Structural basis of SETD6-mediated regulation of
the NF-kB network via methyl-lysine signaling
Yanqi Chang1, Dan Levy2, John R. Horton1, Junmin Peng3, Xing Zhang1,
Or Gozani2and Xiaodong Cheng1,*
1Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322,
2Department of Biology, Stanford University, Stanford, CA 94305 and3Department of Human Genetics,
Emory Proteomics Service Center, Center for Neurodegenerative Diseases, Emory University School of
Medicine, 615 Michael Street, Atlanta, GA 30322, USA
Received January 25, 2011; Revised March 30, 2011; Accepted April 1, 2011
SET domain containing 6 (SETD6) monomethylates
the RelA subunit of nuclear factor kappa B (NF-iB).
The ankyrin repeats of G9a-like protein (GLP) recog-
nizes RelA monomethylated at Lys310. Adjacent to
Lys310 is Ser311, a known phosphorylation site
of RelA. Ser311 phosphorylation inhibits Lys310
methylation by SETD6 as well as binding of
Lys310me1 by GLP. The structure of SETD6 in
complex with RelA peptide containing the methyla-
tion site, in the presence of S-adenosyl-L-methio-
suggests a model for NF-iB binding to SETD6. In
addition, structural modeling of the GLP ankyrin
repeats bound to Lys310me1 peptide provides
insight into the molecular basis for inhibition of
Lys310me1 binding by Ser311 phosphorylation.
Together, these findings provide a structural explan-
ation for a key cellular signaling pathway centered
on RelA Lys310 methylation, which is generated
by SETD6 and recognized by GLP, and incorporate
cent lysine and serine residues. Finally, SETD6 is
structurally similar to the Rubisco large subunit
methyltransferase. Given the restriction of Rubisco
to plant species, this particular appearance of the
protein lysine methyltransferase has been evolu-
tionarily well conserved.
Mammalian nuclear factor kB (NF-kB) is a critical
mediator of inducible transcription in the control of key
physiological and pathological states, from immunity and
inflammation to cancer [reviewed in (1)]. The NF-kB
family of transcription factors consists of five members:
p65 (RelA), p50 (NF-kB1), p52 (NF-kB2), c-Rel and RelB
(2). The area of greatest homology among the NF-kB
homology region, which is composed of a DNA binding
domain and a dimerization domain. Through the dimer-
ization domain, different NF-kB members form a variety
of homo- and hetero-dimers, with the p65/p50 combin-
ation being the most abundant. In addition, p65, but not
p50, possesses a C-terminal transactivation domain
(TAD) that is required for promoting transcription (see
Supplementary Figure S1a). In unstimulated cells, the
majority of NF-kB, including the p65/p50 heterodimer
ankyrin-repeat-containing IkB proteins. NF-kB activation
by stimulants-like cytokines triggers a signaling cascade
that results in degradation of IkB and releasing NF-kB
to translocate into the nucleus and function as a transcrip-
tion factor at target genes (3).
Recently, we described a previously uncharacterized
mechanism in which, under basal conditions, the protein
methylates chromatin-associated RelA at lysine 310, a
residue located within the linker region between the dimer-
ization and activation domains of RelA (4). Another
PKMT, G9a-like protein (GLP), via its ankyrin-repeat
domain, binds RelA methylated at Lys310 (Lys310me1)
and acts to locally condense chromatin at several
NF-kB-dependent target genes. The repressed chromatin
state is terminated upon stimulating cells with TNFa, due
to phosphorylation of RelA at serine 311 (4). Here we
determine the SETD6–RelA peptide complex structure,
which provides insight into the molecular basis for RelA
peptide recognition by SETD6 as well as an understanding
of the effects of modifications at nearby residues on
SETD6-mediated Lys310 methylation. In addition, we
*To whom correspondence should be addressed. Tel: +1 404 727 8491; Fax: +1 404 727 3746; Email: email@example.com
Nucleic Acids Research, 2011, Vol. 39, No. 15Published online 22 April 2011
? The Author(s) 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
investigate the molecular basis of RelA Lys310me1
peptide recognition by the GLP ankyrin repeats. Finally
we generate a model that connects histone peptide-bound
GLP with a DNA-bound NF-kB, using the existing struc-
tural information. Our study suggests that the methyl-
phospho switch between two adjacent residues of RelA,
Lys310 and Ser311, regulates localized chromatin state to
influence NF-kB-target gene expression.
MATERIALS AND METHODS
Protein expression and purification
(Figure 1a): the longer 473 residue variant (isoform a)
and the shorter 449 residue variant (isoform b), which
lacks an in-frame segment (residues 40–63) and was used
here for crystallography (residue numbering is based on
the longer variant). SETD6 isoform b (residues 17–449)
was sub-cloned into a His6-SUMO vector (generating
plasmid pXC862) and confirmed by sequencing. All of
the proteins were overexpressed in Escherichia coli strain
BL21 (DE3) RIL-Codon plus strain (Stratagene). Cells
expressing His6-SUMO-tagged SETD6 were induced
(IPTG) for 16 h at 16?C. Cells were collected, pelleted
and then resuspended in 50mM sodium phosphate,
aretwo splicevariants ofhumanSETD6
pH7.4, 300mM NaCl, 5% glycerol. The cells were lysed
by two passes through a French pressure cell press and
then centrifuged at 23000g for 1h. The soluble His6-
SUMO fusion protein was first purified using the
HisTrap HP column (GE Healthcare). The fusion
protein was cleaved by Ulp-1 protease in overnight
dialysis at 4?C. Only two extraneous N-terminal amino
acids (HisAsn) were left as a result of a restriction site.
The cleaved SUMO tag was removed by ion exchange
SETD6 protein was further purified by gel-filtration chro-
matography (Superdex 200, GE Healthcare). All protein
purification was performed at 4?C. For crystallization, the
purified protein was concentrated to ?16mgml?1in the
presence of 100mM S-adenosyl-L-methionine (AdoMet).
The human RelA dimerization domain plus the linker
Figure S1a) was expressed as a His6-SUMO tagged con-
struct in E. coli BL21(DE3)-Gold cells (Stratagene) with
RIL-Codon plus plasmid (pXC875). For the RelA:p50
heterodimer, His6-SUMO-tagged human RelA (residues
1–325 including DNA binding domain, dimerization
domain and linker region; pXC914) and non-tagged
human p50 dimerization domain (residues 243–366;
pXC902) in pET21b (Novagen) were coexpressed in
E. coli BL21(DE3). Similar purification strategies were
used for both complexes. The soluble fraction was
isolated using a nickel-charged HiTrap chelating HP
column (GE Healthcare). The fused SUMO tag was
removed by Ulp-1 protease in overnight dialysis at 4?C.
The product protein was further purified by cation-
exchange and gel-filtration chromatography. Size exclu-
sion chromatography was employed to evaluate dimer
Crystallization of the SETD6–AdoMet–RelA peptide
vapor-diffusion method at 16?C after mixing the protein
with peptide in a ratio of ?1:1.2, and then mixing with an
equal amount of well solution (1.5ml). Both native and
selenium-substituted SETD6–AdoMet, in the presence of
RelA peptide, were crystallized using well solutions con-
taining 15% (w/v) polyethylene glycol (PEG) 3350, 0.1M
di-ammonium hydrogen citrate, pH4.6. The crystals
belonged to space group P1 with two SETD6–AdoMet–
peptide complexes per asymmetric unit. For data collec-
tion, the crystals were equilibrated in a cryoprotectant
ethylene glycol. The native and selenium SAD data sets
were collected at SER-CAT beamline 24ID of Advanced
Photon Source (APS) at Argonne National Laboratory.
All the data sets were processed using the program
HKL2000. The structure was determined and refined
utilizing components of the SGXPRO (5) and PHENIX
(6) program packages. The program COOT (7) was used
for building peptide and manual model manipulation
between rounds of refinement with PHENIX. Structural
figures were generated by the program MacPyMol
(DeLano Scientific). The RelA peptide was synthesized
Figure 1. Structure of SETD6. (a) Schematic representation of human
SETD6, with the long and short isoforms. (b) Two views of SETD6
with a V-cleft appearance, colored magenta (N-terminal helix), yellow
(SET), orange (i-SET), and green (C-SET). Dashed lines indicate the
disordered loops, residues 230–236 (yellow) and 387–394 (green). The
RelA K310 peptide and AdoMet are in stick model.
Nucleic Acids Research, 2011,Vol.39, No. 156381
at the W.M. Keck Foundation Biotechnology Resource
Laboratory (Yale University).
Mass spectrometry-based peptide methylation assay
Human RelA peptide (residues 302–316) was used as sub-
strate for SETD6. A reaction mixture contained 50mM
glycine pH 10.2 (or di-ammonium hydrogen citrate for pH
4.6, Bis–Tris–HCl for pH 6.4, Tris–HCl for pH 7.4, 8.2
and 8.6, and glycine/NaOH for pH 9.0–11.0), 5mM
dithiothreitol, 200–500mM AdoMet, 10mM SETD6 and
30mM peptide. The assays were carried out at room tem-
perature (?25?C) for 1h with a total volume of 20ml. The
reaction was terminated by addition of trifluoroacetic acid
(TFA) to 0.1% (v/v). The resulting peptides were
measured by MALDI-TOF on a Bruker Ultraflex II
Emory University School of Medicine).
Peptide methylation analysis by the LC-MS/MS approach
tandem mass spectrometry (LC–MS/MS) as reported (8).
The peptide mixtures were loaded onto a C18 column,
eluted and monitored in a MS survey scan followed by
data-dependent MS/MS scans on a LTQ-Orbitrap mass
spectrometer (Thermo Finnigan, San Jose, CA, USA).
The acquired MS/MS spectra were searched against a
database containing the synthetic peptides. Modified
methylation sites were determined by dynamic assignment
of mass addition (14Da) to lysine residues during the
search. Finally, all modified peptide assignments were
SETD6 were analyzed
In vitro methylation of RelA by SETD6
Methylation assays were carried out in a 20ml reaction
(45mM substrate, 5.5mM [methyl-3H]AdoMet, with and
without 32mM recombinant SETD6) in 20mM Tris, pH
8.5 and 5mM DTT for 12h at 25?C. Samples were
analyzed by 17% SDS–PAGE gel and fluorography
after 36h of exposure.
Cell lines and transfections
Human embryonic kidney 293T cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM; GIBCO)
supplemented with 10% fetal calf serum (FCS, GIBCO),
100Uml?1penicillin and L-glutamine. Cells were trans-
fected with TransIT 293 transfection reagent (Mirus) ac-
cording to the manufacturer’s protocols.
Plasmids and mutagenesis
For overexpression in mammalian cells, the plasmids used
were: pcDNA-RelA, pCAG-Flag-SETD6 WT, pCAG-
Flag-SETD6N283A. The pGEX-derived plasmids used
Mutants were constructed
site-directed mutagenesis kit (Stratagene).
Immunoblot analysis and antibodies
Cell extracts and immunoblot analyses were done as
described (4). The antibodies used were as follows:
(Abcam). RelAK310me1 and SETD6 rabbit polyclonal
antibodies were described (4).
Peptide pull-down assays
Peptide pull-down assays were performed as previously
described (9). Peptides were synthesized at the W.M.
Keck Foundation Biotechnology Resource Laboratory
used were Biotin-E-K-R-K-R-T-Y-E-T-F-Km-S-I-M-K-
K-S-P-F-S-G for RelAamino
Biotin for H3 amino acids 1–22. RelAK310 and H3K9
were either unmodified (me0), monomethylated (me1) or
dimethylated (me2). GLP ankyrin repeats (GLPANK)
WT (residues 734–968) and mutants were purified as
of the peptides
Overall structure of SETD6
We determined the structure of SETD6 by producing
wild-type protein in complex with a RelA Lys310
peptide in the presence of AdoMet at a resolution of
2.2A˚(Supplementary Table S1). There are two complexes
in the crystallographic asymmetric unit. The protein com-
ponents of the two complexes are highly similar, with a
root mean squared deviation of ?0.7A˚when comparing
410 pairs of Ca atoms.
The overall structure of SETD6 in complex with
AdoMet and the RelA peptide, as viewed in Figure 1b,
resembles a V-shaped cleft. The V-like appearance is
mainly determined by the helical structures of i-SET (an
insertion of about 125 amino acids in the middle of the
SET domain; orange helices aB-aG) and the C-terminal
domain (green) that is mainly helical (aH-aQ) except for
two b strands (6 and 7) (Figure 1b). The two pairs of
helices (one short and one long)—helices aE and aF and
helices aH and aQ—lie next to one another near the
bottom of the cleft and are largely responsible for the
V-like appearance of SETD6 (Supplementary Figure S2a).
We used a RelA peptide encompassing amino acids
302–316 for co-crystallization in the presence of the
methyl donor AdoMet. The complex was crystallized
under the conditions of pH 4.6 (see ‘Materials and
Methods’ section), under which no activity was observed
in vitro (Figure 2a), and an intact AdoMet was present in
the structure (Supplementary Figure S2b). Like other SET
domain proteins DIM-5 (12) and Rubisco large subunit
methyltransferase (LSMT) (13), SETD6 showed maximal
in vitro activity at approximately pH 10. At pH 10, the
e-amino group of target lysine (with a typical pKa value
of 10) may be partially neutralized in the active site.
However, under the low pH conditions, the deprotonation
6382Nucleic Acids Research, 2011,Vol.39, No. 15
Figure 2. Interactions of SETD6 and RelA K310 peptide. (a) SETD6 activity as a function of pH, (b) the surface representation of SETD6, colored
with yellow (SET), orange (i-SET) and green (C-SET). For clarity, the C-SET domain has been sliced away. In addition, the surface nitrogen atoms
are colored blue and surface oxygen atoms are red, (c) electrostatic interactions, hydrogen bonds and van der Waals interactions define SETD6 (in
green) and RelA peptide (in light blue) interactions. The linear conformation of Lys310 of RelA is in cyan, and the bent conformation in light blue,
(d) the bent conformation of the target lysine side chain forms a hydrogen bond with the side chain hydroxyl oxygen of Y297 and the main-chain
carbonyl oxygen of S224, (e) a model of phosphated-Ser311 of RelA (pSer311) with the phosphate group potentially clashing with P228 of SETD6
and (f) In vitro methylation assays by SETD6 on unmodified RelA peptides (left panels) or phosphorylated at Ser311 (pS311) (right panels) followed
by mass spectrometry of the reaction products (0h, before assay; 3h, after assay).
Nucleic Acids Research, 2011,Vol.39, No. 156383
event would not occur and the methyl transfer between the
donor methyl group (S+-CH3) and the acceptor amino
3) would be inhibited.
Interestingly, the side chain of the target lysine 310 of
RelA adopts two conformations: one is linear and the
other is bent (Figure 2c). The linear conformation of
lysine 310 points its e-amino group to the transferable
methyl group of AdoMet (Figure 2c) so that the methyl
donor and acceptor are aligned in a nearly linear geometry
(N...C distance of 3.4A˚and N...C-S angle of 161?) for
SN2 nucleophilic transfer of the methyl group during ca-
talysis. The terminal e-amino group of the bent conform-
ation sits in the carboxyl end of helix aG (Figure 2d and
Supplementary Figure S2c). Thus the positive charge of
the terminal e-amino group of the bent conformation
under the low pH condition is effectively balanced by
the partial negative dipole charge at the carboxyl end of
the helix and stabilized by hydrogen-bonding interactions
with S224 (main chain carbonyl oxygen) and Y297 (side
chain hydroxyl oxygen) of SETD6 (Figure 2d; one-letter
code is used for SETD6 residues).
Specificity for the RelA peptide is determined primarily
through recognition of side chains of RelA (Phe309,
Ser311 and Ile312) before and after the target Lys310
(Figure 2c; three-letter code is used for RelA residues).
The network of interactions includes the following: (i)
the phenyl ring of Phe309 of RelA packs against M296
and Y297 of SETD6, (ii) The target nitrogen atom of
RelA Lys310 forms a water-mediated hydrogen bond
with the main chain carboxyl oxygen of L250 (the linear
conformation) or with Y297 and S224 (the bent conform-
ation); (iii) Ser311 of RelA is involved in a polar inter-
action with the main chain carbonyl oxygen of Q226 and a
van der Waals contact with P228 of SETD6. Adding a
phosphate group to the side chain hydroxyl oxygen of
Ser311 of RelA would result in repulsion from SETD6
(Figure 2e). As shown in Figure 2f, phosphorylation of
Ser311 (14) causes a complete loss of Lys310 methylation
in the context of peptide substrate; and (iv) the side chain
of Ile312 of RelA fits into a surface pocket formed by
F225, L260, N283 and T284 of SETD6.
Four aromatic residues (Y223, F225, Y285, Y297) and
one polar residue (N283) of SETD6 form the largely
hydrophobic active site and wrap around the aliphatic
chain of the target lysine. Interestingly, only one of these
residues (Y285) is conserved in Set7/9 (Figure 3a), another
hydroxyl group of Y285 of SETD6 is hydrogen bonded
to the backbone carbonyl oxygen of L250 and is snug
between the methyl group and adenine ring of AdoMet
(Figure 3b). The Y285A mutation in SETD6 abolished
its enzymatic activity (4), whereas the corresponding
Y283F mutation in DIM-5 (a Neurospora histone H3
lysine 9 tri-methyltransferase) lost its AdoMet binding
Figure 3. Comparison of active sites of SETD6 and related SET domain proteins. (a) Superimposition of active sites of SETD6 (colored) and Set7/
9-ER (estrogen receptor) complex (PDB 3CBM), (b) The hydroxyl group of Y285 of SETD6 is in contact with AdoMet, (c) Superimposition of active
sites of SETD6 (colored) and Dim-5-H3 complex (PDB 1PEG) and (d) Superimpositions of active sites of SETD6 (colored) and LSMT (PDB 2H2J).
6384Nucleic Acids Research, 2011,Vol.39, No. 15
and thus activity (12). In contrast, all four aromatic
residues are conserved and superimposable between
methyltransferase [a mammalian histone H3 lysine 9
mono- and di-methyltransferase (15,16)], while N283 of
SETD6 is in the place of F281 of DIM-5 (Figure 3c) or
the corresponding Phe (F1205) of G9a. There are two dif-
ferences between SETD6 and Rubisco LSMT (an enzyme
that generates a tri-methyl-lysine) in the active site, Y223
and N283 of SETD6 replacing R222 and I285 of LSMT,
respectively (Figure 3d). Despite the high level of conser-
vation among the active site residues of the two
Figure 4. Structural and sequence similarities between SETD6 and LSMT. (a) Superimposition of SETD6 (colored) and LSMT (gray) (PDB 2H2J)
by their respective N-terminal (left panel) or C-terminal halves (right panel) reveals an ?20?rotation between the two lobes. (b) Structure-based
sequence alignment of human SETD6 (AAH22451) and Rubisco methyltransferase (LSMT, PDB 2H2E). Isoform a of SETD6 has 473 residues
(NP_001153777), whereas isoform b has 449 residues (NP_079136) missing 24 amino acids (residues 40–63). Between AAH22451 and isoform a
(NP_001153777), there is a point mutation at position 206 (G or R). Secondary structural elements (arrows for b-strands, and rectangles for
a-helices) are indicated. White-on-black residues are invariant between the two sequences examined, while gray-highlighted positions are conserved
(R and K, E and D, T and S, Q and N, F and Y, V, I, L and M). Positions highlighted are responsible for various functions as indicated
(a=AdoMet binding; s=substrate binding; c=catalysis).
Nucleic Acids Research, 2011,Vol.39, No. 156385
tri-methyltransferases (DIM-5 and LSMT) and SETD6,
SETD6 is a mono-methyltransferase (see Discussion in
Structural and sequence similarities between SETD6
The structure of SETD6 shares high similarity with
Rubisco LSMT (13) (Figure 4a), and the structure-based
sequence alignment between the two enzymes reveals
sequence conservation throughout the entire region
(Figure 4b). SETD6 represents a sub-family of SET
(PKMTs) with an insertion of about 100–200 amino
acids in the middle of the SET domain (Supplementary
Figure S2d). Two sequences share 18% identity (81/449)
and 31% similarity (140/449). Only three regions have
suffered insertions of more than five residues in SETD6:
residues 232–239 (part of a disordered loop connecting
i-SET to SET domain), residues 333–345 (helix aJ) and
residues 440–444 (helix aP). There is one 9-residue
deletion around SETD6 residue 265 (making a shorter
loop between two strands of SET domain).
The SET domain was originally identified in three
Drosophila proteins involved in epigenetic processes: the
suppressor of position-effect variegation 3-9, Su(var)3-9;
an enhancer of the eye color mutant zeste, En(zeste); and
the homeotic gene regulator Trithorax (17). Based on the
sequence similarity of the SET domain to that of plant
methyltransferases including the Rubisco LSMT, the
mammalian homologues of Drosophila Su(var)3-9 and of
Schizosaccharomyces pombe Clr4 were the first histone
lysine methyltransferases identified, and they specifically
methylate lysine 9 of histone H3 (18). Here we show
that human SETD6 shares a striking structural similarity
to the Rubisco LSMT (13) throughout the entire protein.
The unexpected resemblance of these two PKMTs, given
the restriction of Rubisco to plant species, suggests that
this particular appearance of the PKMT has been evolu-
Model of the SETD6–RelA complex
Based on the fact that the RelA linker region—containing
the methylation site Lys310—has a simple secondary
structure (either a single helix or a flexible loop;
Supplementary Figure S1b and c), we performed a rigid
body docking of the RelA/p50 heterodimer into the
V-cleft of SETD6, resulting in a very good overall fit
(Figure 5a). In our docking, the RelA linker helical
region localizes to the bottom of the cleft and the helical
axis is nearly perpendicular to the longest dimension of
SETD6, with the dimerization and the activation domains
on either side of the V-cleft (Figure 5a). In this view,
SETD6 grips RelA like a dumbbell. The corresponding
linker region from the p50 subunit of the heterodimer is
positioned near the helical rim of the C-terminal domain,
away from the active site.
In vitro methylation assays indicated that the p50
subunit of NF-kB is not a substrate of SETD6
(Figure 5b; lanes 4 and 5) (4). There are two disordered
internal loops of the SETD6 structure: residues 230–236
(yellow) and 387–394 (green), located in the inner surface
of the V-cleft and the rim of the C-terminal domain. We
hypothesize that on association with the RelA/p50
heterodimer orRelA homodimer
Figure S1d), the unstructured SETD6 loops adopt stable
conformations that include contacts with the linker
regions of RelA/p50.
Recognition of RelA K310me1 by GLP ankryin repeats
Methylation of RelA by SETD6 represses NF-kB signal-
ing via the recognition of Lys310me1 by the ankryin
repeats of GLP (G9a-like protein) with a dissociation
constant (KD) of ?5mM (4). G9a and GLP are
euchromatin-associated methyltransferases that repress
transcription by mono- and di-methylating histone H3
lysine 9 (H3K9me1/2) via their C-terminal SET domains
(19). Previously, we showed that the centrally located
ankyrin repeat domains of G9a and GLP bind to histone
Figure 5. Hypothetical complex model of SETD6/NF-kB. (a) The
RelA subunit resembles a dumbbell with the dimerization and the
SETD6-driven methylation site, Lys310, is part of the linker region
that also harbors nuclear localization signal (see Supplementary
Figure S1a). The truncated NF-kB heterodimer structure [PDB 1FNI;
p50 (gray)/RelA (cyan)] (24) is docked onto the V-cleft of SETD6,
which grips the linker region. (b) In vitro methylation of RelA, either
as a RelA homodimer (lanes 2 and 3) or a RelA/p50 heterodimer (lanes
4 and 5), by SETD6. The top panel shows the Coomassie stain and the
fluorography is presented at the bottom.
bya linker region.The
6386Nucleic Acids Research, 2011,Vol.39, No. 15
H3 peptides containing H3K9me2/1, and phosphorylation
peptide binding (10). Analogous to H3S10 phosphoryl-
ation (see sequence alignment in Figure 6a), phosphoryl-
ation of Ser311 of RelA blocks the binding of Lys310me1
by GLP in vitro and in cells (4), probably due to steric
hindrance from the Ser-interacting glutamate (E874 of
human GLP) (Figure 6a). Mutations of the GLP residues
involved in forming the methyl-lysine cage (W843A,
E851A and W881A) abrogate the binding of GLP to the
Lys310 -monomethylated RelA peptide (Figure 6b).
proteins are involved in controlling NF-kB signaling.
Figure 6. Recognition of RelA Lys310me1 by GLP. (a) The model of GLP ankyrin repeats with the RelA peptide was built based on the structure of
GLP with a bound H3K9 peptide (residues 1–15) (PDB 3B95). The side chains of RelA Lys310me1 and Ser311 were modeled graphically without
repulsive clashing between GLP and the RelA peptide, (b) Biotinylated peptide pull-down assay with the indicated GLPANKconstructs (WT and
mutants) using the indicated biotinylated peptides, (c) A model of DNA-NF-kB-GLP (ANK-SET)-H3 peptide. The RelA and p50 are in cyan and
gray, respectively; GLP is in green, the H3 peptide is in magenta stick model and the DNA is in orange (phosphate backbone) and blue (bases). The
structures of DNA-NF-kB (PDB 2I9T) and IkB-NF-kB (PDB 1NFI) were superimposed via their respective dimerization domains, generating a
ternary complex of DNA-IkB-NF-kB (data not shown). The IkB was then replaced by GLP (PDB 3B95) via superimposing their respective ankyrin
repeats. In this model, the Lys310-contaning helix or the flexible loop of RelA might undergo intradomain movement to position the Lys310 in the
methyl-lysine binding cage of the GLP ankyrin repeat domain, as shown in a. The GLP SET-H3 peptide structure (PDB 3HNA) was manually
connected to the GLP ankyrin repeat domain with a short dotted stretch and (d) A cartoon illustration of the proposed model of signaling cross-talk
between the Lys310 methylated RelA/p50 heterodimer, the GLP/G9a heterodimer (29), and repressive chromatin with H3K9 methylation.
Nucleic Acids Research, 2011,Vol.39, No. 156387
The NF-kB precursor protein p105 possesses a N-terminal
p50 amino acid sequence and its own inhibitory ankyrin
repeats within its carboxy-terminal region (20). Once pro-
cessed, the NF-kB dimer RelA/p50 exists in the cytoplasm
of resting cells by its association with an IkB inhibitor
protein (21). IkB uses its entire ankyrin repeat domain
for interacting with the RelA linker region, which
includes Lys310 [reviewed in (22)]. Active NF-kB accumu-
lates in the nucleus where it preferentially binds a specific
DNA sequence in the promoter regions of target genes to
activate transcription. In addition, Levy et al. (4) sug-
gested that SETD6-mediated Lys310 methylation and its
recognition by the ankyrin repeat domain of GLP renders
NF-kB inert due to downstream silencing events mediated
by GLP-associated histone H3 lysine 9 methylation.
Encouraged by the known structures of (i) the RelA/p50
heterodimer bound to DNA (23), (ii) the RelA/p50
heterodimer bound to IkB (24) and (iii) the GLP
ankyrin repeat domain (10) and SET domain of GLP
bound with H3 peptide (25), we modeled a quaternary
complex involving DNA, NF-kB (RelA/p50), and GLP
(ankyrin repeats and SET domain) (Figure 6c). Our
model supports the notion that the SETD6–RelA–GLP–
H3K9me2/1 network constitutes a lysine methylation sig-
naling cascade, initiated by SETD6-mediated RelA
methylation at Lys310, followed by recruitment of a
histone-modifying enzyme (GLP), which subsequently
generates a repressive mark (H3K9me2/1).
Finally, it is interesting to note that a different class of
nuclear, ankyrin repeat-containing IkB proteins (26,27)
bind homodimers of p50 (28) that lack tranactivation
domains, and the nuclear IkB·NF-kB complexes can
bind DNA as repressors of transcription. In this regard,
we speculate that the ankyrin repeat-containing GLP
might function as an interaction competitor of nuclear
IkB for the RelA subunit, and the resulting H3K9 methy-
lation may reinforce silencing. It is possible that the re-
pressive GLP and G9a heterodimer (29) allows one of
these methyltransferases to interact with RelA and the
other to interact with histone H3 (Figure 6d).
Protein Data Bank: The coordinates and structure factors
of the SETD6–RelA peptide–AdoMet complex have been
deposited with accession numbers 3QXY (with the target
lysine in alternative bent and linear conformations) and
3RC0 (with the target lysine in bent conformation).
Supplementary Data are available at NAR Online.
Y.C. performed SETD6 enzyme purifications for both
spectrometry-based methylation assays, crystallization
and X-ray data collection; D.L. performed the cell
culture and the peptide pull-down experiments; J.R.H.
participated in collecting X-ray data, determined struc-
tures and performed structural refinements; J.P. per-
formed peptide methylation analysis by the LC–MS/MS
approach; D.L. and O.G. provided initial expression con-
structs and the knowledge of specificities of SETD6 and
RelA peptides. X.Z. developed and optimized the mass
spectrometry-based assay; X.C. and O.G. organized and
designed the scope of the study, and all were involved in
analyzing data and helped in writing and revising the
manuscript. The authors thank Ruth Tennen for reading
and editing the manuscript
The Department of Biochemistry at the Emory University
School of Medicine supported the use of the SER-CAT
synchrotron beamline at the Advanced Photon Source of
Argonne National Laboratory, local X-ray facility and
Institutes of Health (Grants GM068680 to X.C. and
DA025800 to O.G.); (NIH/NCRR R21RR025822 to
Human Frontier Science Program, and the Machiah
Foundation Fellowship (to D.L., partial); X.C. is a
Georgia Research Alliance Eminent Scholar; O.G. is an
Ellison Senior Scholar. Funding for open access charge:
National Institutes of Health.
1. Ghosh,S. and Hayden,M.S. (2008) New regulators of NF-kB in
inflammation. Nat. Rev. Immunol., 8, 837–848.
2. Hoffmann,A., Natoli,G. and Ghosh,G. (2006) Transcriptional
regulation via the NF-kB signaling module. Oncogene, 25,
3. Oeckinghaus,A. and Ghosh,S. (2009) The NF-kB family of
transcription factors and its regulation. Cold Spring Harbor
Perspect. Biol., 1, a000034.
4. Levy,D., Kuo,A.J., Chang,Y., Schaefer,U., Kitson,C., Cheung,P.,
Espejo,A., Zee,B.M., Liu,C.L., Tangsombatvisit,S. et al. (2011)
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. Nat. Immunol.,
5. Fu,Z.Q., Rose,J. and Wang,B.C. (2005) SGXPro: a parallel
workflow engine enabling optimization of program performance
and automation of structure determination. Acta Crystallogr.
D Biol. Crystallogr., 61, 951–959.
6. Adams,P.D., Afonine,P.V., Bunkoczi,G., Chen,V.B., Davis,I.W.,
Echols,N., Headd,J.J., Hung,L.W., Kapral,G.J., Grosse-
Kunstleve,R.W. et al. (2010) PHENIX: a comprehensive
Python-based system for macromolecular structure solution.
Acta Crystallogr. D Biol. Crystallogr., 66, 213–221.
7. Emsley,P. and Cowtan,K. (2004) Coot: model-building tools for
molecular graphics. Acta Crystallogr. D Biol. Crystallogr., 60,
8. Xu,P., Duong,D.M., Seyfried,N.T., Cheng,D., Xie,Y., Robert,J.,
Rush,J., Hochstrasser,M., Finley,D. and Peng,J. (2009)
Quantitative proteomics reveals the function of unconventional
ubiquitin chains in proteasomal degradation. Cell, 137, 133–145.
9. Bua,D.J., Kuo,A.J., Cheung,P., Liu,C.L., Migliori,V., Espejo,A.,
Casadio,F., Bassi,C., Amati,B., Bedford,M.T. et al. (2009)
Epigenome microarray platform for proteome-wide dissection of
chromatin-signaling networks. PLoS ONE, 4, e6789.
10. Collins,R.E., Northrop,J.P., Horton,J.R., Lee,D.Y., Zhang,X.,
Stallcup,M.R. and Cheng,X. (2008) The ankyrin repeats of G9a
and GLP histone methyltransferases are mono- and
6388 Nucleic Acids Research, 2011,Vol.39, No. 15
dimethyllysine binding modules. Nat. Struct. Mol. Biol., 15, Download full-text
11. Hendrickson,W.A., Horton,J.R. and LeMaster,D.M. (1990)
Selenomethionyl proteins produced for analysis by
multiwavelength anomalous diffraction (MAD): a vehicle for
direct determination of three-dimensional structure. EMBO J., 9,
12. Zhang,X., Tamaru,H., Khan,S.I., Horton,J.R., Keefe,L.J.,
Selker,E.U. and Cheng,X. (2002) Structure of the Neurospora
SET domain protein DIM-5, a histone H3 lysine
methyltransferase. Cell, 111, 117–127.
13. Trievel,R.C., Beach,B.M., Dirk,L.M., Houtz,R.L. and Hurley,J.H.
(2002) Structure and catalytic mechanism of a SET domain
protein methyltransferase. Cell, 111, 91–103.
14. Duran,A., Diaz-Meco,M.T. and Moscat,J. (2003) Essential role
of RelA Ser311 phosphorylation by zPKC in NF-kB
transcriptional activation. EMBO J., 22, 3910–3918.
15. Peters,A.H., Kubicek,S., Mechtler,K., O’Sullivan,R.J.,
Derijck,A.A., Perez-Burgos,L., Kohlmaier,A., Opravil,S.,
Tachibana,M., Shinkai,Y. et al. (2003) Partitioning and plasticity
of repressive histone methylation states in mammalian chromatin.
Mol. Cell, 12, 1577–1589.
16. Rice,J.C., Briggs,S.D., Ueberheide,B., Barber,C.M.,
Shabanowitz,J., Hunt,D.F., Shinkai,Y. and Allis,C.D. (2003)
Histone methyltransferases direct different degrees of methylation
to define distinct chromatin domains. Mol. Cell, 12, 1591–1598.
17. Jenuwein,T., Laible,G., Dorn,R. and Reuter,G. (1998) SET
domain proteins modulate chromatin domains in eu- and
heterochromatin. Cell Mol. Life Sci., 54, 80–93.
18. Rea,S., Eisenhaber,F., O’Carroll,D., Strahl,B.D., Sun,Z.W.,
Schmid,M., Opravil,S., Mechtler,K., Ponting,C.P., Allis,C.D.
et al. (2000) Regulation of chromatin structure by site-specific
histone H3 methyltransferases. Nature, 406, 593–599.
19. Tachibana,M., Sugimoto,K., Nozaki,M., Ueda,J., Ohta,T.,
Ohki,M., Fukuda,M., Takeda,N., Niida,H., Kato,H. et al. (2002)
G9a histone methyltransferase plays a dominant role in
euchromatic histone H3 lysine 9 methylation and is essential for
early embryogenesis. Genes Dev., 16, 1779–1791.
20. Basak,S., Kim,H., Kearns,J.D., Tergaonkar,V., O’Dea,E.,
Werner,S.L., Benedict,C.A., Ware,C.F., Ghosh,G., Verma,I.M.
et al. (2007) A fourth IkB protein within the NF-kB signaling
module. Cell, 128, 369–381.
21. Baeuerle,P.A. and Baltimore,D. (1988) IkB: a specific inhibitor of
the NF-kB transcription factor. Science, 242, 540–546.
22. Huxford,T. and Ghosh,G. (2009) A structural guide to proteins
of the NF-kB signaling module. Cold Spring Harbor Perspect.
Biol., 1, a000075.
23. Escalante,C.R., Shen,L., Thanos,D. and Aggarwal,A.K. (2002)
Structure of NF-kB p50/p65 heterodimer bound to the PRDII
DNA element from the interferon-beta promoter. Structure, 10,
24. Jacobs,M.D. and Harrison,S.C. (1998) Structure of an IkBa/
NF-kB complex. Cell, 95, 749–758.
25. Wu,H., Min,J., Lunin,V.V., Antoshenko,T., Dombrovski,L.,
Zeng,H., Allali-Hassani,A., Campagna-Slater,V., Vedadi,M.,
Arrowsmith,C.H. et al. (2010) Structural biology of human H3K9
methyltransferases. PLoS ONE, 5, e8570.
26. Yamazaki,S., Muta,T. and Takeshige,K. (2001) A novel IkB
protein, IkB-z, induced by proinflammatory stimuli, negatively
regulates nuclear factor-kB in the nuclei. J. Biol. Chem., 276,
27. Michel,F., Soler-Lopez,M., Petosa,C., Cramer,P., Siebenlist,U.
and Muller,C.W. (2001) Crystal structure of the ankyrin repeat
domain of Bcl-3: a unique member of the IkB protein family.
EMBO J., 20, 6180–6190.
28. Trinh,D.V., Zhu,N., Farhang,G., Kim,B.J. and Huxford,T. (2008)
The nuclear IkB protein IkB z specifically binds NF-kB p50
homodimers and forms a ternary complex on kB DNA. J. Mol.
Biol., 379, 122–135.
29. Tachibana,M., Ueda,J., Fukuda,M., Takeda,N., Ohta,T.,
Iwanari,H., Sakihama,T., Kodama,T., Hamakubo,T. and
Shinkai,Y. (2005) Histone methyltransferases G9a and GLP form
heteromeric complexes and are both crucial for methylation of
euchromatin at H3-K9. Genes Dev., 19, 815–826.
Nucleic Acids Research, 2011,Vol.39, No. 156389