]; however,
in vivo, in the absence of Tat, p300 overexpression
enhances p50 acetylation [17,18]. One possible explanation
for this discrepancy is that, under physiological conditions,
p300-mediated p50 acetylation relies on a Tat-like cofactor.
The acetylated form of p50 binds to target sites with higher
affinity than the unacetylated form in vitro does [16]. In
agreement with these data, enhanced in vivo p50 acetylation correlates with increased binding of p50 to two NFkB-regulated promoters: cyclooxygenase-2 (COX-2) and
inducible NO synthase (iNOS) [17,18]. Three p50 lysines,
Lys431, Lys440 and Lys441, are acetylated in vitro [16],
but the specific role for each acetylated residue has not
been demonstrated (Figure 2).
p65 is acetylated by p300 and is deacetylated through
specific interactions with HDAC-3 [19,20] or SIRT1 [21,22]
(Figure 2; Table 1). p65 acetylation is functionally important; indeed, endogenous p65 is acetylated in response to
many different stimuli, including exposure to tumor necrosis factor a (TNFa), phorbol 12-myristate 13-acetate (PMA)
or transforming growth factor 1 (TGFb1) [19,20,24,49].
Accordingly, inhibition of TNFa-induced IKK- and Aktkinase activity correlates with suppression of p65
acetylation [50,51]. Furthermore, doxorubicin-dependent
NF-kB induction, which does not affect p65 phosphorylation or acetylation, is not associated with NF-kB-dependent transcriptional activation [52]. The Greene group [23]
demonstrated that p65 acetylation occurs on three lysine
residues: Lys218, Lys221 and Lys310. p300-mediated
acetylation at Lys221 enhances p65–DNA-binding activity
and, together with Lys218 acetylation, impairs the p65–
IkBa interaction, thus impeding IkBa-dependent nuclear
export of NF-kB complexes and prolonging the NF-kB
response [19,23]. Thus, HDAC-3-mediated p65 deacetylation at Lys221 and Lys218 is involved in terminating the
NF-kB response by promoting IkBa–p65 interaction and
NF-kB nuclear export. Lys310 acetylation is required for
full p65 transcriptional activity on the E-selectin promoter
IL-1b-induced derepression involves MEKK1 (MAP/ERK kinase kinase 1)-mediated TAK1-binding protein 2 (TAB2; pink) phosphorylation, which causes the nuclear export of
the N-CoR–TAB2–HDAC-3 complex (ii) and the recruitment of the HAT Tip60 (brown) to the promoter (iii). This recruitment is accompanied by histones H3 (light purple) and
H4 (blue) acetylation and subsequent transcription initiation [41]. Adapted, with permission, from Refs [24,41].
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Figure 2. Acetylation and deacetylation of the NF-kB p50 and p65 subunits. The NF-kB p65 (green) subunit is acetylated by p300 or PCAF and deacetylated through specific
interactions with HDAC-3 [19,20] or SIRT1 [5,21]. Lys221 acetylation enhances p65 DNA-binding activity and, together with acetylation at Lys218, impairs its assembly with
IkBa (brown), thus impeding IkBa-dependent nuclear export of the NF-kB complex and enabling prolongation of the NF-kB response [23]. p65 acetylation at Lys310 is
required for its full transcriptional activation [23]. Although a specific role for p65 Lys314 and Lys315 acetylation has not been demonstrated, these modifications are not
involved in NF-kB–DNA binding or intracellular shuttling [53]. Lys431, Lys440 and Lys441 of p50 (red) are acetylated in vitro, but the specific functional role of these
modifications has not been demonstrated; however, in vitro, the acetylated form of p50 binds target sequences with higher affinity than the unacetylated form does [16]. In
agreement with these data, enhanced p50 acetylation correlates with increased p50 binding to some NF-kB-regulated promoters in vivo [17,18]. Green arrows represent
increased DNA-binding affinity, red lines pointing toward DNA represent decreased DNA-binding affinity, red lines pointing toward acetylated residues represent
deacetylation events, and red lines pointing toward IkBa represent decreased p65–IkBa interaction.
[23]. TNFa-dependent phosphorylation of p65 at either
Ser276 or Ser536 increases its interaction with p300 and
its subsequent acetylation at Lys310, indicating that p65
phosphorylation regulates subsequent acetylation [24,25]
(Figure 3). By contrast, Benkirane and colleagues [20] have
shown that Lys122 and Lys123 are the only acetylated
residues in p65 and that p300- or PCAF-mediated p65
acetylation lowers its binding affinity for target sequences.
Recently, two additional acetyl acceptor sites (Lys314 and
Lys315) were identified by the Hottiger group [53], who
Figure 3. NF-kB-dependent transcription requires histone acetylation. When NF-kB enters the nucleus after cell stimulation, it activates two subsets of target promoters:
those that were heavily acetylated before stimulation (i.e. constitutively and immediately accessible) (a) and those requiring stimulus-dependent modifications in chromatin
structure to make NF-kB sites accessible (b) [60]. (a) The subset of NF-kB target genes, of which the promoter chromatin is immediately accessible to NF-kB (green), is
transcribed immediately after NF-kB activation. (b) By contrast, for NF-kB target genes that are not immediately accessible (i), stimulus-dependent chromatin remodeling
and histone hyperacetylation are prerequisites for NF-kB recruitment. As an example, mcp-1 (also known as ccl2) transcription requires binding of AP-1 (light blue)inducible transcription factors to the promoter and subsequent ATP-dependent chromatin remodeling by the SWI/SNF (switch/sucrose nonfermentable) complex (orange)
(ii). This remodeling enables NF-kB-binding-site accessibility, HAT (purple) recruitment and activity and, thus, mcp-1 gene activation (iii). Green arrows represent gene
transcription and red arrows represent transcriptional repression. Adapted, with permission, from Ref. [79].
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also confirmed the presence of Lys310 acetylation. They
found that Lys314 and Lys315 acetylation is not involved
in DNA-binding activity or shuttling of NF-kB. The discrepancies between the conclusions reached by the Greene,
Benkirane and Hottiger groups regarding p65 acetylation
might result from differences in the experimental
approaches, stimuli (e.g. none, PMA and/or HDAC inhibitor, and TNFa and/or HDAC inhibitor, respectively) and
cell lines (i.e. Jurkat, Cos-7 or HEK 293T) used. Importantly, the study by the Hottiger group [53] also provides
evidence that, after TNFa stimulation, specific subsets of
genes are either stimulated or represssed by p300mediated p65 Lys310, Lys314 and Lys315 acetylation.
Thus, distinct p65 acetylation events regulate the specificity of NF-kB-dependent gene expression [53].
Taken together, the results from several laboratories
demonstrate that NF-kB acetylation regulates NF-kB
transcriptional potential, DNA-binding activity and
protein–protein interactions with several transcription
cofactors. Indeed, the unique combinations of NF-kB
post-translational modifications form a recognition code
that regulates the interactions between NF-kB and these
cofactors.
IkBa- and IKK-dependent gene regulation through
HAT and/or HDAC recruitment
In addition to their cytoplasmic role in the NF-kB-activating cascade, IkBa and some members of the IKK complex
have a nuclear function involving HAT and HDAC recruitment. IkBa interacts with HDAC-1, -3 and -5 [6,7].
Although IkBa was first identified as the specific cytoplasmic inhibitor of NF-kB, it now is clear that it shuttles
between the nucleus and the cytoplasm [54]. In the
nucleus, IkBa represses NF-kB-dependent and -independent transcription. Indeed, on the one hand, IkBa induces
NF-kB nuclear export, leading to the termination of NF-kB
transcriptional activation [55]. On the other hand, transcription from the promoter of the Notch-target gene Hes1
(which is not a classical NF-kB target gene) is repressed by
IkBa, which is recruited to this promoter together with NCoR, HDAC-1 and HDAC-5 [7]. TNFa treatment causes a
temporary release of IkBa from the Hes1 promoter, which
is correlated with increased histone acetylation and transcriptional activation. Because IkBa is not detected on the
IL-6 or RANTES (regulated upon activation, normally T
cell expressed and presumably secreted) NF-kB-dependent
promoters, this regulatory mechanism probably does not
operate on classical NF-kB target genes [7]. Thus, IkBa
probably represses transcription by tethering HDACs to
promoter regions, a mechanism that could be involved in
the termination of NF-kB-independent transcriptional
activation.
In addition to its cytoplasmic role in the IKK complex,
IKKa is recruited to NF-kB-dependent promoters after
TNFa treatment, where it phosphorylates histone H3
Ser10. Because IKKa interacts with CBP [8] and histone
phosphorylation is often associated with histone acetylation [56,57], the association of a kinase such as IKKa
with an acetyltransferase (e.g. CBP) could enable the
removal of a repressive chromatin structure associated
with NF-kB-dependent genes. It recently was proposed
Trends in Biochemical Sciences
Vol.33 No.7
that IKKa could promote p65 acetylation at some
promoters via both CBP recruitment and p65 phosphorylation [24,58]. Furthermore, IKKa modifies HDAC-3
recruitment on NF-kB-dependent genes in a promoterspecific manner [58]. Indeed, although this kinase is
necessary for the removal of SMRT and HDAC-3 from
some promoters where HDAC-3 inhibits p65 binding
[39], IKKa also might function as a repressor that tethers
small quantities of HDAC-3 to the IkBa promoter, thereby
preventing excessive p65–DNA binding [39,58]. Moreover,
trichostatin A (TSA), an HDAC inhibitor, treatment
impairs IKKa recruitment to the IkBa promoter, indicating a role for HDACs in this recruitment [59].
IKKg (also called NEMO), the IKK complex regulatory
subunit, shuttles between the cytoplasm and the nucleus
and interacts directly with CBP [9]. In addition to the key
role of IKKg in regulating cytokine-induced IKK activity,
this regulatory subunit competes with p65 and IKKa for
binding to the CBP N terminus, thereby inhibiting
p65- and IKKa-induced CBP-dependent transcriptional
activation [9]. These findings could explain the downregulation of NF-kB activity that occurs under basal conditions
and the post-induction repression of the NF-kB pathway
[9].
Thus, the recruitment of HATs and HDACs by IkBa,
IKKa and IKKg adds a level of complexity to the interplay
between acetylation-dependent transcriptional regulation
and NF-kB-dependent transcriptional regulation.
Acetylation-dependent crosstalk between the NF-kB
pathway and other signaling pathways
The cell must integrate the functions of different pathways
to generate a coherent biological response to environmental changes. In agreement with this, the NF-kB response
results from the integration of the NF-kB and other signaling pathways, which can modulate either kB-site accessibility or NF-kB activity through histone and non-histone
protein post-translational modifications. In this section we
discuss the prominent role of acetylation and deacetylation
events and HAT and/or HDAC recruitment in the crosstalk
between the NF-kB and other signaling pathways.
In unstimulated cells, NF-kB binding sites in the promoter regions of genes such as RANTES, chemokine (C-C
motif) ligand 2 (ccl2) also known as monocyte chemoattractant protein-1 (mcp-1), and IL-6 are in a repressedchromatin environment that prevents NF-kB–DNA binding. These sites become accessible after histone acetylation
mediated by the activation of other signaling pathways [60]
(Figure 3). IKKa, after cytokine stimulation or contextual
conditioned fear memory retrieval, and p38 MAPK (mitogen-activated protein kinase), after LPS stimulation, are
recruited to specific NF-kB-responsive promoters where
they phosphorylate histone H3 at Ser10, thus promoting
subsequent H3 Lys14 acetylation and transcriptional activation [8,56,57,61,62]. Importantly, after cytokine stimulation IKKa also phosphorylates CBP at Ser1382 and
Ser1386, thus enhancing CBP-binding affinity for p65
and CBP HAT activity and, consequently, histone H3
acetylation [63]. Similarly, MAPK p38- or Akt-mediated
p300 phosphorylation enhances its acetyltransferase
activity and might facilitate p65–p300 complex formation,
345
Review
resulting in p65 Lys310 acetylation and optimal p65
transactivation activity [64,65].
Different signaling pathways can interfere with one
another by modulating the availability of HATs or HDACs
for a particular transcription complex. For example, p65
and p53 directly compete for CBP or p300 [63], and this
competition is responsible for their cross-repression. After
IKKa-mediated CBP phosphorylation, the CBP–p65 interaction increases, whereas the CBP–p53 interaction
decreases [63]. A consequence of the functional relationship between p53 and p65 is the ability of p65 to facilitate
p53-induced cell death, probably because p53 converts NFkB to a repressor of antiapoptotic gene expression. Glycogen synthase kinase 3b (GSK3b), a constitutively active
downstream kinase in the phosphoinositide 3-kinase (PI 3kinase) pathway, regulates the NF-kB-mediated inflammatory response by affecting the amount of nuclear CBP
that is complexed with p65 or CREB. Indeed, GSK3b
phosphorylates and inactivates CREB, thereby disrupting
the CREB–CBP interaction. Thus, GSK3b inactivation
releases CREB repression, promotes CREB–CBP interaction and, as a consequence, reduces CBP access to NFkB, thereby dampening NF-kB transcriptional potential
[66]. PPARg is a nuclear receptor in the peroxisome proliferator-activated receptor (PPAR) family that inhibits
inflammatory responses by repressing NF-kB-mediated
gene expression [67]. It is likely that PPARg suppresses
Eotaxin (an NF-kB target) gene expression by direct inhibition of p65-associated HAT activity, for example, by
competing with p65 for limited amounts of CBP and/or
by recruiting HDAC to the p65–HAT complex [67].
Acetylation and deacetylation events regulate the interactions between NF-kB and other transcription factors,
including the glucocorticoid receptor (GR) and STAT1
(signal transducer and activator of transcription 1). Recent
studies indicate that the GR acetylation status modulates
its interaction with NF-kB [68], which is probably responsible for the inhibitory effect on inflammatory and immune
genes by glucocorticoids, the most effective class of antiinflammatory agents [69]. More specifically, HDAC-2mediated deacetylation of the GR, which is acetylated after
ligand binding, enables GR–p65 interaction and the subsequent attenuation of NF-kB-mediated, but not GRmediated, gene transcription [68]. This mechanism could
provide a molecular explanation for the repression of NFkB-mediated IL-8 transcription induced by GR-associated
steroid receptor coactivator-2 (SRC-2), a member of the
p160 family of coactivators [70]. Indeed, when tethered to
DNA by protein–protein interactions, GR-associated SRC2 represses transcription via its unique corepression
surface; by contrast, GR interaction with SRC-2 at glucocorticoid response elements (GREs) results in transcriptional activation [70]. Thus, because the GR acetylation
status regulates GR–NF-kB binding, HDAC-2 might be
crucially involved in regulating the SRC-2 coactivator–
corepressor switch.
Similarly to GR acetylation, direct acetylation of STAT1,
which is a mediator of interferon signaling, modulates its
ability to regulate NF-kB activity [71]. Interferon-a-induced
STAT1 acetylation, within its DNA-binding domain,
enables its interaction with p65. As a consequence, the level
346
Trends in Biochemical Sciences Vol.33 No.7
of nuclear p65 strongly decreases and NF-kB–DNA binding
is inhibited, thereby leading to the downregulation of antiapoptotic NF-kB target genes [71]. These findings offer an
explanation for the induction of apoptosis observed in a
human melanoma cell line after a combined treatment with
HDAC inhibitors and interferon a [71].
Interference between signaling modules also can terminate signal-induced gene expression. Indeed, although
activating protein-1 (AP-1)-mediated histone H4 acetylation is required for NF-kB–DNA binding at promoters
with regulated and late NF-kB accessibility [60] (Figure 3),
AP-1 also can be recruited to some activated NF-kB target
promoters at a later time in induction and can inhibit
transcription by recruiting a specific HDAC complex to
modify local histone acetylation patterns [72]. AP-1mediated HDAC recruitment to specific NF-kB target
promoters represses only the promoters containing AP1-binding sites and, because most AP-1 transcription factors are synthesized after activation of the transcriptional
response, this mechanism also provides a sufficient time
delay for transcriptional termination [72].
In conclusion, NF-kB activity is regulated at multiple
levels by acetylation and deacetylation events that occur in
the NF-kB-activation pathway or regulate the crosstalk
between the NF-kB pathway and other signaling pathways.
Concluding remarks and future perspectives
NF-kB was discovered in 1986 [73]; ten years later, CBP
and p300 were demonstrated to possess HAT activity [5].
Shortly thereafter, NF-kB-dependent transcription was
shown to require multiple coactivators that possess HAT
activity, including CBP and p300. The interactions between NF-kB and these HATs indicated the existence of
a link between histone acetylation and NF-kB-mediated
transactivation.
It is now well established that lysine acetylation plays a
crucial part in regulating NF-kB-dependent gene transcription. Indeed, interactions between several members
of the NF-kB pathway with HATs and/or HDACs, direct
acetylation of NF-kB family members, and acetylation and
deacetylation events regulating the crosstalk between the
NF-kB pathway and other signaling pathways, modulate
NF-kB activation and transcriptional potential both at
the chromatin level and at the non-histone protein level.
The specificity of these regulatory events depends on the
inducer, the promoter and the cell line considered. An
important issue for future investigations is to further
elucidate how this specificity is achieved, and how it relates
to what we have termed the ‘NF-kB-signaling code’.
Much remains to be learned about the role of nonhistone protein acetylation in the NF-kB pathway. Indeed,
the specific role of p50 Lys431, Lys440 and Lys441 acetylation in addition to the acetylation target site(s) in p52
remain undefined. Moreover, future studies will probably
identify acetylation-dependent regulation of other members of the NF-kB pathway. In this regard, we have
reported that TSA prolongs TNFa-induced IKK activity,
thus suggesting that unidentified acetylation events
regulate IKK activity [27,74]. Because temporal control
of NF-kB activation can direct specific gene-expression
Review
patterns [75], stimulus-specific temporal control of IKK
activity could promote distinct biological responses and
could introduce an additional level of complexity to
the known intricate regulation of the NF-kB-activating
pathway.
To gain a complete understanding of the multi-level and
multi-dimensional network that regulates NF-kB activation and activity in a given cell type in response to a
given inducer will be a huge challenge in the coming years.
Abnormal NF-kB activation is observed in several inflammatory diseases, neurodegenerative disorders and cancers
[76]. However, molecules that block NF-kB activation have
shown weak clinical efficacy, and their lack of specificity
causes side effects by interfering with NF-kB physiological
roles in immunity, inflammation and cellular homeostasis
[76]. One of the major challenges in clinical research is to
develop a series of NF-kB inhibitors aimed at treating
different diseases, based on their ability to target specific
pathways or cells, thereby avoiding the risk of undesired
side effects. To this end, it is important to investigate the
molecular mechanisms that govern the differential association of NF-kB with coactivator and corepressor proteins
that have profound consequences on many cellular processes and are at least partially responsible for NF-kB
response specificity. Several HDAC inhibitors have been
developed by the pharmaceutical industry and are promising agents for cancer treatment [77]. However, NF-kB
provides a strong protective role against HDAC-inhibitormediated apoptosis; thus, a combined molecular strategy
that inhibits both NF-kB and HDAC activities might lead
to the design of an efficient anticancer therapy [78]. Thus,
further investigation of the underlying molecular mechanisms of HAT- and HDAC-mediated NF-kB-pathway regulation, and the identification of additional specific NF-kB
and HDAC inhibitors should provide us with new therapeutic opportunities.
Acknowledgements
We thank the reviewers for helpful comments on this manuscript and we
apologize to those whose work could not be cited directly because of space
limitations. We acknowledge grant support from the Belgian Fund for
Scientific Research (FRS-FNRS, Belgium), the Télévie-Program of the
FRS-FNRS, the Action de Recherche Concertée du Ministère de la
Communauté Française (Université Libre de Bruxelles, ARC program no.
04/09–309), the Internationale Brachet Stiftung, the Fondation contre le
Cancer, the Interreg III program (Intergenes project), the Région
Wallonne (Program WALEO2 616295) and the Theyskens-Mineur
Foundation. M.C. is a fellow of the Belgian Fonds pour la Recherche
dans l’Industrie et l’Agriculture. A.D. and C.V.L. are Chargé de
Recherches and Directeur de Recherches of the FRS- FNRS, respectively.
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Articles of interest in other Cell Press journals
Human Alu RNA Is a Modular Transacting Repressor of mRNA Transcription during Heat
Shock
Peter D. Mariner,Ryan D. Walters, Celso A. Espinoza, Linda F. Drullinger, Stacey D. Wagner,
Jennifer F. Kugel, and James A. Goodrich
Molecular Cell February 29, 2008
Molecular Dissection of Mammalian RNA Polymerase II Transcriptional Termination
Steven West, Nicholas J. Proudfoot, and Michael J. Dye
Molecular Cell March 14, 2008
Poised Polymerases: On Your Mark. . .Get Set. . .Go!
David Price
Molecular Cell April 11, 2008
Molecular Evolution of RNA Polymerase II CTD
Rob D. Chapman, Martin Heidemann, Corinna Hintermair and Dirk Eick
Trends in Genetics June 2008
Deciphering the (expanding) RNA polymerase II CTD code
Sylvain Egloff and Shona Murphy
Trends in Genetics June 2008
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