Cell, Vol.
113, 127–137, April 4, 2003, Copyright 2003 by Cell Press
The Polycomb Protein Pc2 Is a SUMO E3
Michael H. Kagey, Tiffany A. Melhuish,
and David Wotton*
Department of Biochemistry and Molecular
Genetics
Center for Cell Signaling
University of Virginia
Charlottesville, Virginia 22908
Summary
Polycomb group (PcG) proteins form large multimeric
complexes (PcG bodies) which are involved in the sta-
ble repression of gene expression. The human PcG
protein, Pc2, has been shown to recruit the transcrip-
tional corepressor, CtBP, to PcG bodies. We show that
CtBP is sumoylated at a single lysine. In vitro, CtBP
sumoylation minimally requires the SUMO E1 and E2
(Ubc9) and SUMO-1. However, Pc2 dramatically en-
hances CtBP sumoylation. In vivo, this is likely due to
the ability of Pc2 to recruit both CtBP and Ubc9 to
PcG bodies, thereby bringing together substrate and
E2, and stimulating the transfer of SUMO to CtBP.
These results demonstrate that Pc2 is a SUMO E3, and
suggest that PcG bodies may be sumoylation centers.
Polycomb group (PcG) proteins were identified in Dro-
sophila as regulators of homeotic gene expression (Ken-
nison, 1995; Simon, 1995). PcG proteins maintain the
repressed state of these genes, and in PcG mutant flies,
expression of homeotic genes is seen outside their nor-
mal segment boundaries, resulting in altered body part
identity (Simon, 1995; Schumacher and Magnuson,
1997). PcG proteins appear to act as an epigenetic mark
on the chromatin template that maintains the silent state
at specific target loci (Cavalli and Paro, 1998). PcG pro-
teins comprise a family of functionally related proteins,
many of which interact with each other to form PcG
complexes (Pirrotta, 1998; Sewalt et al., 1998; Francis
et al., 2001; Gunster et al., 2001).
Mammalian homologs of many Drosophila PcG pro-
teins are known (Brock and van Lohuizen, 2001), and
it appears that multiple core PcG complexes exist in
different cell types (Sewalt et al., 1998; Francis et al.,
2001; Gunster et al., 2001). Although direct DNA binding
of individual PcG proteins has not been demonstrated,
PcG complexes can bind to DNA (Francis et al., 2001).
In addition, PcG complexes can be recruited to DNA by
interactions with specific DNA binding proteins, includ-
ing YY1, E2F, and Rb (Dahiya et al., 2001; Satijn et al.,
2001; Ogawa et al., 2002). Direct tethering of PcG pro-
teins, such as Drosophila Pc, to DNA reveals that they
can mediate repression of gene expression (Muller,
1995; Poux et al., 2001). Once recruited to DNA, PcG
proteins appear to exert their repressive effects over
large distances, either by spreading along the DNA tem-
plate, or by bringing together distant DNA-bound com-
*Correspondence: dw2p@virginia.edu
plexes (Pirrotta, 1998; Jacobs and van Lohuizen, 2002).
PcG complexes form discrete foci within the nucleus,
which have been termed PcG bodies (Gerasimova and
Corces, 1998; Saurin et al., 1998), which are generally
located near centromeres and may result from the cluster-
ing of multiple DNA-associated PcG complexes. Recruit-
ment of other transcriptional regulators to PcG bodies
may play a role in mediating transcriptional repression
(Sewalt et al., 1999; van der Vlag and Otte, 1999; Espinas
et al., 2000; Simon and Tamkun, 2002). Recent work
has demonstrated that PcG complexes contain histone
methyl transferase activity, which likely plays an impor-
tant role in transcriptional regulation (Cao et al., 2002;
Czermin et al., 2002; Kuzmichev et al., 2002; Muller et
al., 2002; Sewalt et al., 2002). However, much remains
to be determined with respect to the function of PcG
complexes.
SUMO (small ubiquitin-related modifier) is similar to
ubiquitin in terms of its structure and mechanism of
attachment to target proteins (Mayer et al., 1998;
Jentsch and Pyrowolakis, 2000; Melchior, 2000). Three
mammalian SUMO isoforms exist: SUMO-2 and 3, which
are highly related and the more divergent SUMO-1
(Boddy et al., 1996; Mannen et al., 1996; Okura et al.,
1996; Shen et al., 1996; Lapenta et al., 1997). It is not
clear whether there are important functional differences
between the three SUMOs. Indeed, the functional signifi-
cance of sumoylation is less well understood than that
of ubiquitination. Unlike poly-ubiquitination, sumoyla-
tion of a protein does not target it for degradation, but
may play a role in targeting proteins to specific sub-
cellular locations (Melchior, 2000; Hay, 2001; Muller et
al., 2001). For example, sumoylation of PML is required
for its localization to PML bodies (Muller et al., 1998;
Lallemand-Breitenbach et al., 2001). Sumoylation of
RanGAP targets it to the nuclear pore complex (NPC)
(Matunis et al., 1996). Sumoylation has also been shown
the regulate the activity of a variety of transcription fac-
tors including p53 and Sp3 (Rodriguez et al., 1999; Ross
et al., 2002; Sapetschnig et al., 2002).
Conjugation of SUMO or ubiquitin to a target protein
occurs by a similar mechanism: SUMO/ubiquitin is
cleaved at two conserved glycines and is then attached
by the carboxyl-terminal residue to a lysine within the
target protein (Hershko and Ciechanover, 1998; Mel-
chior, 2000; Pickart, 2001). Ubiquitination involves an
activating enzyme (E1), a conjugating enzyme (E2), and
a ubiquitin ligase (E3) (Hershko and Ciechanover, 1998).
A single ubiquitin E1, and multiple E2 and E3 proteins
are known (Jackson et al., 2000). It is the E3 ligases
which largely determine substrate specificity. For
SUMO, a single E1 (a heterodimer of Aos1 and Uba2)
and E2 (Ubc9) have been identified (Dohmen et al., 1995;
Desterro et al., 1997; Johnson and Blobel, 1997; John-
son et al., 1997). However, until recently, no SUMO E3
ligases were known, raising the question of how speci-
ficity is controlled.
Many SUMO target proteins contain the amino acid
motif, ⌿KxE (⌿ is hydrophobic) (Melchior, 2000). Muta-
tional and structural evidence suggest that this motif
Cell
128
plays an important role in binding Ubc9 and directing
sumoylation to the lysine (Sampson et al., 2001; Bernier-
Villamor et al., 2002). Recent evidence has demon-
strated the presence of two families of SUMO E3s. In S.
cerevisiae, Siz1p is required for sumoylation of septins in
vivo and enhances in vitro sumoylation (Johnson and
Gupta, 2001; Takahashi et al., 2001). Siz1p, and the
related Siz2p, contain a divergent RING-like motif similar
to those found in a class of ubiquitin E3s. Mammalian
PIAS (protein inhibitor of activated STAT) proteins act
as SUMO E3s for LEF1, p53, and c-Jun (Kahyo et al.,
2001; Sachdev et al., 2001; Schmidt and Muller, 2002).
Siz and PIAS proteins share a conserved RING-like do-
main, suggesting they are members of the same family
of SUMO E3s (Hochstrasser, 2001; Jackson, 2001).
RanBP2 is an NPC-associated protein which has SUMO
E3 activity toward Sp100 and a class II histone deacety-
lase (Kirsh et al., 2002; Pichler et al., 2002). RanBP2 also
contains a RING domain, but it is dispensable for E3
activity, suggesting that RanBP2 represents a second
class of SUMO E3s.
We demonstrate that the transcriptional corepressor
CtBP (carboxyl-terminus binding protein) is modified by
SUMO-1 or SUMO-3 on a single lysine. CtBP was identi-
fied by its interaction with the adenovirus E1A protein
and this interaction is required for transcriptional repres-
sion by E1A (Boyd et al., 1993; Schaeper et al., 1995).
CtBP has since been shown to bind many gene-specific
and general transcriptional repressors via a PLDLS (or
related) motif (Schaeper et al., 1998; Turner and Cross-
ley, 1998, 2001; Chinnadurai, 2002). CtBP interacts with
Pc2, a polycomb group protein, resulting in the recruit-
ment of CtBP to PcG bodies (Sewalt et al., 1999). We
show that Pc2 is a SUMO E3 which recruits both CtBP
and Ubc9 to PcG bodies. This work demonstrates a
novel activity at PcG bodies, and we speculate that PcG
complexes may exert their effects on transcription in
part by mediating the sumoylation of specific target pro-
teins.
Results
CtBP Is Sumoylated, In Vitro and In Vivo
CtBP contains a single sumoylation motif (see Figure
2A), suggesting that it may be a target for sumoylation.
To determine whether CtBP is a potential substrate,
purified bacterially expressed T7-CtBP was incubated
with recombinant Aos1/Uba2 (E1), Ubc9, and SUMO-1
in the presence of ATP. Reactions were terminated by
the addition of SDS-PAGE loading buffer and analyzed
by Western blot with a T7-specific antibody to detect
CtBP. When recombinant E1, Ubc9, and SUMO-1 were
all present, a slower migrating band was observed that
is consistent with the conjugation of SUMO-1 to CtBP
(Figure 1A). This band was not present when either the
E1, Ubc9, or SUMO-1 was absent from the reaction,
suggesting that CtBP can be sumoylated and indicating
that CtBP minimally requires these proteins for sumo-
ylation.
To determine whether CtBP can be sumoylated in
vivo, COS-1 cells were cotransfected with expression
constructs encoding T7-tagged CtBP and six-histidine-
(H6)-tagged SUMO-1 or -3. Cell lysates were separated
by SDS-PAGE and Western blotted to detect T7-CtBP.
In addition to unmodified CtBP, a slower migrating band
was also observed in the presence of H6-SUMO-1 or -3
(Figure 1B). The mobility of this band is consistent with
the attachment of H6-SUMO to CtBP. The intensity of
the slower migrating band was clearly enhanced by
coexpression of Ubc9, suggesting that Ubc9 may be
limiting. As further evidence that the slower migrating
CtBP was sumoylated, COS-1 cells were transiently
transfected with H6-CtBP and Flag-tagged SUMO-1 or
-3. Cells were lysed in 6 M guanidine-HCl and proteins
were affinity purified on cobalt agarose, separated by
SDS-PAGE, and Western blotted with a Flag antibody
to detect the CtBP-conjugated SUMO. Multiple slower
migrating bands corresponding to SUMO conjugated
CtBP were observed (Figure 1C). Multiple sumoylated
forms of CtBP were also evident by direct Western blot
of the lysates (Figure 1C, lower panel). To determine
whether CtBP was subject to SUMO modification at
endogenous expression levels, HeLa cell proteins were
immunoprecipitated with an antibody specific for CtBP.
When immunoprecipitates were analyzed by SDS-PAGE
and Western blotted with a SUMO-1-specific antibody,
a band of the appropriate mobility for SUMO-1-conju-
gated CtBP was clearly visible (Figure 1D). This band
was not detected when proteins were first immunopre-
cipitated with a CtBP2-specific antibody, or with a con-
trol antibody (Flag). Together, these data clearly demon-
strate that CtBP is a substrate for sumoylation, both in
vivo and in vitro.
CtBP Is Sumoylated at Lysine 428
To determine the importance of the sumoylation motif
surrounding lysine 428, we created a version of CtBP
in which lysine 428 was altered to arginine (K428R; Fig-
ure 2A). COS-1 cells were cotransfected with Flag-
CtBPK428R, T7-Ubc9, and either H6-SUMO-1 or -3 and
analyzed by Western blot. No sumoylated CtBPK428R was
detected, indicating that lysine 428 is the principal lysine
utilized for SUMO conjugation (Figure 2B). This lysine
is present in the related CtBP2, but the surrounding
motif is absent (Figure 2A). No sumoylation of CtBP2
was detected when lysates from COS-1 cells trans-
fected with Fl-CtBP2, T7-Ubc9, and H6-SUMO-1 or -3
were analyzed by Western blot (Figure 2B). Thus, it ap-
pears that the motif surrounding lysine 428 is important
for efficient sumoylation. Since lysine 428 appeared to
be the major sumoylation site in CtBP, the additional
bands observed in Figure 1C are likely the result of poly
sumoylation of CtBP at lysine 428. However, we cannot
rule out the possibility that an initial sumoylation event
at lysine 428 is required for subsequent modification of
other lysines within CtBP.
To determine whether either CtBP or CtBP2 was able
to interact with Ubc9, COS-1 cells were transfected with
expression vectors encoding Fl-CtBP, Fl-CtBP2 or Fl-
CtBPK428R, and T7-Ubc9. Flag proteins were precipitated
from cell lysates and Western blotted to detect copreci-
pitating T7-Ubc9. Surprisingly, CtBP, CtBP2, and CtBPK428R
all associated with Ubc9, even though only CtBP is mod-
ified by SUMO (Figure 2C). Thus, it appears that determi-
nants outside the sumoylation motif contribute to inter-
action with Ubc9.
Polycomb and SUMO
129

Sumoylated CtBP Is Associated with Pc2

CtBP may have both nuclear and non-nuclear functions,
and localizes to nucleus and cytoplasm (Criqui-Filipe et
al., 1999; Weigert et al., 1999; Turner and Crossley,
2001). The polycomb group protein, Pc2, can recruit
CtBP to PcG bodies within the nucleus (Sewalt et al.,
1999). We, therefore, wished to determine where in the
cell sumoylated CtBP was localized. We created tagged
versions of CtBP and Pc2, which were fused to color-
shifted enhanced green fluorescent protein variants (ei-
ther cyan or yellow; CFP or YFP). As shown in Figure
3A, when visualized by fluorescence microscopy, CFP-
CtBP was present in both the nucleus and cytoplasm
of COS-1 cells. However, in the presence of coex-
pressed T7-Pc2, a significant proportion of the CFP-
CtBP localized to nuclear foci. Visualization of CFP-
CtBP and YFP-Pc2 in the same cells clearly revealed
that the two proteins colocalize (Figure 3B). Thus, in
live cells, CFP-CtBP was recruited to PcG bodies by
coexpression of Pc2.
Since only a proportion of the CtBP is modified by
SUMO, we first sought to determine biochemically the
localization of this fraction of CtBP. Cells were treated
with digitonin to selectively permeabilize the plasma
membrane. After removal of the cytosol cells were
treated with 1% NP40 to dissolve the nuclear membrane
and release soluble nuclear proteins. The remaining in-
soluble cellular material was designated as the pellet
fraction. Digitonin soluble, NP40 soluble, and pellet (in-
soluble) fractions were analyzed by Western blot with
a T7 antibody to detect transfected CtBP. CtBP was
primarily localized in the digitonin and NP40 soluble
fractions, with some CtBP in the pellet (Figure 3C), con-
sistent with its localization to nucleus and cytoplasm.
When COS-1 cells were transfected with T7-CtBP, Fl-
Ubc9, and H6-SUMO-3, the majority of SUMO-3-conju-
gated CtBP was in the NP40 fraction, suggesting that
it was present in a soluble nuclear fraction (Figure 3C).
When Flag-Pc2 was coexpressed with T7-CtBP, Fl-
Ubc9, and H6-SUMO-3, virtually all of the SUMO-3-con-
jugated CtBP was present in the insoluble nuclear frac-
tion (pellet), together with the majority of the Pc2 (Figure
3D). Interestingly, coexpression of Pc2 also appeared
to increase the amount of SUMO-3 conjugated to CtBP.
These results suggest that sumoylated CtBP is associ-

analyzed by Western blot with a T7 antibody. The positions of un-

Figure 1. CtBP Is Sumoylated
(A) Purified T7-CtBP was incubated in the presence or absence of
recombinant Aos1/Uba2 (E1), Ubc9, and SUMO-1. Proteins were
modified and SUMO-1 modified CtBP are shown.
(B) COS-1 cells were transfected with T7-CtBP, Flag-Ubc9, and H6-
SUMO-1 or -3 expression constructs. Proteins were separated by
SDS-PAGE and analyzed by Western blot with a T7 antibody. The
positions of sumoylated forms of CtBP are shown.
(C) COS-1 cells were transfected with H6-CtBP, T7-Ubc9, and Flag-
SUMO-1 or -3 expression constructs as indicated. Cells were lysed
in 6 M guanidine-HCl, proteins were purified on cobalt agarose and
Western blotted with a Flag antibody. The positions of sumoylated
forms of CtBP are indicated. The white arrow shows where unmodi-
fied CtBP would be expected to migrate. Lower panel: 10% of the
cells were analyzed by direct Western blot using a Flag antibody.
(D) Proteins were precipitated from HeLa cell lysates with antibodies
specific for CtBP, CtBP2, or Flag (as indicated) and Western blotted
with a SUMO-1-specific antibody. The positions of CtBP-SUMO-1
and the immunoglobulin heavy chain (HC) are shown. The positions
of molecular weight markers (kD) are shown.
Cell
130

Figure 2. CtBP Is Sumoylated at Lysine 428

(A) An amino acid alignment of the carboxy termini of CtBP and CtBP2 is shown, with the lysine to arginine mutant (K428R). The sumoylation
consensus is boxed.
(B) COS-1 cells were transfected with Fl-CtBP, Fl-CtBP2, or the K428R mutant and T7-Ubc9 and H6-SUMO as indicated. Proteins were
separated by SDS-PAGE and analyzed by Western blot using a Flag antibody.
(C) COS-1 cells were cotransfected with T7-Ubc9 and the indicated Flag-tagged CtBP expression vector. Proteins were precipitated with Flag
agarose and analyzed by Western blot with a T7 antibody. Expression of transfected proteins in the lysates is shown below. The bar denotes
immunoglobulin light chain.

ated with Pc2, and raise the possibility that Pc2 en-
hances CtBP sumoylation.
Pc2 Is Sumoylated
Coexpression of SUMO-3 and Ubc9 with Flag-Pc2 re-
sulted in the presence of multiple slower migrating Flag-
reactive bands, indicating that Pc2 may be sumoylated
(Figure 3D). To test this possibility, whole cell extracts
from COS-1 cells that had been transfected with T7-
Pc2 and H6-SUMO-1 or -3 were Western blotted for
T7-Pc2. In addition to detecting Pc2, multiple slower
migrating bands were observed, suggesting the conju-
gation of multiple SUMOs to Pc2 (Figure 4A). The inten-
sity of these bands was not significantly enhanced by
coexpression of Ubc9, suggesting that endogenous
Ubc9 levels were sufficient to sumoylate Pc2.
As further evidence that the slower migrating bands
were sumoylated forms of Pc2, COS-1 cells were trans-
fected with T7-Pc2 and Fl-SUMO-1 or -3, and lysed in
6 M guanidine-HCl. Pc2 contains 12 consecutive histi-
dine residues, making it possible to purify T7-Pc2 with
cobalt agarose (Figure 4B, lower panel). After purifica-
tion on cobalt agarose, proteins were Western blotted
to detect Flag-SUMO conjugated to Pc2. As expected,
unmodified Pc2 was not detected with the Flag anti-
body, but multiple slower migrating forms of Pc2 were
present (Figure 4B). In a parallel experiment, when cells
were transfected with T7-Pc2 alone and proteins puri-
fied on cobalt agarose, conjugation of endogenous
SUMO-1 to T7-Pc2 was detected (Figure 4B, right panel).
To further demonstrate that Pc2 is modified by SUMO,
bacterially expressed T7-Pc2 was purified on T7 aga-
rose and incubated with GST-SUMO-3, Ubc9, E1, and
ATP. As shown in Figure 4C, slower migrating forms of
Pc2, corresponding to attachment of multiple SUMOs
to Pc2, were detected. When either SUMO-3, Ubc9, or
the E1 was absent, no modified Pc2 was observed.
Together, these results demonstrate that both CtBP and
Pc2 are sumoylated and that sumoylated CtBP appears
to be preferentially associated with Pc2.
Pc2 Localizes SUMO and Ubc9 to Nuclear Foci
Since Pc2 is sumoylated, we decided to use fluores-
cence microscopy to determine the effect of coexpres-
sion of Pc2 on the subcellular localization of CFP-
SUMO-1. COS-1 cells transfected with CFP-SUMO-1
showed a dispersed nuclear distribution of CFP-
SUMO-1, with some fluorescent signal present outside
the nucleus (Figure 5A). Coexpression of Fl-Pc2 resulted
in the localization of much of the CFP-SUMO-1 to nu-
clear foci characteristic of PcG bodies (Figure 5A). We
obtained similar results with CFP-SUMO-3 to those ob-
served with SUMO-1 (Figure 5D and data not shown).
Fluorescence microscopy of COS-1 cells transfected
with YFP-Ubc9 indicated that it was localized primarily
to the nucleus, and coexpression of Pc2 resulted in the
recruitment of YFP-Ubc9 to nuclear foci (Figure 5B). To
confirm that the foci observed with YFP-Ubc9 are PcG
bodies, we coexpressed YFP-Ubc9 and CFP-Pc2. As
shown in Figure 5C, a significant proportion of both
Polycomb and SUMO
131

Figure 3. CtBP-SUMO Associates with Pc2

(A) COS-1 cells were transfected with a CFP-CtBP expression vector. After 24 hr, cells were stained with Hoechst and live cells were visualized.
CFP-CtBP was expressed in the absence (upper) or presence (lower) of T7-Pc2. CFP and Hoechst images are shown. Scale bar ⫽ 10 ␮m.
(B) CFP-CtBP was coexpressed with YFP-Pc2. Hoechst, cyan, and yellow images are shown, with the merged image of CFP and YFP
fluorescence (overlay).
(C and D) COS-1 cells were transfected with the indicated expression constructs and partitioned into digitonin soluble (“D”), NP40 soluble
(“N”), and pellet (“P”) fractions, which were analyzed for T7-CtBP or Fl-Pc2 by Western blot.

proteins clearly colocalized to nuclear foci. Together,
these data suggest a novel subnuclear localization of
SUMO and Ubc9 to PcG bodies, dependent on Pc2.
PML localizes at subnuclear domains (PML bodies)
and is modified by SUMO-1 (Muller et al., 1998; Lalle-
mand-Breitenbach et al., 2001). We, therefore, wanted
to know whether Pc2 localized SUMO to nuclear do-
mains that were distinct from PML bodies. COS-1 cells
were cotransfected with CFP-Pc2 and a YFP-PML ex-
pression construct. As shown in Figure 5D, YFP-PML
and CFP-Pc2 both form subnuclear foci, but do not
colocalize. In contrast, cotransfection with YFP-PML
and CFP-SUMO-3 resulted in clear colocalization of
SUMO-3 with PML bodies. We then wanted to determine
whether PML and Pc2 recruited SUMO to separate sites.
CFP-SUMO-3 localized to an increased number of foci
within the nucleus of cells expressing Pc2, and only a
proportion of these foci colocalized with YFP-PML (Fig-
ure 5D). Thus, both PML and PcG bodies may be distinct
sites of SUMO modification within the nucleus.
Pc2 Is a SUMO E3
SUMO E3 ligases, like RING domain ubiquitin E3s, do not
possess intrinsic enzymatic activity, but act as adapters
which bring together the E2 (Ubc9) and the substrate
(Hochstrasser, 2001; Jackson, 2001). As shown in Figure
6A, CFP-CtBP and YFP-Ubc9 show only partial colocali-
zation. However, when Pc2 is also coexpressed, both
CFP-CtBP and YFP-Ubc9 become colocalized to PcG
bodies. Thus, Pc2 is recruiting substrate and E2 to the
same subnuclear compartment. As an E3, Pc2 would
be expected to enhance CtBP sumoylation. To test this,
COS-1 cells were transfected with T7-CtBP and H6-
SUMO-1 or 3, with and without Fl-Pc2, and cell lysates
were Western blotted to detect T7-CtBP. Pc2 clearly
enhanced the amount of SUMO-1 or SUMO-3 conju-
gated to CtBP and was able to further increase sumoyla-
tion over that seen with Ubc9 coexpression (Figure 6B).
In addition, when these lysates were probed for Pc2,
sumoylated forms of Pc2 were detected. Interestingly,
Pc2 is a chromodomain protein, which shares no signifi-
cant homology with known E3 ligases. Removal of the
chromodomain of Pc2 (construct 68–558) did not affect
enhancement of CtBP sumoylation, whereas, further
truncation to amino acid 401 abolished this activity (Fig-
ure 6C). Since both constructs interact with CtBP (data
not shown), the region between amino acids 68 and 401
may be required for Ubc9 interaction.
To further demonstrate that Pc2 increased sumoyla-
tion of CtBP, COS-1 cells were cotransfected with H6-
CtBP, T7-Pc2, and Fl-SUMO, lysed in 6 M guanidine-HCl
and proteins were purified on cobalt agarose. Western
blotting with a Flag antibody to detect sumoylated CtBP
demonstrated that Pc2 expression increased the
amount of purified sumoylated H6-CtBP (Figure 6D).
Together, these results show that Pc2 recruits both
Ubc9 and CtBP to PcG bodies and enhances sumoyla-
tion of CtBP, suggesting that Pc2 is at least a component
of a SUMO E3 complex.
To determine whether Pc2 is a SUMO E3, we per-
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132
Figure 4. Pc2 Is Sumoylated
(A) COS-1 cells were transfected with the indicated expression con-
structs and cell lysates were analyzed by Western blot with a T7
antibody.
(B) Transfected COS-1 cells were lysed in guanidine-HCl, proteins
purified on cobalt agarose, and analyzed by Western blot with Flag
or T7 antibodies, or a SUMO-1 specific antibody (right panel). The
positions of Pc2 and sumoylated Pc2 are indicated, together with
molecular weight (kD) markers. The white arrow indicates the ex-
pected mobility of unmodified Pc2.
(C) Bacterially expressed T7-Pc2 was incubated in the presence or
absence of recombinant E1, Ubc9, and GST-SUMO-3 for 2 hr at
30⬚C. Proteins were analyzed by Western blot with a T7 antibody.
The positions of Pc2 and SUMO-3 modified Pc2 are shown.
formed in vitro sumoylation assays with CtBP, using
limiting levels of Ubc9 in the presence or absence of
purified bacterially expressed Pc2. As shown in Figure
7A, with Ubc9, only a small amount of sumoylated CtBP
was visible. However, addition of recombinant Pc2 to
the reaction resulted in a clear increase in sumoylated
CtBP. No modified CtBP was observed in the absence
of Ubc9, irrespective of Pc2 addition. We had observed
that although CtBP2 interacted with Ubc9, no sumo-
ylated CtBP2 was detected in vivo (see Figure 2). Sur-
prisingly, CtBP2 was sumoylated in vitro in the presence
of E1 and Ubc9 and this modification was significantly
enhanced by the addition of Pc2 (Figure 7B). We next
determined whether Pc2 expression would allow detec-
tion of CtBP2 sumoylation in vivo, by enhancing the
level of this modification. As shown in Figure 7C, coex-
pression of H6-SUMO-1 with either Ubc9 or Pc2 resulted
in efficient sumoylation of CtBP, but not of the CtBPK428R
mutant. Interestingly, although no sumoylated CtBP2
was observed in the presence of H6-SUMO-1 and Ubc9,
the coexpression of Pc2 and H6-SUMO-1 clearly in-
duced CtBP2 sumoylation (Figure 7C). Taken together,
these results demonstrate that Pc2 is a SUMO E3.
Discussion
We show that CtBP can be sumoylated, both in vivo
and in vitro, at a SUMO consensus motif. Importantly,
Pc2 clearly enhances sumoylation of CtBP, suggesting
that Pc2 represents a novel class of SUMO E3s. In addi-
tion, Pc2 can recruit both CtBP and Ubc9 to PcG bodies,
suggesting that these structures may function as cen-
ters of sumoylation within the nucleus.
A SUMO E3
Ubc9 is necessary and sufficient to attach SUMO to
numerous substrates. However, by analogy with ubiqui-
tination, SUMO E3s are likely to help determine sub-
strate specificity and enhance efficiency of sumoylation.
Ubiquitin E3s fall into two classes: HECT domain pro-
teins are true enzymes, which form a thioester bond
with ubiquitin prior to transferring it to the substrate. In
contrast, RING domain proteins act as adapters, bring-
ing together E2 and substrate (Laney and Hochstrasser,
1999). Two classes of SUMO E3 have been identified,
both of which contain RING-like domains and appear
to act as nonenzymatic adapters. In Siz1p and PIAS
proteins, the RING-like domains are required for E3 ac-
tivity (Johnson and Gupta, 2001; Kahyo et al., 2001;
Sachdev et al., 2001; Schmidt and Muller, 2002) whereas
the RING domain of RanBP2 is dispensable for E3 activ-
ity (Pichler et al., 2002).
Here, we demonstrate a novel function for Pc2, namely
that it acts as a SUMO E3 for CtBP: Pc2 interacts with
both Ubc9 and with CtBP, concentrates both proteins
into PcG bodies and stimulates the transfer of SUMO
to CtBP. Importantly, purified Pc2 also enhanced CtBP
sumoylation in vitro. Pc2 is analogous to other SUMO
E3s, in that it acts as a nonenzymatic adaptor, but may
also have a more specialized function, since it causes
a stable colocalization of E2 and substrate. RanBP2,
acts as an E3 for Sp100, a protein which localizes to PML
bodies. In contrast to our data with Pc2, this suggests a
transient interaction of E2, E3, and substrate at the NPC,
possibly while the substrate is en route to its final loca-
tion in the nucleus (Wu et al., 1995; Sternsdorf et al.,
1997; Pichler et al., 2002). Pc2 recruits both Ubc9 and
CtBP to PcG bodies, suggesting that PcG bodies may
be a specific site of CtBP sumoylation. A second possi-
bility is that CtBP is itself part of an E3 complex which
Polycomb and SUMO
133
recruits other proteins to PcG bodies to be sumoylated.
It is also possible that other PcG proteins may be part
of a Pc2-containing E3 complex. It will now be of interest
to determine the importance of other PcG proteins for
sumoylation of CtBP or other substrates.
Our data demonstrate that Pc2 represents a novel
class of SUMO E3, which has no obvious sequence
similarity to other known E3s, perhaps suggesting the
existence of other unrelated SUMO E3s. It is likely that
multiple E3s will compete for interaction with Ubc9 and
recruit it to specific subcellular locations, resulting in
a high local concentration of E2 and substrate. Thus,
proteins like Pc2 may be important rate-limiting factors
in specific sumoylation pathways.
Nonconsensus SUMO Substrates
Many SUMO substrates, including CtBP, contain a con-
sensus targeting motif, which is clearly an important
determinant of substrate specificity. We show that
CtBP2, which does not contain this motif, interacts with
Ubc9 and is sumoylated in vitro. However, in vivo su-
moylation of CtBP2 is much more dependent on Pc2
than is sumoylation of CtBP, suggesting that even in the
Figure 5. Ubc9 and SUMO Localize to Poly-
comb Bodies
COS-1 cells were transfected with the indi-
cated expression vectors and fluorescence
visualized as in Figure 3.
(A) CFP-SUMO-1 was expressed alone or
with Flag-Pc2.
(B) YFP-Ubc9 was expressed alone or in the
presence of Flag Pc2. CFP or YFP and
Hoechst images are shown.
(C) YFP-tagged Ubc9 was coexpressed with
CFP-Pc2. The images are shown individually
and merged (overlay).
(D) YFP-tagged PML was coexpressed with
CFP-Pc2 (upper), or with CFP-SUMO-3 (mid-
dle). YFP-PML and CFP-SUMO-3 were coex-
pressed with Flag-tagged Pc2 (lower). Indi-
vidual YFP, CFP, and Hoechst images are
shown, together with the merged YFP and
CFP image (overlay). Scale bar ⫽ 10 ␮m.
absence of a targeting motif, a SUMO E3 can determine
substrate specificity. Thus, it appears that two factors
can contribute to substrate specificity: The presence
of a sumoylation motif within the substrate, and the
presence of an appropriate E3 factor. That Pc2 can
enhance sumoylation of CtBP2 by Ubc9, suggests that
other potential SUMO substrates, which lack the tar-
geting motif may indeed be sumoylated in vivo, provided
the correct E3 is present.
A Novel Function for Polycomb Bodies
We have demonstrated a novel enzymatic activity at
PcG complexes, which results in the sumoylation of at
least two proteins. Although purified recombinant Pc2
has E3 activity toward CtBP, it is possible that, since
Pc2 is part of a multimeric complex, other associated
PcG proteins may contribute to full E3 activity in vivo.
Pc2 recruits Ubc9 and SUMO, resulting in Pc2 sumoyla-
tion. This E2-E3 complex (Ubc9 and Pc2) efficiently su-
moylates CtBP, and may also modify other PcG proteins
within the complex. For example, Bmi-1 contains a
SUMO consensus site, is a component of PcG com-
plexes, and Bmi-1 interacts with Pc2 (Gunster et al.,
Cell
134

Figure 6. Pc2 Enhances the Sumoylation of CtBP

(A) COS-1 cells were transfected and imaged as described in Figure 3. CFP-CtBP and YFP-Ubc9 were coexpressed in the absence or presence
of Flag-Pc2. Individual CFP and YFP images and the merged (overlay) are shown. Scale bar ⫽ 10 ␮m.
(B) COS-1 cells were transfected with T7-CtBP and expression constructs encoding Fl-Ubc9, Fl-Pc2, and H6-SUMO-1 or -3 as indicated and
analyzed for T7-CtBP by Western blot. Expression of Pc2 and Ubc9 is shown below.
(C) COS-1 cells were transfected with T7-CtBP, H6-SUMO-1 with the indicated Flag-Pc2 expression constructs, encoding amino acids 1–558
(full length), 68–558, or 401–558, and analyzed for T7-CtBP by Western blot.
(D) COS-1 cells were transfected with the indicated expression constructs, lysed in guanidine-HCl and H6-CtBP was purified on cobalt agarose.
Proteins were analyzed by Western blot with a Flag antibody. Expression of CtBP and Pc2 was monitored by direct Western.

1997). It is possible that sumoylation of a proportion of
PcG proteins is required for the integrity of PcG bodies.
In this context, sumoylation of PML is required for its
localization to PML bodies (Muller et al., 1998). Thus,
the formation of specific macromolecular complexes
within the nucleus may require SUMO modification of
the proteins within them.
Alternatively, it is possible that one role of Pc2 is to
recruit and modify specific target proteins involved in
transcriptional regulation. CtBP is recruited to PcG bod-
ies by Pc2, and sumoylated CtBP preferentially associ-
ates with Pc2. Thus, CtBP may simply represent a sub-
strate for recruitment and sumoylation at PcG bodies.
However, CtBP has been suggested to bridge interac-
tions between multiple proteins involved in transcrip-
tional regulation (Turner and Crossley, 2001; Chinna-
durai, 2002). It is, therefore, tempting to speculate that
CtBP is part of the PcG sumoylation machine, which
recruits specific binding partners, to the larger PcG E3
complex. These may then be sumoylated by Pc2-associ-
ated Ubc9.
In summary, we show that Pc2 is a SUMO E3, and
that sumoylation is likely to occur specifically at PcG
bodies. This identifies a novel function of this subnuclear
compartment, and has implications for the regulation of
nuclear function and gene expression.
Experimental Procedures
Cell Culture and Transfection
COS-1 and HeLa cells were maintained in DMEM with 10% fetal
bovine serum and transfected in 6-well plates or 60 mm dishes
using LipofectAMINE (Life Technologies, Inc.).
Plasmids
CtBP, CtBP2, Ubc9, SUMO-1, SUMO-3, and Pc2 were expressed
from modified pCMV5 plasmids that contained Flag, T7, or six histi-
dine tags. Fluorescent protein fusions were expressed from pCS2
with amino terminal fusions to eCFP or eYFP. The CtBPK428R mutation
was created by PCR and verified by sequence analysis. GST fusions
were expressed from pGEX-5TK or pGEX-4T1 (Amersham Phar-
macia) and T7 and six histidine fusions from pET21a (Novagen).
Bacterial histidine tagged CtBP2 was expressed from pParallel. A
Pc2 construct was engineered by PCR which lacked the internal
Polycomb and SUMO
135
Figure 7. Pc2 Is a SUMO E3
(A) Purified T7-CtBP was incubated with recombinant Aos1/Uba2
(E1) and SUMO-1, with or without 75 ng Ubc9, with 0, 3, or 15 ng
of purified Pc2, as indicated. Proteins were analyzed by Western
blot with a T7 antibody. The position of CtBP and sumoylated CtBP
are indicated.
(B) Bacterial H6-CtBP2 was incubated with E1 and SUMO-3 with or
without 75 ng Ubc9 and Pc2 (0.5 or 1 ng) was added as indicated.
The positions of modified and unmodified CtBP2 are shown.
(C) COS-1 cells were transfected with expression vectors encoding
either Flag-CtBP, Flag-CtBPK428R, or Flag-CtBP2 together with H6-
SUMO-1, and either T7-Ubc9 or T7-Pc2, as indicated. Cell lysates
poly-histidine stretch (aa 381–399) and was truncated at codon 529,
and was expressed from within pParallel.
Coimmunoprecipitation and Western Blot
COS-1 cells were lysed by sonication in phosphate-buffered saline
with 1% NP40 (PBS-N) and protease inhibitors (protease inhibitor
mixture; Roche). Following removal of cell debris by centrifugation,
lysates were immunoprecipitated with Flag-agarose (Sigma). After
washing, proteins were separated by SDS-PAGE and transferred to
Immobilon-P (Millipore). Blots were incubated with anti-T7 antisera
(Novagen) and HRP-conjugated goat anti-mouse antibody (Pierce).
Proteins were visualized by ECL (Amersham Pharmacia). For direct
Western, COS-1 cells were lysed in SDS-loading buffer and analyzed
with the appropriate antisera (anti-Flag M2, Sigma; anti-T7 tag, No-
vagen). For analysis of endogenous CtBP, HeLa cells (one confluent
150 mm plate per immunoprecipitation) were lysed in 500 ␮l PBS-N
with 1% SDS, protease inhibitors and 10 mM N-ethylmaleimide.
Samples were diluted with 4.5 ml PBS-N. Following centrifugation,
lysates were pre-cleared with protein A and protein G Sepharose
(Pierce). Proteins were precipitated with 1 ␮g antibody (anti-CtBP
or CtBP2; BD Biosciences or anti-Flag; Sigma), washed four times
alternating between RIPA buffer (150 mM NaCl, 1% NP40, 50 mM
Tris-HCl [pH 8.0], and 0.1% SDS) and PBS-N, and Western blotted
with a SUMO-1 antibody (Zymed).
Cobalt Affinity Purification
COS-1 cells were lysed in 6 M Guanidine-HCl, 50 mM NaH2PO4 (pH
8.0), 10 mM Tris-HCl (pH 8.0), and 100 mM NaCl. H6 tagged proteins
were bound to Talon Resin (Clontech) by rocking for 2 hr. Beads
were washed 4 times for 30 min with 8 M Urea, 50 mM NaH2PO4
(pH 7.0), and 100mM NaCl.
Recombinant Proteins and In Vitro Sumoylation
T7-CtBP-H6 was expressed in BL21-CodonPlus-RP (Stratagene);
H6-CtBP2 was expressed in BL21 and proteins were purified on a
1 ml Hi-Trap CoCl2 column (Amersham Pharmacia), using a continu-
ous imidazole gradient. GST-SUMO-1 and -3 and GST-Ubc9 were
purified from BL21 on glutathione agarose. GST was removed by
cleavage with Factor X or Thrombin and the protease removed by
affinity chromatography. Alternatively, bound proteins were eluted
using 10 mM glutathione. The SUMO E1 (Aos1/Uba2 dimer) was
expressed in BL21-CodonPlus-RIL (Stratagene) and purified via an
H6 tag on Uba2. Proteins were purified as for H6-CtBP2, followed
by size exclusion chromatography on a Superdex 200 column
(Amersham Pharmacia). T7-tagged Pc2 was purified from BL21-
CodonPlus-RP on T7-agarose (Novagen). H6 tagged Pc2 (amino
acids 2-381:400-529) was expressed in BL21 and purified by cobalt
affinity chromatography and size exclusion chromatography as for
Aos1/Uba2. In vitro sumoylation reactions were performed essen-
tially as described (Schmidt and Muller, 2002): 200 ng of T7-CtBP-
H6 were incubated for 2 hr at 30⬚C in 20 ␮l (50 mM Tris [pH 7.5], 5
mM MgCl2, and 2 mM ATP) with 2 ␮g of SUMO-1, 500 ng of Ubc9,
and 100 ng of Aos1/Uba2. Reactions were analyzed by Western
blot. For analysis of the effect of Pc2 on CtBP sumoylation, reactions
were as above except that 75 ng Ubc9 was used and Pc2 (0.5–15ng)
was added.
Biochemical Fractionation
Transfected COS-1 cells were permeabilized with transport buffer
(TB: 20 mM HEPES [pH 7.4], 110 mM potassium acetate, 2 mM
magnesium acetate, and 0.5 mM EGTA) with 0.005% digitonin, pro-
tease inhibitors, and 10 mM N-ethylmaleimide, for 5 min. The cytosol
fraction was removed and cells were washed twice with TB, prior
to solubilization in TB with 1% NP40. NP40 soluble and pellet frac-
tions were separated by centrifugation.
were analyzed by Western blot with a Flag antibody. The position
of modified and unmodified forms of CtBP (upper), CtBPK428R (mid-
dle), and CtBP2 (lower) are shown. The positions of molecular weight
markers are indicated.
Cell
136
Live Cell Imaging
COS-1 cells were split onto 4-well chamber slides (Nunc) and trans-
fected with cyan or yellow fluorescent protein (eCFP or eYFP; BD
Biosciences) tagged fusion proteins using Fugene 6 (Roche). After
22–26 hr, cells were stained with 0.5 ␮g/ml Hoechst 33342 (Sigma)
for 20 min at 37⬚C. Cells were imaged using a Zeiss Axiovert 135T
inverted fluorescence microscope on a heated stage with CFP and
YFP filter sets (Omega Opticals). Hoechst staining was visualized
with a DAPI filter set. Images were visualized and captured with a
Zeiss 32⫻/0.40 objective and a Hamamatsu Orca II cooled CCD
camera controlled by Isee software (Inovision). Images were converted
to 8 bit .tif files using Isee and manipulated in Photoshop 6.0.
Acknowledgments
The authors thank M. Matunis for the Ubc9 cDNA, the E1 expressing
cells, and the kind gift of recombinant E1 and E2. We thank L.F.
Pemberton and B.M. Paschal for advice and L.F. Pemberton for
critical reading of this manuscript. This work was supported by an
NIH grant to D.W. (HD 39926).
Received: June 20, 2002
Revised: February 11, 2003
Accepted: February 13, 2003
Published: April 3, 2003
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