SETD6 lysine methylation of RelA couples GLP activity at chromatin to tonic repression of NF-κB signaling

Article (PDF Available)inNature Immunology 12(1):29-36 · January 2011with37 Reads
DOI: 10.1038/ni.1968 · Source: PubMed
Abstract
Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-κB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-ζ (PKC-ζ) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs.
nature immunology VOLUME 12 NUMBER 1 JANUARY 2011 2 9
A R T I C L E S
Chromatin dynamics regulate key cellular functions that influence
survival, growth and proliferation, and disruption of chromatin homeo-
stasis has been linked to diverse pathologic processes
1
. Methylation of
lysine residues of histone, which is catalyzed by protein lysine methyl-
transferases (PKMTs), is a principal chromatin-regulatory mecha-
nism involved in directing fundamental DNA-templated processes
such as transcription and DNA repair
1
. Histone methylation plays a
central part in orchestrating proper programming of the genome in
response to various stimuli, and aberrant signaling via lysine methyl-
ation has been linked to the initiation and progression of many human
diseases
2
. Many non-histone proteins are also regulated by lysine
methylation, which indicates that this modification is probably a
common mechanism for the modulation of protein-protein inter-
actions and signaling pathways
3
.
NF-κB is a transcription factor and key inducer of inflammatory
responses
4,5
. One of the principal subunits of NF-κB is RelA (p65
(A001645)), which forms either a homodimer or a heterodimer
with the structurally related p50 protein (A002937). Under basal
conditions, most RelA is sequestered in the cytoplasm because of
its association with members of the inhibitor of NF-κB family of
proteins
4,5
. Stimulation of cells with NF-κB-activating ligands such
as the cytokine tumor necrosis factor (TNF) results in degradation
of these inhibitors of NF-κB and translocation of the released NF-κB
to the nucleus, where it directs various transcriptional programs
5,6
.
In addition to that canonical pathway, there are several additional
mechanisms that regulate and fine-tune NF-κB signaling and activa-
tion of target genes
7
. For example, various post-translational modifi-
cations of RelA influence the specificity, transcriptional activity and
activation kinetics of RelA target genes. Furthermore, even in resting
conditions, a population of RelA is present in the nucleus, bound at
chromatin; however, the functional relevance of this constitutively
nuclear population is not known.
Deregulation of NF-κB signaling is linked to many human diseases,
including cancer and autoimmune disorders
8
. Thus, understanding
the full range of molecular mechanisms that modulate this factor in
response to diverse conditions has important biological and clinical
implications. Here we screened over 40 known and candidate human
PKMTs for methylation of RelA in vitro. We identify SETD6 (SET
domain–containing protein 6) as a PKMT that monomethylated
RelA at Lys310 (RelAK310me1). The ankryin repeat of the PKMT
1
Department of Biology, Stanford University, Stanford, California, USA.
2
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia,
USA.
3
Laboratory of Lymphocyte Signaling, The Rockefeller University, New York, New York, USA.
4
EpiNova DPU, Immuno-Inflammation group, GlaxoSmithKline,
Stevenage, UK.
5
Department of Carcinogenesis, M.D. Anderson Cancer Center, Smithville, Texas, USA.
6
Department of Molecular Biology, Princeton University,
Princeton, New Jersey, USA.
7
Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, California,
USA.
8
Department of Endocrinology, Gerontology, and Metabolism Medicine, Stanford University School of Medicine, Stanford, California, USA.
9
Center for Cancer
Epigenetics, University of Texas M.D. Anderson, Houston, Texas, USA.
10
Geriatric Research, Education, and Clinical Center, VA Palo Alto Health Care System,
Palo Alto, California, USA. Correspondence should be addressed to O.G. (ogozani@stanford.edu).
Received 15 October; accepted 9 November; published online 5 December 2010; doi:10.1038/ni.1968
Lysine methylation of the NF-kB subunit RelA by
SETD6 couples activity of the histone methyltransferase
GLP at chromatin to tonic repression of NF-kB signaling
Dan Levy
1
, Alex J Kuo
1
, Yanqi Chang
2
, Uwe Schaefer
3
, Christopher Kitson
4
, Peggie Cheung
1
,
Alexsandra Espejo
5
, Barry M Zee
6
, Chih Long Liu
1,7
, Stephanie Tangsombatvisit
7
, Ruth I Tennen
8
,
Andrew Y Kuo
1
, Song Tanjing
9
, Regina Cheung
7
, Katrin F Chua
8,10
, Paul J Utz
7
, Xiaobing Shi
9
, Rab K Prinjha
4
,
Kevin Lee
4
, Benjamin A Garcia
6
, Mark T Bedford
5
, Alexander Tarakhovsky
3
, Xiaodong Cheng
2
& Or Gozani
1
Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the
catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened
over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription
factor NF-kB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated
RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized
by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at
RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation–linked phosphorylation of RelA
at Ser311 by protein kinase C-z (PKC-z) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene.
Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs.
© 2011 Nature America, Inc. All rights reserved.
3 0 VOLUME 12 NUMBER 1 JANUARY 2011 nature immunology
A R T I C L E S
GLP (G9A-like protein) functioned as a recognition module for
RelAK310me1, which links this mark to localized methylation of
histone H3 Lys9 (H3K9) and repressed chromatin at RelAK310me1-
occupied genes
9,10
. The SETD6-initiated lysine-methylation signaling
cascade acted to restrain activation of NF-κB-mediated inflammatory
responses in diverse cell types. This repressive pathway was terminated
by phosphorylation of RelA at Ser311 by the atypical protein kinase
PKC-ζ (A001934)
11
, which blocked recognition of RelAK310me1 by
GLP to promote the expression of target genes of RelA. Together our
findings identify SETD6 as a previously uncharacterized regulator
of the NF-κB network, identify the ankryin-repeat domain of GLP
as an effector of methylated RelA, describe a metazoan example of a
methylation-phosphorylation switch on a non-histone proteins, and
demonstrate a new paradigm for how integrated crosstalk between
modifications on transcription factors and histones modulates key
physiological and pathological programs.
RESULTS
Monomethylation of RelA at Lys310 by SETD6
To identify additional activities for predicted PKMT enzymes and
lysine-methylation events, we screened most of the SET domain–
containing proteins present in the human proteome for in vitro
catalytic activity on various histone and non-histone candidate
substrates (Supplementary Table 1 and data not shown). SETD6,
a previously uncharacterized PKMT, methylated an N-terminal
RelA polypeptide encompassing amino acids 1-431 (RelA(1–431))
but not a C-terminal polypeptide (residues 430–531; Fig. 1a and
Supplementary Fig. 1a,b). Substitution of individual lysine residues
with arginine in RelA(1–431) identified Lys310 as the target site of
SETD6 (Fig. 1b). In contrast, SET7-SET9, which methylates RelA
at several lysine residues
12,13
, was active on the RelA mutant with
replacement of lysine with arginine at position 310 (RelA(K310R);
Supplementary Fig. 1c). Mass-spectrometry analysis of SETD6-
catalyzed methylation of RelA peptides spanning Lys310 (amino
acids 300–320) demonstrated that SETD6 added only a single methyl
moiety to RelA at Lys310 (Fig. 1c and Supplementary Fig. 2).
We raised antibodies to SETD6 (Supplementary Fig. 3) and the
RelAK310me1 epitope (anti-RelAK310me1). Anti-RelAK310me1
specifically recognized RelAK310me1 peptides and did not detect
unmodified RelA, RelA dimethylated or trimthrylated at Lys310,
or numerous methylated histone peptides (Supplementary Fig. 4).
Furthermore, anti-RelAK310me1 detected RelA(1431) that had
been methylated in vitro by SETD6 but failed to detect unmethylated
RelA(1431) or RelA(K310R) (Fig. 1d).
RelA(1–431)
RelA(1–431)
RelA(300–320) RelA(300–320) + SETD6
RelA(1–431)
555.896
555.896
556.096
556.096
555.696
555.695
556.296
556.296
556.497
556.497
+ me1
556.697
556.697
558.699
559.098
559.297
558.498
SETD6
SETD6
RelA
3
H
3
H
RelA
Coomassie
3
H
Coomassie
250
100
100
80
60
40
20
0
100
80
60
40
20
0
554 554555 555556 556557 557558 558559 559560 560
m/z m/z
SETD6 WT
SETD6 WT
+
+
SETD6
SETD6
SETD6(Y285A)
SETD6(Y285A)
RelAK310me1
RelA
RelA
SETD6
RelA WT
+
+
+
+
+
+ +
WT K310R
SETD6
SETD6
SETD6
SETD6
SETD6 siRNA:
293T
C 1 2
ChIP
293T U2OS
+ + + +
RelA(K310R)
RelA(1–431)
RelAK310me1
RelAK310me1
RelAK310me1
RelAK310me1
RelAK310me1
RelA
RelA
RelA
RelA
RelA
RelA
TNF
Cyt CytChrom Chrom
H3
H3
H3
Input
lgG
RelA
Cyt
Nuc
Chrom
Tubulin
Tubulin
Tubulin
Tubulin
RelA
WCE
IP: anti-RelA
WCE
IP: anti-RelA
Abundance (%)
Abundance (%)
(kDa)
37
75
50
150
SETD2(SET)
WT
K122R
K123R
K218R
K310R
K221R
SETD5(SET)
SETMAR(SET)
ASH1(SET)
SUV39H2
PRDM4(SET)
PRDM2(SET)
SETD4
G9a(SET)
GLP(SET)
SETDB2
SETDB1
SETD6
SETD8
SETD2(SET)
SETD5(SET)
SETMAR(SET)
ASH1(SET)
SUV39H2
PRDM4(SET)
PRDM2(SET)
SETD4
G9a(SET)
GLP(SET)
SETDB2
SETDB1
SETD6
SETD8
RelA(1–431)
a b
c
d e f
g h i j k
Figure 1 SETD6 monomethylates RelA at Lys310. (a) Methylation reactions (
3
H autoradiogram; left) with recombinant RelA(1–431) as substrate and
recombinant PKMT enzymes (full-length or SET domains only; above lanes) and Coomassie staining (right) of recombinant enzymes (left margin, molecular
size in kilodaltons (kDa)). Top band, automethylated SETD6. (b) SETD6-catalyzed methylation assay (autoradiogram; top) with wild-type or mutant
RelA(1–431) (above lanes), and Coomassie staining (below) of proteins used. (c) Mass spectrometry analysis of methylation assays of RelA peptide (amino
acids 300–320; RelA(300–320)) with (right) or without (left) SETD6; results are presented relative to those of the most abundant ion, set as 100%.
Numbers above peaks indicate the mass/charge (m/z) ratio. (d) Immunoblot analysis of methylation reactions with (+) or without (−) SETD6 on wild-
type (WT) RelA(1–431) or RelA(1–431) with the K310R substitution. (e) Immunoblot analysis of whole-cell extracts (WCE; 5% of total) of 293T cells
transfected with Flag-tagged SETD6 and either RelA or RelA(K310R), probed with antibodies to various molecules (left margin). (f) In vitro methylation
reaction (top) with wild-type SETD6 or SETD6(Y285A) on RelA(1–431) and Coomassie staining (below) of recombinant proteins used. (g) Immunoblot
analysis of immunoprecipitated RelA (IP) or WCE (5% of total) from 293T cells transfected with wild-type SETD6 or SETD6(Y285A). (h) Immunoblot
analysis (as in g) of 293T cells treated with control (C) or SETD6-specific siRNA (two independent siRNAs: 1 and 2). (i) Immunoblot analysis of RelA or
control immunoglobulin G (IgG) protein-protein ChIP, probed with antibodies to various molecules (left margin). Input, 5% of total. (j) Immunoblot analysis
of 293T cells biochemically separated into cytoplasmic (Cyt), nucleoplasmic (Nuc) or chromatin-enriched (Chrom) fractions; tubulin and H3 signals serve
as controls for fractionation integrity. (k) Immunoblot analysis of cytoplasmic and chromatin-enriched fractions (as in j) from 293T and U2OS cells with (+)
or without (−) treatment with TNF (10 ng/ml for 1 h). Data are representative of three (a,b,e,g,h) or two (c,d,f,ik) independent experiments.
© 2011 Nature America, Inc. All rights reserved.
nature immunology VOLUME 12 NUMBER 1 JANUARY 2011 3 1
A R T I C L E S
In cotransfection experiments, wild-type RelA was monomethyl-
ated by overexpressed SETD6, but RelA(K310R) was not (Fig. 1e).
Structure-based homology modeling indicated that SETD6 was most
similar to the plant enzyme LSMT (Rubisco large subunit methyltrans-
ferase)
14
(Supplementary Fig. 5). On the basis of that homology, we
identified SETD6 with replacement of tyrosine with alanine at posi-
tion 285 (SETD6(Y285A)) as a catalytic SETD6 mutant in vitro (Fig. 1f
and Supplementary Figs. 5 and 6) and found that overexpression of
SETD6 led to more monomethylation of endogenous RelA at Lys310,
but overexpression of SETD6(Y285A) did not (Fig. 1g). Finally, deple-
tion of endogenous SETD6 protein in 293T cells by RNA-mediated
interference (RNAi) with two independent small interfering RNAs
(siRNAs) resulted in less endogenous RelAK310me1 (Fig. 1h). On the
basis of these data, we conclude that SETD6 monomethylates RelA
at Lys310 in vitro and is required for maintenance of physiological
concentrations of RelAK310me1 in cells.
Chromatin association of RelAK310me1 under basal conditions
In unstimulated cells, most RelA is localized to the cytoplasm, but
a population of RelA is present in the nucleus
15
. In this context,
protein-protein chromatin-immunoprecipitation (ChIP) assays in
the absence of stimulation demonstrated association of RelA with
histone H3 (Fig. 1i). Moreover, we detected RelA by ChIP at the
promoters of several target genes in unstimulated cells of many types
(Supplementary Fig. 7). SETD6 was also present in the nucleus
(Supplementary Fig. 3b). RelA and SETD6 interacted in vitro and
were coimmunoprecipitated from cells (Supplementary Fig. 8ad).
Thus, we reasoned that in contrast to most of RelA, which is localized
to the cytoplasm in unstimulated cells
4
, the population of RelA that is
monomethylated at Lys310 might reside and function in the nucleus.
In support of our hypothesis, RelAK310me1 was biochemically frac-
tionated almost exclusively with chromatin in unstimulated 293T
human embryonic kidney cells and U2OS human osteosarcoma cells
(Fig. 1j,k). Treatment with TNF, which activates NF-κB
4
, resulted
in much less RelAK310me1 at chromatin than did no stimulation
(Fig. 1k). On the basis of these data, we conclude that RelAK310me1
is present at chromatin in unstimulated cells.
Next we did ChIP assays with U2OS cells under basal conditions to
determine if RelAK310me1 is bound to chromatin at the promoters of
RelA target gene. RelAK310me1 occupied the promoters of several RelA
target genes (IL8, IL1A, MYC and CCND1), and detection of this RelA
species at target promoters was lost in U2OS cells treated with either of
two independent siRNAs targeting SETD6 (Fig. 2a and Supplementary
Fig. 9a), as well as in U2OS cells stably expressing a short hairpin RNA
U2OS U2OS THP-1
0.045
0.030
0.015
0
0.045
0.030
0.015
0
0.24
0.16
0.08
0
RelAK310me1 (% input)
RelAK310me1 (% input)
RelAK310me1 (% input)
RelAK310me1 (% input)
RelAK310me1 (% input)
RelAK310me1 (% input)
0.12
0.08
0.04
0
siRNA: C
SETD6 C SETD6
1 2
siRNA:
C
SETD6
C
SETD6
C
SETD6
siRNA: C SETD6
1 2
C SETD6
1 2
C SETD6
1 2
siRNA: C SETD6
1 2
C SETD6
1 2
C
SETD6
1 2
C
SETD6
1 2
1 2 1 2 1 2
siRNA: C SETD6 C SETD6
1 2 1 2
1 2
0.24
0.16
0.08
0
0.045
0.030
0.015
0
0.045
0.030
0.015
0
0.045
0.030
0.015
0
0.045
0.030
0.015
0
0.12
0.08
0.04
0
TNF:
+
+
TNF:
+ +
0.03
0.02
0.01
0
0.03
0.02
0.01
0
1.6
1.2
0.8
0.4
0
Luciferase
activity (relative)
SETD6 mRNA (relative)
IL8 mRNA (relative)
IL1A mRNA (relative)
SETD6
SETD6
WT Y285A
Actin
1.2
0.8
0.4
0
480
320
160
0
21
14
7
0
U2OS
U2OS
THP-1 mBMDM
IL1A mRNA (relative)
TNF mRNA (relative)
IL8 mRNA (relative)
II1a mRNA (relative)
II1a mRNA (relative)
Tnf mRNA (relative)
Tnf mRNA (relative)
300
200
100
0
7.5
5.0
2.5
0
36
24
12
0
45
30
15
0
90
60
30
0
210
140
70
0
900
600
300
0
Primary mBMDM
THP-1
a c
e
f
b
g
h
IL8
GAPDH
IL1A
GAPDH
IL8
GAPDH
IL1A
GAPDH
IL8
GAPDH
IL1A
GAPDH
CCND1
GAPDH
MYC
GAPDH
CCND1
GAPDH
CCND1
GAPDH
– TNF + TNF
– TNF + TNF
+ TNF
– TNF
+ LPS
– LPS
MYC
GAPDH
MYC
GAPDH
siRNA:
C
SETD6
1 2
Luciferase activity
(relative)
12
8
4
0
TNF
d
Figure 2 Monomethylation of RelA by SETD6 inhibits the transactivation activity of RelA.
(a) Occupancy of RelAK310me1 at the promoters of IL8, IL1a, MYC, CCND1 and GAPDH (control)
in U2OS cells treated with control or SETD6 siRNA, assessed by real-time PCR analysis of ChIP
samples; enrichment is presented as (ChIP/input) × 100. (b) ChIP assays (as in a) of U2OS cells
(left) and THP-1 cells (right) with (+) or without (−) TNF stimulation (20 ng/ml for 1 h). ChIP with
negative control antibody (a,b), Supplementary Figure 9. (c) Activity of a κB-Luc luciferase reporter
(top) 24 h after transfection of U2OS cells with increasing amounts (wedges) of wild-type SETD6 or
SETD6(Y285A); results are normalized to those of renilla luciferase and are presented relative to those
of cells transfected with control vector. Below, immunoblot analysis of SETD6 and SETD6(Y285A).
(d) Luciferase activity (as in c) in 293T cells transfected with control or SETD6-specific siRNA with (right) or without (far left) TNF treatment (10 ng/ml
for 1 h). (e) Real-time PCR analysis of the efficiency of knockdown of SETD6 mRNA by SETD6-specific siRNA in U2OS cells, THP-1 cells and mouse
BMDMs (mBMDM), presented relative to its expression in cells transfected with control siRNA (C). (fh) Real-time PCR analysis of various mRNAs
(vertical axes) in U2OS cells (f), THP-1 cells (g) and primary mouse BMDMs (h) transfected with control or SETD6-specific siRNA with or without TNF
(20 ng/ml for 1 h) or LPS (100 ng/ml for 1 h). Data are from at least three experiments (error bars, s.e.m.).
© 2011 Nature America, Inc. All rights reserved.
3 2 VOLUME 12 NUMBER 1 JANUARY 2011 nature immunology
A R T I C L E S
(shRNA) targeting SETD6 (Supplementary Fig. 10). Consistent with
the results obtained with the cellular fractionation assays (Fig. 1j,k),
treatment with TNF resulted in less occupancy by RelAK310me1 at
promoters of target genes both in U2OS cells and in the THP-1 acute
monocytic leukemia cell line (Fig. 2b and Supplementary Fig. 9b).
Thus, monomethylation of RelAK310 is a chromatin-associated modi-
fication and is inversely correlated with activation of NF-κB by TNF.
SETD6 attenuates transcription of RelA target genes
To investigate the relationship between SETD6 and the transcriptional
activity of RelA, we cotransfected U2OS cells with an NF-κB-driven
reporter
16
and either SETD6 or SETD6(Y285A). The activity of this
reporter was repressed by SETD6 in a dose-dependent manner and
required that the catalytic activity of SETD6 be intact (Fig. 2c). In addi-
tion, depletion of SETD6 resulted in more reporter activity in unstimulated
cells (Supplementary Fig. 11) and after exposure to TNF (Fig. 2d). These
data suggested that SETD6 represses physiological transactivation by RelA.
To test that hypothesis, we depleted U2OS cells, THP-1 cells and primary
mouse bone marrow–derived macrophages (BMDMs) of SETD6 (Fig. 2e)
and measured mRNA for canonical NF-κB targets in the presence or
absence of NF-κB stimulation
4
(Fig. 2fh). In response to TNF, knockdown
of SETD6 led to more expression of RelA target genes than their expres-
sion in control cells for all three cell types (Fig. 2fh). We obtained similar
results with mouse BMDMs stimulated with lipopolysaccharide (LPS;
Fig. 2h, right). In addition, depletion of SETD6 resulted in higher basal
expression of a subset of RelA target genes (Supplementary Fig. 12). We
noted that SETD6 did not methylate the RelA partner p50 (Supplementary
Fig. 1d) and, in contrast to known histone lysine methyltransferases such as
G9a and GLP
9,17
, SETD6 did not methylate nucleosomes (Supplementary
Fig. 1e). Finally, genome-wide gene-expression analysis of Rela
−/−
mouse
embryonic fibroblasts (MEFs)
18
reconstituted with wild-type mouse RelA
or mutant mouse RelA(K310R) showed that in the absence of stimulation,
cells complemented with RelA(K310R) expressed more RelA-regulated
genes and expression of these genes was higher than that of cells comple-
mented with wild-type RelA (Supplementary Fig. 13 and Supplementary
Data), which indicated involvement of Lys310 in regulating the expression
of RelA target genes. Together these results indicate that SETD6-mediated
methylation of RelAK310 has an inhibitory effect on expression of many
RelA-regulated genes.
Attenuation of RelA-driven inflammatory responses by SETD6
Hyperactive NF-κB has been linked to the development and progres-
sion of many types of cancer
8
. To investigate potential roles for SETD6
and the interaction of SETD6 and RelA in cellular transformation,
we established U2OS cells with stable expression of shRNAs targeting
SETD6 alone, RelA alone or SETD6 and RelA, or a control shRNA,
and assessed cell transformationassociated properties (Fig. 3a,b
and Supplementary Fig. 14). Knockdown of SETD6 accelerated the
proliferation rate of cells in a RelA-dependent way relative to the pro-
liferation of control cells (Fig. 3a). In addition, depletion of SETD6
conferred a 25-fold greater ability of cells to form colonies in soft
agar than that of control cells or cells depleted of RelA, and deple-
tion of both RelA and SETD6 reversed the anchorage-independent
growth advantage provided by knockdown of SETD6 alone (Fig. 3b).
Depletion of SETD6 also led to higher cell proliferation rates in wild-
type 3T3 mouse embryonic fibroblasts but not in Rela
−/−
3T3 cells
(Fig. 3c). Next, we depleted human U2OS cells and mouse 3T3 cells of
endogenous SETD6 through the use of shRNA targeting the 3 untrans-
lated region of human or mouse SETD6, respectively (Fig. 3d,e). We
reconstituted the cells depleted of SETD6 with exogenous SETD6
or exogenous SETD6(Y285A) that lacked the 3 untranslated region
and was therefore resistant to shRNA. Complementation with wild-
type SETD6 reestablished a normal proliferative rate, whereas com-
plementation with SETD6(Y285A) failed to do so (Fig. 3d,e). These
data suggest that the enzymatic activity of SETD6 regulates a RelA-
dependent effect on cell proliferation.
In addition to being linked to cancer, NF-κB—as a key regulator of
inflammation—has also been linked to the etiology of inflammatory
and autoimmune diseases
5,8
. Analysis of published gene-expression
data sets comparing peripheral blood mononuclear cells from
patients suffering from rheumatoid arthritis, septic shock or juvenile
idiopathic arthritis with control samples showed downregulation
of SETD6 mRNA in the disease state (Fig. 4a and Supplementary
Fig. 15). In addition, SETD6 expression was lower in patients with
rheumatoid arthritis who responded to TNF inhibitors than in
patients who were not responsive to this treatment (Supplementary
Fig. 15b), which suggested a role for NF-κB in their rheumatoid
arthritis disease.
The inverse correlation between SETD6 expression and NF-κB-
linked inflammatory diseases and the observation that SETD6 attenu-
ates RelA-dependent transactivation of cytokines such as interleukin
1α (IL-1α) and TNF suggest that SETD6 might have a role in mitigat-
ing NF-κB-driven inflammatory responses. Consistent with a negative
role for SETD6 in NF-κB signaling, in monocytic THP-1 cells exposed
to TNF, knockdown of SETD6 led to more production of the secreted
cytokines TNF and IL-6 than that of cells treated with control siRNA
Figure 3 SETD6 attenuates RelA-driven
cell proliferation. (a) Growth of U2OS cells
treated with control shRNA (Ctrl sh) or
SETD6-specific shRNA (SETD6sh) and/or
RelA-specific shRNA (RelAsh), assessed daily
for 7 d. (b) Colonies of U2OS cells, treated
as in a, in soft agar. (c) Growth of Rela
+/+
and Rela
−/
MEFs treated and assessed as in
a (right), and immunoblot analysis of WCE
of Rela
+/+
and Rela
−/
3T3 fibroblasts left
untreated () or treated with SETD6-specific
shRNA (+). (d,e) Growth curves (right) and
immunoblot analysis (of WCE; left) of U2OS
cells (d) and 3T3 fibroblasts (e) treated
with control or SETD6-specific shRNA and
reconstituted (Recon vec) with SETD6 or
SETD6(Y285A) or not reconstituted (C).
Data are from at least three independent
experiments (error bars, s.e.m.).
30
30
18
12
6
0
0
8
16
24
0
20
20
SETD6sh + RelAsh
SETD6sh
SETD6sh: + +
SETD6
RelA
Rela
+/+
Rela
+/+
SETD6sh
Rela
–/–
SETD6sh
Rela
–/–
Ctrl sh
SETD6(Y285A)
SETD6
SETD6sh
U2OS
3
2
1
0
3T3
Rela
–/–
3T3
Actin
SETD6
+
RelAsh
RelAsh
SETD6sh
U2OS
RelAsh
10
10
0
0
0 1 1
Time (d) Time (d)
Cells (×10
6
)
Cells (×10
6
)
Cells (×10
5
)
Cells (×10
5
)
Colonies per (field)
7
C
65 54 43 32
0 1
Time (d)
5432
2
0 1
Time (d)
5432
a b c
d e
SETD6(Y285A)
Recon. Vec:
SETD6
SETD6CtrlshRNA:
RelA
Actin
SETD6
C
C
SETD6(Y285A)
SETD6
C C
Recon. Vec:
SETD6
SETD6CtrlshRNA:
RelA
Actin
Rela
+/+
Ctrl sh
Ctrl sh
Ctrl sh
SETD6(Y285A)
SETD6
SETD6sh
Ctrl sh
© 2011 Nature America, Inc. All rights reserved.
nature immunology VOLUME 12 NUMBER 1 JANUARY 2011 3 3
A R T I C L E S
(Fig. 4b). We next assessed the relationship between SETD6 depletion
and cytokine production in mouse BMDMs. First, kinetic analysis
showed that in response to TNF and LPS, the abundance of Il1a and
Tnf mRNA was higher in cells depleted of SETD6 than in control cells
across a range of time points (Fig. 4c and Supplementary Fig. 16a;
SETD6-knockdown efficiency; Fig. 2e). Furthermore, multiplex
cytokine analysis of supernatants of these cells demonstrated upregula-
tion of nearly 20 secreted NF-κB-regulated cytokines in cells depleted
of SETD6 relative to their expression in control cells in response to
TNF (Fig. 4d) and LPS (Supplementary Fig. 16b). Finally, depletion
of SETD6 with two independent siRNAs in primary monocyte-derived
dendritic cells isolated from human donors (Fig. 4e) conferred a
Figure 4 SETD6 attenuates RelA-driven
inflammatory responses. (a) SETD6 expression
in patients with rheumatoid arthritis (RA; n = 8)
and healthy controls (n = 15), presented
as the normalized log
2
ratio of the sample
compared with a common reference (left);
and SETD6 expression in patients with septic
shock (n = 30) and healthy controls (n = 15),
presented relative to the median of the results
obtained with healthy controls (right). Each
symbol (in boxes) represents an individual
sample. *P = 0.0015 and **P = 0.00036
(two-tailed t-test). (b) Enzyme-linked
immunosorbent assay of cytokines in
supernatants of THP-1 cells transfected with
control siRNA or SETD6-specific siRNA with
or without TNF (20 ng/ml). (c) Real-time
PCR analysis of Il1a and Tnf mRNA in mouse
BMDMs transfected with control siRNA
(Ctrl si) or SETD6-specific siRNA (SETD6si).
(d) Multiplex enzyme-linked immunosorbent
assay of RelA-regulated cytokines in super-
natants of primary mouse BMDMs treated for
2 h as in c, presented as (SETD6 siRNA /
control siRNA – 1) × 100. (e) Real-time PCR
analysis of SETD6 mRNA in primary human monocyte-derived dendritic cells (hMDDC; n = 3 donors) transfected with control siRNA (C) or SETD6-
specific siRNA (1,2). (f) Secretion of TNF and IL-6 by human monocyte-derived dendritic cells transduced with siRNA as in e and treated for 6 or 24 h
with LPS (left; n = 3 donors) or at 24 h after treatment with 10 or 100 ng/ml of LPS (right; n = 1 donor). Data are an analysis of published studies (a;
error bars indicate minimum and maximum values within 1.5 interquartile range of the lower and upper quartile, respectively), are from at least three
independent experiments (b,c,e,f; error bars, s.e.m.) or are representative of two independent experiments (d).
c
d
h
e f g
b
RelA(300–320)
RelA(1–431)
RelAK310me1
RelAK310me1
RelAK310me1
RelAK310me1
RelAK310me1
RelAK310me1
RelAK310me1
RelA
RelA
RelA
RelA
RelA
RelA
0.045
0.030
0.05
0.02
0.06 0.45
0.30
0.15
0
0.04
0.015
0
0
0
0.15
0.10
RelA
RelA
RelA
RelA
RelA WT
GLP(ANK)
GLP
GLP
GLP
GLP
+
+
+
+
+
+
+
+
+
++
++
+
+
SETD6
SETD6
SETD6
SETD6
SETD6
–TNF
+TNF
GLP (% input)
H3K9me2 (% input)
H3K9me2 (% input)
IL8
SETD6
SETD6
MYC
SETD6
siRNA:
siRNA:
C C
C C
1 2 1 2
1 2 1 2
SETD6
SETD6si
SETD6
GLP
GLP
GLP
GLP
GLP
TNF
Ctrl si
GLP
GLP
GLP
GLP
(ANK)
ReIA
(K310R)
GLP(ANK)
Input
Input
GLP (% input)
IP: ReIA
IP: anti-ReIA
IP: anti-ReIA
IP: anti-ReIA
IP: anti-GLP
WCE
WCE
WCE
WCE
Input
Input
No protein
No peptide
No peptide
ReIA(300–320)
ReIA K310me1
ReIA K310me1
ReIA K310me1
ReIA K310me2
ReIA K310me3
H3K9me2
H3K9me1
H3K23me1
H3K27me1
H3K36me1
H4K20me1
H3K4me1
H3K14me1
H3K18me1
a
Figure 5 GLP(ANK) binds specifically to RelAK310me1. (a) CADOR microarray analysis of the binding
of proteins with 268 unique domains (Supplementary Fig. 17) to RelA(300–320) and RelAK310me1.
(b) Peptide-binding assay of the precipitation of various biotinylated peptides (above lanes) with glutathione
S-transferase-linked GLP(ANK). (c) Anti-RelA immunoprecipitation of RelA(1-431), either mock-methylated
(−) or methylated by SETD6 (+), then incubated with GLP(ANK), analyzed by immunoblot with anti-GLP or
anti-RelA. Below, immunoblot analysis of input (10% of starting material). (d) Immunoprecipitation (with
anti-Flag) of proteins from 293T cells transfected with plasmids encoding Flag-tagged GLP and wild-type RelA or RelA(K310R), followed by immunoblot
analysis of immunoprecipitates and WCE (10% of total). (e) Immunoprecipitation (with anti-RelA) of proteins from 293T cells left untransfected (−) or
transfected with plasmid encoding SETD6, followed by immunoblot analysis of immunoprecipitates and WCE (10% of total). (f) Immunoprecipitation
and immunoblot analysis as in e of 293T cells treated with control or SETD6-specific siRNA. (g) Immunoprecipitation and immunoblot analysis as in e of
U2OS cells transfected with plasmid encoding GLP, with or without TNF treatment (10 ng/ml). (h) Occupancy of GLP and H3K9me2 at the IL8 and MYC
promoters in U2OS cells treated with control siRNA (C) or SETD6-specific siRNA (1,2), with or without TNF treatment (20 ng/ml), assessed as in Figure 2a
(ChIP with negative control antibody, Supplementary Fig. 19). Data are representative of three (a,b) or two (cg) independent experiments or are from at
least three experiments (h; error bars, s.e.m.).
0.50
0.25
0
–0.25
–0.50
–0.75
–1.00
Ctrl
Donor 244
TNF (fold)
Ctrl
*
Septic
shock
THP-1
200
150
100
50
0
Eotaxin
G-CSF
GM-CSF
IL-10
IL-12p40
IL-13
IL-17
IL-2
IL-23
IL-1a
IL-1b
IP-10
KC
MCP-1
MCP3
IMIP1a
RANTES
VEGF
TNF
SETD6
(normalized expression)
**
SETD6
(log
2
expression)
1.4
1.2
1.0
0.8
0.6
0.4
Primary mBMDM
Primary mBMDM
Cytokine secretion
% increase
Primary hMDDC
Primary hMDDC
TNF (h)TNF (h)
1.6
RA
a b
c
e f
d
C C
SETD6
SETD6
TNF (pg/ml)
0.6
siRNA:
0.4
0.2
0 0
1
2
3
IL-6 (pg/ml)
2.7
1.8
0.9
0
0
1
2
3
IL-6 (fold)
6 24 6 24
LPS (h) LPS (h) LPS (ng/ml) LPS (ng/ml)
0
10 100
10 100
1
2
3
4.5
TNF (ng/ml)
IL-6 (ng/ml)
3.0
1.5
0
1.2
0.8
0.4
0
1
SETD6
SETD6 mRNA
(relative)
siRNA: C
2
–TNF +TNF
SETD6siCtrl si
Ctrl si SETD6si (1) SETD6si (2)
60
40
20
1
0 1 2 3 4 0
1
25
50
75
1 2 3 4
II1a mRNA (fold)
Tnf mRNA (fold)
© 2011 Nature America, Inc. All rights reserved.
3 4 VOLUME 12 NUMBER 1 JANUARY 2011 nature immunology
A R T I C L E S
time- and dose-dependent increase in secretion of the cytokines
TNF and IL-6 in response to LPS stimulation (Fig. 4f). Together
these experiments indicate that SETD6 inhibits the production of a
broad array of NF-κB-regulated cytokines in diverse cell types, includ-
ing antigen-presenting cells, which suggests that SETD6 is a critical
repressor of RelA-mediated inflammatory responses.
GLP ankyrin repeat is a RelAK310me1 effector domain
To understand the molecular basis of the repressive function
associated with RelAK310me1, we screened CADOR microarrays
19
for protein motifs that could potentially act as transducers of this
mark (Supplementary Fig. 17). Of the 268 proteins on the array, the
RelAK310me1 peptide bound specifically to only one: the ankyrin-repeat
domain of GLP (GLP(ANK); Fig. 5a). Peptide-precipitation assays and
measurement of dissociation constants independently confirmed and
characterized the interaction between GLP(ANK) and RelAK310me1
(Fig. 5b and Table 1). These data also demonstrated that except for the
positive-control monomethylated and dimethylated H3K9 peptides
20
,
other methylated histone peptides did not bind to GLP(ANK) (Fig. 5b)
and that GLP(ANK) had similar binding affinities for RelAK310me1
and monomethylated H3K9 (ref. 20; Table 1 and Supplementary
Fig. 18). In addition, GLP(ANK) did not bind nonmethylated or tri-
methylated RelAK310 peptides (Fig. 5b and Table 1), which indicated
that the recognition of RelAK310 requires mono- or dimethylation.
Finally recombinant GLP(ANK) bound recombinant RelA(1–431) in
coimmunoprecipitation experiments, but only after RelA(1–431) was
methylated in vitro by SETD6 (Fig. 5c). From these data we conclude
that GLP(ANK) binds specifically to RelAK310me1 in vitro.
Next we investigated the ability of RelAK310me1 to be recognized
by GLP in cells. Exogenous GLP coimmunoprecipitated overexpressed
wild-type RelA but not RelA(K310R) (Fig. 5d and Supplementary
Fig. 8e). In addition, SETD6 expression enhanced the interaction
between endogenous GLP and RelA (Fig. 5e). Decreasing the
abundance of RelAK310me1 by depleting SETD6 via RNAi or TNF
treatment inhibited the association of GLP with RelA (Fig. 5f,g).
These data suggest that in the absence of NF-κB activation, RelA
and GLP directly interact and that this interaction requires SETD6-
dependent monomethylation of RelA at Lys310.
GLP and its heterodimeric partner G9a generate mono- and
dimethylated H3K9 at euchromatin to repress transcription
9,21
, and
methylation of H3K9 suppresses the expression of inducible inflam-
matory genes
10
. Our observations that methylation of RelA by SETD6
inhibited the expression of NF-κB target genes and that GLP bound
to RelAK310me1 suggested a model in which the content of histone
H3 dimethylated at Lys9 (H3K9me2) is greater at RelAK310me1-
occupied NF-κB target genes in unstimulated cells because of the
stabilization of GLP through its interaction with RelAK310me1. Two
predictions of this model are as follows: first, under basal conditions
and in a SETD6-dependent manner, the chromatin of distinct RelA-
regulated gene promoters should be enriched for GLP and H3K9me2;
and second, GLP should be required for SETD6 to inhibit expression
of these RelA-regulated genes. In support of the first prediction, ChIP
assays demonstrated less occupancy by GLP and H3K9me2 at the
promoters of IL8 and MYC in response to TNF stimulation (Fig. 5h
and Supplementary Fig. 19), and knockdown of SETD6 with two
independent siRNAs largely eliminated the baseline enrichment of
GLP and H3K9me2 at these promoters (Fig. 5h and Supplementary
Fig. 19). We obtained similar results with cells stably depleted of
SETD6 by an shRNA approach (Supplementary Fig. 20). In addi-
tion, induction of RelAK310me1 via SETD6 overexpression resulted
in more occupancy by GLP and H3K9me2 at two RelA target promot-
ers (Supplementary Fig. 21a). Thus, these data demonstrate a role
for SETD6 and RelAK310me1 in stabilizing GLP activity at specific
RelA target genes.
Table 1 Binding affinity of GLP(ANK)
Peptide Ligand K
d
(µm)
RelA(300–320) GLP NB
RelAK310me1 GLP 4.8 ± 0.4
RelAK310me2 GLP 5.4 ± 0.5
RelAK310me3 GLP NB
RelAK310me1S311ph GLP NB
H3K9me1 GLP 5.0 ± 0.3
Isothermal titration calorimetry analysis of the affinity of the binding of GLP(ANK) to
various peptides (left column), presented as the dissociation constant (K
d
). NB, no
binding. RelAK310me2 and RelAK310me3, RelA di- and trimethylated, respectively,
at Lys310; RelAK310me1S311ph, RelAK310me1 with phosphorylation of Ser311.
Data are representative of three independent experiments (mean ± s.e.m.).
Figure 6 Phosphorylation of RelA at
S311 by PKC-ζ blocks GLP recognition of
RelAK310me1. (a) Model of the mechanism
by which a methylation-phosphorylation switch
at RelA Lys310 and Ser311 (bolded residues)
regulates the recognition of RelAK310me1
by GLP. (b) Peptide-binding assay of the
precipitation of various biotinylated peptides
(above lanes) with glutathione S-transferase–
linked GLP(ANK). (c) Immunoprecipitation
(with anti-RelA) of proteins from 293T cells left
untransfected (−) or transfected with plasmid
encoding PKC-ζ(ca), followed by immunoblot
analysis of immunoprecipitates and WCE
(5% of total). Bottom row, blot probed antibody
to V5-tagged PKC-ζ(ca). (d) Immunoblot
analysis of chromatin-enriched fractions
(Chrom) isolated from 293T cells with or
without treatment with TNF or transfection of
V5-tagged PKC-ζ(ca) (above lanes). (e) Dot-
blot analysis of in vitro kinase reactions with
recombinant PKC-ζ plus various peptides (left margin), spotted at a concentration of 0.25 µg/µl followed by 5× serial dilutions (wedges), probed with
anti-RelAS311ph, anti-RelAK310me1, anti-PKC-ζ or horseradish peroxidase (HRP)-conjugated streptavidin (loading control). (f) Immunoblot analysis of
RelA immunoprecipitated from 293T cells transfected with V5-tagged PKC-ζ(ca) and left untreated (−) or treated for 1 h (+) with calf intestinal alkaline
phosphatase (CIP). Data are representative of two (b,c,e,f) or three (d) independent experiments.
SETD6
PKC-ζ
+
V5-
PKC-ζ(ca)
GLP
RelA
GLP
RelA
P
GLP
(ANK)
Chrom
TNF +
+
Input
RelA(300–320)
RelAK310me1
RelAS311ph
RelAK310me1S311ph
H3K9me1
WCE
IP: RelA
me1
me1
GLP
GLP
PKC-ζ(ca)
RelA(300–320) + PKC-ζ
Anti-RelAS311ph
Anti-PKC-ζ
Anti-RelAK310me1
Streptavidin-HRP
RelA(300–320) + PKC-ζ
RelAK310me1 + PKC-ζ
RelAK310me1 + PKC-ζ
RelAK310me1
RelAK310me1
RelAS311ph
RelA
RelAK310me1
RelAK310me1
RelA(300–320)
250 ng 250 ng
IP: RelA
CIP +
RelA300–320
RelAS311ph
RelA
H3
a
d e f
b c
© 2011 Nature America, Inc. All rights reserved.
nature immunology VOLUME 12 NUMBER 1 JANUARY 2011 3 5
A R T I C L E S
To investigate the functional interaction between GLP and SETD6-
mediated attenuation of RelA transcriptional activity, we depleted cells
of GLP and challenged them with TNF and found they had more
IL8 and MYC mRNA than did cells treated with control shRNA
(Supplementary Fig. 22a). Moreover, the ability of SETD6 over-
expression to suppress baseline expression of IL8 and MYC mRNA was
largely abrogated in cells depleted of GLP (Supplementary Fig. 22b).
These data indicate that inhibition of NF-κB signaling by SETD6
occurs at chromatin and is mediated by a lysine-methylation network
that connects SETD6 activity on RelA to GLP activity on H3K9.
RelA phosphorylation blocks GLP-RelAK310me1 interaction
TNF stimulation initiates several activating phosphorylation events
on RelA
7
, including phosphorylation at Ser311 by the atypical
protein kinase PKC-ζ
11
. The molecular mechanism by which
phosphorylation of Ser311 activates RelA is not known
11
. Because
methylation of Lys310 and phosphorylation of Ser311 are coupled to
opposing biological outcomes and the two modifications are in close
physical proximity, we postulated that phosphorylation of Ser311
functionally inhibited the recognition of RelAK310me1 by GLP
(Fig. 6a). The ability of GLP(ANK) to bind RelAK310me1 peptides
was abolished when Ser311 was phosphorylated, as determined by
isothermal titration calorimetry (Table 1) and peptide-precipitation
assays (Fig. 6b). Moreover, overexpression of constitutively active
V5-tagged PKC-ζ (PKC-ζ(ca))
22
disrupted the endogenous inter-
action between RelA and GLP (Fig. 6c). Consistent with the idea of
a physiological role for RelA phosphorylated at S311 (RelAS311ph)
in regulating the binding of GLP to RelA at chromatin, treatment
with TNF (and overexpression of V5-tagged PKC-ζ(ca)) resulted
in a stronger RelAS311ph signal at chromatin, but a weaker
RelAK310me1 signal (Fig. 6d). Thus, we propose that phosphor-
ylation of Ser311 masks RelAK310me1 to prevent its recognition by
GLP(ANK) (Fig. 6a).
Antibody to the RelAS311ph epitope recognized the RelAS311ph
mark regardless of methylation at Lys310 (Supplementary Fig. 23).
In contrast, recognition of RelAK310me1 by anti-RelAK310me1
was disrupted by phosphorylation of Ser311, as observed in dot-blot
assays with a dually modified peptide containing monomethylation
of Lys310 and phosphorylation of Ser311 (Supplementary Fig. 23b)
or with a RelAK310me1 peptide phosphorylated at Ser311 in vitro
by recombinant PKC-ζ (Fig. 6e). These results also demonstrate that
PKC-ζ phosphorylated Ser311 regardless of the monomethylation
status of Lys310 (Fig. 6e). Furthermore, detection of endogenous
RelAK310me1 on RelA immunoprecipitated from cells overex-
pressing V5-tagged PKC-ζ(ca) was much greater after in vitro
dephosphorylation of the immunoprecipitated protein (Fig. 6f and
Supplementary Fig. 23c), which indicated that the RelAK310me1
epitope becomes exposed after removal of RelAS311ph and there-
fore demonstrated that the two marks probably co-occupy the same
molecule. Together these findings suggest that TNF-induced phos-
phorylation of chromatin-bound RelAK310me1 at Ser311 disrupts
the association of GLP with RelA, thereby promoting activation of the
population of NF-κB target genes occupied by RelAK310me1.
A RelA methylation-phosphorylation switch in NF-kB signaling
Consistent with the hypothesis outlined above, coexpression of V5-
tagged PKC-ζ(ca) with SETD6 abrogated the greater occupancy of RelA
target genes by RelAK310me1, GLP and H3K9me2 induced by SETD6
expression alone (Supplementary Fig. 21a). In addition, SETD6
failed to inhibit the expression of IL8 and MYC when coexpressed
with PKC-ζ(ca) (Supplementary Fig. 22b). Indeed, expression of
V5-tagged PKC-ζ(ca) induced more expression of IL8 and MYC above
baseline, whereas the ability of V5-tagged PKC-ζ(ca) to antagonize
SETD6 and stimulate the activation of RelA target genes was abro-
gated in cells depleted of GLP (Supplementary Fig. 22b). Finally, the
occupancy by RelAK310me1, GLP and H3K9me2 at promoters of four
different RelA target genes involved in cell proliferation and inflam-
mation was lower after TNF stimulation in PKC-ζ-sufficient MEFs,
but we did not observe these changes in PKC-ζ-deficient (Prkcz
−/−
)
MEFs
23
(Fig. 7a and Supplementary Fig. 24). In agreement with the
ChIP results and as reported before
23
, TNF induction of the expres-
sion of Il1a and Il6 was largely attenuated in Prkcz
−/−
cells (Fig. 7b).
These results support a model in which the chromatin environment
of NF-κB target genes can be regulated by competing modifications
of RelA by SETD6 and PKC-ζ, with the inert state linked to SETD6-
mediated binding of GLP to RelAK310me1 and the active state linked
to PKC-ζ-mediated disassociation of GLP from RelAK310me because
of phosphorylation of Ser311 (Supplementary Fig. 25).
DISCUSSION
We have reported here the discovery of a previously unknown lysine-
methylation event that occurs on the transcription factor RelA,
–TNF +TNF
–TNF +TNF
Actin
WT
PKC-ζ
Anti-
H3K9me2
WT
Prkcz
–/–
II1a mRNA
II6 mRNA
a b
Anti-
RelAK310me1
0.06
0.04
0.02
0
WT Prkcz
–/–
II6 (% of input)
Anti-
RelAK310me1
0.045
0.030
0.015
0
WT
Prkcz
–/–
Ccnd1 (% of input)
Anti-
GLP
Ccnd1 (% of input)
0.045
0.030
0.015
0
WT
Prkcz
–/–
Anti-
H3K9me2
Ccnd1 (% of input)
0.21
0.14
0.07
0
WT
Prkcz
–/–
Anti-
RelAK310me1
Myc (% of input)
0.09
0.06
0.03
0
WT
Prkcz
–/–
Anti-
GLP
Myc (% of input)
0.3
0.2
0.1
0
WT
Prkcz
–/–
Myc (% of input)
0.36
0.24
0.12
0
WT
Prkcz
–/–
24
16
8
0
WT
Prkcz
–/–
4.5
3.0
1.5
0
WT
Prkcz
–/
Anti-
GLP
0.03
0.02
0.01
0
WT Prkcz
–/–
II6 (% of input)
Anti-
RelAK310me1
0.03
0.02
0.01
0
Prkcz
–/–
Tnf (% of input)
Anti-
GLP
0.045
0.015
0
0.030
WT Prkcz
–/–
Tnf (% of input)
Anti-
H3K9me2
0
1.0
1.5
0.5
WT Prkcz
–/–
Tnf (% of input)
Anti-
H3K9me2
0.6
0.4
0.2
0
WT Prkcz
–/–
II6 (% of input)
Figure 7 RelA methylation-phosphorylation switch at chromatin regulates NF-κB signaling. (a) ChIP assay of RelAK310me1, GLP and H3K9me2 at
the promoters of Il6, Tnf, Ccnd1 and Myc in Prkcz
+/+
(WT) and Prkcz
−/−
MEFs
23
with or without TNF treatment (20 ng/ml), presented as in Figure 2a
(ChIP with negative control antibody, Supplementary Fig. 24). (b) Real-time PCR analysis (below) of Il1a and Il6 mRNA in Prkcz
+/+
and Prkcz
−/−
cells
with or without TNF treatment (20 ng/ml), and immunoblot analysis (above) of PKC-ζ in Prkcz
+/+
and Prkcz
−/−
MEFs
23
. Data are from at least three
experiments (error bars, s.e.m.).
© 2011 Nature America, Inc. All rights reserved.
3 6 VOLUME 12 NUMBER 1 JANUARY 2011 nature immunology
which is catalyzed by SETD6 and regulates the clinically important
NF-κB pathway. Monomethylation of nuclear RelA at Lys310 by
SETD6 attenuated NF-κB signaling by docking GLP (via its ankyrin
repeats) at target genes to generate a silent chromatin state, effec-
tively rendering chromatin-bound RelA inert. As deregulation of
NF-κB is linked to pathological inflammatory processes and cancer
8
and SETD6 inhibits NF-κB signaling in diverse cell types, including
primary human cells, SETD6 provides another link by which methyl-
ation of lysine residues of proteins and regulation of chromatin influ-
ence tumor suppression and anti-inflammatory responses
2,5
.
SET7-SET9 is a well-characterized PKMT with many protein sub-
strates
3
, including TNF-dependent methylation of RelA at three dif-
ferent lysine residues
12,13
. In contrast, SETD6 methylated RelA at a
single residue (Lys310), and this occurred in the absence of stimula-
tion and was functionally suppressed by TNF-induced phosphoryla-
tion of RelA at Ser311. Thus, RelAK310me1 represents a specialized
population of RelA that is not sequestered in the cytoplasm under
basal conditions but is instead bound and quiescent at target pro-
moters. We postulate that methylation of transcription factors such
as RelA can aid in the rapid and dynamic modulation of specific
gene-expression programs by establishing transcriptional memory
at marked genes
24
. In addition, the SETD6-RelA-GLP-H3K9me2
network delineated here constitutes the first description to our
knowledge of a lysine-methylation signaling cascade. We have also
demonstrated that TNF-induced phosphorylation of RelA Ser311
terminated SETD6 action by blocking recognition of RelAK310me1
by GLP, which in turn led to chromatin relaxation and expression of
RelA target genes. These findings provide the first example to our
knowledge of a metazoan regulatory methylation-phosphorylation
switch on a non-histone protein
25–27
. Together our results emphasize
how the convergence and integration of multiple signals at chromatin
can modify important biological and disease pathways.
METHODS
Methods and any associated references are available in the online version
of the paper at http://www.nature.com/natureimmunology/.
Accession codes. UCSD-Nature Signaling Gateway (http://www.
signaling-gateway.org): A001645, A002937 and A001934.
Note: Supplementary information is available on the Nature Immunology website.
ACKNOWLEDGMENTS
We thank R. Kingston and M. Simon (Harvard Medical School) for recombinant
nucleosomes; J. Smith (University of Alabama Birmingham) for the PKC-ζ(ca)
plasmid; W.C. Greene (University of California San Francisco) for RelA(1–431)
cDNA and the κB-Luc luciferase reporter plasmid; D. Reinberg (New York
University) for the NSD1(SET) plasmid; M. Covert (Stanford University) and
T.D. Gilmore (Boston University) for the wild-type and Rela
−/−
mouse 3T3 cells;
J. Moscat (University of Cincinnati College of Medicine) for the wild-type and
Prkcz
−/−
MEFs; E. Engleman (Stanford University) for FL-B16 cells; E. Green for
critical reading of the manuscript; and A. Alizadeh for comments. Supported by
the National Institutes of Health (DA025800 to O.G. and M.T.B.; GM068680 to
X.C.; and F32AI080086 to C.L.L.), the American Society for Mass Spectrometry
(B.A.G.), the National Heart, Lung and Blood Institute (HHSN-268201999934C
to P.J.U.), the National Institute of Allergy and Infectious Diseases (U19-AI082719
to P.J.U.), the Floren Family Trust (P.J.U.), the Genentech Foundation (A.J.K.), the
European Molecular Biology Organization (D.L.), the Human Frontier Science
Program (D.L.), the Machiah Foundation (D.L.), the Georgia Research Alliance
(X.C.) and the Ellison Medical Foundation (O.G.)
AUTHOR CONTRIBUTIONS
D.L. did most of the molecular biology and cellular studies; Y.C. did binding affinity
studies and modeling; A.J.K., P.C. and X.S. generated the PKMT library; A.J.K.
identified and initially characterized the activity of SETD6 on RelA Lys310; B.Z.
did mass spectrometry analysis; U.S. and C.K. did the primary cells experiments;
A.E. did CADOR array experiments; C.L.L. analyzed gene expression data sets;
R.I.T., S.T., A.Y.K., R.C. and S.T. provided technical support; X.S., P.J.U., K.C., B.G.,
R.P., M.B., A.T., X.C. and O.G. discussed studies; D.L. and O.G. designed studies,
analyzed data, and wrote the paper; D.L. and A.J.K. contributed independently to
the work; and all authors discussed and commented on the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare competing financial interests: details accompany the full-text
HTML version of the paper at http://www.nature.com/natureimmunology/.
Published online at http://www.nature.com/natureimmunology/.
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/.
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A R T I C L E S
© 2011 Nature America, Inc. All rights reserved.
nature immunology
doi:10.1038/ni.1968
ONLINE METHODS
Plasmids and reagents. For overexpression in mammalian cells, the plasmids
were as follows: pCAG Flag-SETD6 wt, pCAG Flag-SETD6(Y285A), pCAG
Flag-Smyd1, pCAG Flag-Smyd2, pCAG Flag-NSD2, pcDNA-RelA, pcDNA-
RelA(K310R) and pcDNA Flag-GLP. The plasmid pcDNA3.1-GSV5-
PKC-ζ(ca) was a gift from J. Smith. For in vitro assays, RelA(1–431) and
RelA(430–551) were subcloned into pGEX-6P1. Single or double mutation
of the sequence encoding RelA(1–431) was generated with the QuikChange
site-directed mutagenesis kit (Stratagene), and sequences were confirmed by
DNA sequencing. The pGEX-derived plasmids generated by mutagenesis were
as follows: pGEX–RelA(K122R), pGEX-RelA(K123R), pGEX-RelA(K218R),
pGEX-RelA(K221R), pGEX-RelA(K310R), pGEX-RelA(K314R,K315R) and
pGEX-RelA(430–551). Sequence encoding p50 was subcloned into pGEX-6P1
by standard methods. All enzymes in the PKMT library are in Supplementary
Table 1; enzymes used in Figure 1a and Supplementary Figure 1 are in
Supplementary Table 2. NSD1(SET) was a gift from D. Reinberg.
For expression in insect cells, cDNA encoding full-length SETD6 or
various additional PKMTs was first cloned into pENTR3C and then recombined
into Gateway pDEST20 with the Gateway LR Clonase II system (Invitrogen).
Recombinant baculovirus were generated according to the manufacturer’s
protocol (Invitrogen). DH10Bac Escherichia coli cells were transfected with
pDEST20 to generate recombinant baculovirus shuttle vector DNA. Sf9
Spodoptera frugiperda cells were then transfected with 2 µg baculovirus
shuttle vector DNA with Cellfection II reagent (Invitrogen), and the baculovirus
was amplified the times to obtain the optimal viral titer. For protein expression,
baculovirus stocks were added to Sf9 cells grown in suspension at a density
of 1 × 10
6
cells per ml, and transduced Sf9 cells were collected 2 d after trans-
duction. Sf9 cells were maintained in Sf-900 II SFM media supplemented with
0.5% (vol/vol) penicillin-streptomycin.
Mouse RelA cDNA (Open Biosystems) was first cloned into the pENTR3C
vector (Invitrogen) and then was recombined into the pBABE-FLAG-HA
vector with the Gateway system as described above. RelA(K310R) was then
generated with a QuikChange site-directed mutagenesis kit (Stratagene).
Cell lines, transfection and transduction of retrovirus or lentivirus. Human
293T and U2OS cells, mouse 3T3 cells (wild-type and Rela
−/−
; a gift from
M. Covert and T.D. Gilmore) and MEFs (wild-type and Prkcz
−/−
; a gift from
J. Moscat) were grown in DMEM (Gibco) supplemented with 10% (vol/vol) FCS
(Gibco) and 100 units/ml of penicillin and -glutamine. THP-1 cells (American
Type Culture Collection) were cultured in RPMI-1640 medium (Gibco) supple-
mented with 10% (vol/vol) FCS (Gibco), 100 U/ml of penicillin and -glutamine,
0.05 mM β-mercaptoethanol and 1× streptomycin. All cells were cultured at
37 °C in a humidified incubator with 5% CO
2
. Cells were transfected with the
TransIT transfection reagent (for plasmids; Mirus) or DharmaFECT reagent
(for siRNA; Dharmacon), according to the manufacturer’s protocols. Human
SETD6–specific siRNA sequences were 5-ACCTATGCCACAGACTTATT-3
(1) and 5-GACCACCACACTAAAGGTATT-3 (2).
Mouse primary cells were isolated according to protocol 07064 of Rockefeller
University and Institutional Animal Care and Use Committee protocol 9982
of Stanford University. Human biological samples were sourced ethically and
their research use was in accordance with the terms of the informed consent
received from each donor according to the Hertfordshire Ethics Committee
Code 07/H0311/ 103. Mouse primary BMDMs were generated as described
28
.
C57BL/6 bone marrow cells from femur and tibia were cultured for 7–8 d at
37 °C in 5% CO
2
in presence of 5 ng/ml of recombinant macrophage colony-
stimulating factor and IL-3 (Peprotech). For knockdown experiments, siRNA
directed against mouse SETD6 or control siRNA was transfected into cells with
the HiPerFect transfection reagent according to the manufacturer’s protocol
(Qiagen), followed by stimulation experiments 48 h later. SETD6-specific
siRNA sequences for BMDMs were 5-GAACAAAGGATGAAACTGA-3
(1) and 5-GTGAGGAGGTGCTGACTGA-3 (2). For human primary
monocyte-derived dendritic cells, CD14
+
cells were separated with MACS
CD14 beads (positive selection) according to the manufacturer’s protocols
(Miltenyi). After separation, CD14
+
cells were resuspended at a density of 1 ×
10
6
cells per ml in RPMI-1640 medium (plus -glutamine and 10% (vol/vol)
heat-inactivated FCS) containing human recombinant granulocyte-monocyte
colony-stimulating factor (30 ng/ml) and human recombinant IL-4 (20 ng/ml).
Cells were differentiated for 5 d before transfection of siRNA by nucleofection
according to the manufacturer’s protocol (Amaxa) with the following minor
alterations: siRNA was preplated into a 96-well U-bottomed plates with a final
concentration of 2 µM. Monocyte-derived dendritic cells were resuspended in
Amaxa Nucleofecter buffer at a density of 1 × 10
6
cells per 20 µl, and 20 µl of
the suspension was added to the plate containing siRNA. The plate was placed
into the Amaxa device and the monocyte program EA-100 was applied to all
wells. After removal of the Amaxa plate, 100 µl prewarmed RPMI medium
(plus 10% (vol/vol) heat-inactivated FCS, penicillin and -glutamine) was
added to each well, then cells were immediately removed from the Amaxa
plate and added to a second flat-bottomed plate containing an additional
100 µl of prewarmed media. SETD6-specific siRNA sequences for mono-
cyte-derived dendritic cells were 5-TAATGCTGCCTCACGAACTGT-3 (1)
and 5-TAGGAAATCCCAGCGCTCGTA-3 (2). Mouse dendritic cells were
isolated as described
29
. The FL-B16 mouse melanoma cells used to make the
conditioned media were a gift from E. Engleman.
Cells were transduced with retrovirus and lentivirus as described
30
.
Lentivirus for control, SETD6-specific and GLP-specific shRNA was from
Santa Cruz Biotechnology. The human RelA shRNA target sequence is
5-GATTGAGGAGAAACGTAAA-3. For generation of the reconstituted cell
lines, shRNA directed against the 3 untranslated region of SETD6 was cloned
into the shRNA vector pLentiLox3.7, and shRNA directed against wild-type
SETD6 or SETD6(Y285A) was cloned into pWZL-3FLAG-hygro as an AscI-
PacI cassette. U2OS and 3T3 cells were first transduced with pLentiLox SETD6
shRNA plasmid, followed by selection with puromycin (2 µg/ml). Puromycin-
resistant cells were than transduced with pWZL-3Flag-SETD6 shRNA plasmid
(wild-type SETD6 or SETD6(Y285A)) or with empty pWZL-hygro, followed
by selection for 4 d with hygromycin B (250 µg/ml; Invitrogen). The SETD6
shRNA target sequences were 5-CCTGTTCCCTGAAGGAACAGCAATA-3
(human) and 5-TGCTATTTGGCAGTTAGAATCAAAG-3 (mouse). Where
indicated, cells were stimulated with mouse TNF (10–20 ng/ml; R&D Systems)
or LPS (10–100 ng/ml; Sigma).
Enzyme-linked immunosorbent and Luminex bead-based cytokine assays.
Enzyme-linked immunosorbent assays were done as described
31
with anti-
IL-6 (MP5-20F3; eBioscience) and anti-TNF (1F3F3D4; eBioscience). Plates
were scanned with a SpectraMax 190 (Molecular Devices). Luminex standards
were analyzed by multiplex bead-based arrays with the Mouse 26-Plex Multi-
Cytokine Detection system (Panomics-Affymetrix) and with a Luminex 200
according to the manufacturer’s protocol. Cytokine arrays were done at the
Human Immune Monitoring Center of Stanford.
Gene-expression profiling. Gene expression arrays were done at the
Stanford Functional Genomics Facility with a Mouse-Ref8 whole-genome
array, according to the manufacturer’s protocol (Illumina), and included two
independent biological replicates (genes analyzed, Supplementary Fig. 13).
Genes were selected when the mean change (fold) was over 1.5. P values for
Venn and pie diagrams were calculated with Fisher’s exact test and the χ
2
test, respectively.
SETD6 mRNA in rheumatoid arthritis, septic shock and juvenile idiopathic
arthritis. A review of available literature involving microarray studies for
rheumatoid arthritis, septic shock and juvenile idiopathic arthritis was used to
identify four studies that met the criteria described below
32–37
. For rheumatoid
arthritis, data were retrieved from the Stanford Microarray Database with the
filtering criteria implemented before
32
and from the Gene Expression Omnibus
database (accession code, GDS3628)
33
. Data were retrieved from the Gene
Expression Omnibus database for septic shock (accession code, GSE8121)
35,37
and juvenile idiopathic