MOLECULAR AND CELLULAR BIOLOGY, Apr. 2005, p. 2688–2697
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 7
Ikaros SUMOylation: Switching Out of Repression
Pablo Go ´mez-del Arco, Joseph Koipally, and Katia Georgopoulos*
Cutaneous Biology Research Center, Massachusetts General Hospital,
Harvard Medical School, Charlestown, Massachusetts
Received 12 September 2004/Returned for modification 12 October 2004/Accepted 24 December 2004
Ikaros plays a key role in lymphocyte development and homeostasis by both potentiating and repressing gene
expression. Here we show that Ikaros interacts with components of the SUMO pathway and is SUMOylated in
vivo. Two SUMOylation sites are identified on Ikaros whose simultaneous modification results in a loss of
Ikaros’ repression function. Ikaros SUMOylation disrupts its participation in both histone deacetylase
(HDAC)-dependent and HDAC-independent repression but does not influence its nuclear localization into
pericentromeric heterochromatin. These studies reveal a new dynamic way by which Ikaros-mediated gene
repression is controlled by SUMOylation.
Ikaros is a member of a family of Kru ¨ppel-like zinc finger
DNA-binding factors that includes Aiolos, Helios, and Eos (9,
15, 18, 23, 33, 37). The Ikaros gene is composed of seven
translated exons (from two to eight) from which eight identi-
fied isoforms can be generated through alternative splicing
(Ik-1 to -8) (22, 32). All Ikaros isoforms contain a C-terminal
zinc finger dimerization domain encoded by exon 8 that me-
diates interactions with itself and other family members (45).
They differ in their composition of N-terminal DNA-binding
zinc fingers. On the basis of DNA-binding activity, Ikaros iso-
forms can be subdivided into two groups; in the first group are
isoforms with two to four zinc fingers that can bind DNA, and
in the second group are isoforms with fewer than two zinc
fingers that do not bind DNA. The latter group of Ikaros
isoforms maintains the ability to dimerize and exert a domi-
nant-negative effect on the DNA-binding group of isoforms
Genetic studies have established that Ikaros proteins play
critical roles during development and homeostasis of the im-
mune system (8, 47, 48). Ikaros is required in the hematopoi-
etic stem cell and its multipotent progeny to promote cell fate
decisions along the lymphoid pathway (5, 7, 36). In differenti-
ating and mature lymphocytes, Ikaros functions as a tumor
suppressor by negatively regulating proliferation and by pro-
viding homeostasis to this developmental pathway (1, 48).
Reduction of Ikaros DNA-binding activity causes the rapid
development of T-cell leukemias and lymphomas (48). Ikaros-
deficient T cells display an augmented response to activation
signals, whereas Ikaros-overexpressing cells arrest at the tran-
sition from G1to S phase (1, 13). Ikaros’ ability to control the
cell cycle is regulated by phosphorylation at its C-terminal
region, a modification that interferes with its DNA-binding
activity and facilitates cell cycle progression (13).
Ikaros can function as an unconventional potentiator of
gene expression during T-cell development, possibly by recruit-
ing the Swi/Snf chromatin-remodeling complex to appropriate
lineage-specific gene targets like CD8 (3, 17, 28). Ikaros can
also function as a transcriptional repressor (29). Ikaros-depen-
dent repression relies, in some cases, upon its association with
histone deacetylase(HDAC)-containing complexes (NuRD
and Sin3) (29) and in others upon its interaction with the
corepressor CtBP for HDAC-independent mechanisms (26).
Ikaros interacts with the NuRD complex ATPase Mi-2? and
with Sin3 through both its N-terminal and C-terminal regions
(24, 29). Ikaros-CtBP interaction relies on a PEDLS motif
(amino acids 34 to 38) located at the N-terminal region of
SUMOylation is a posttranslational modification that in-
volves conjugation of the small ubiquitin-related modifier
(SUMO) protein (30, 31, 42). SUMOylation of proteins pro-
ceeds via a multienzymatic pathway that is mechanistically
similar to ubiquitination but uses a SUMO-specific enzymatic
machinery: the E1 SUMO-activating enzyme formed by the
heterodimer Aos1/Uba2, the E2 SUMO-conjugating enzyme
Ubc9, and the E3 ligases, which promote SUMO transfer from
Ubc9 to specific protein substrates (25, 30, 31, 42). The bio-
logical implications of protein SUMOylation are broad, re-
flecting the biological activities of the substrates. Unlike ubiq-
uitination, SUMOylation does not mediate protein degradation
and in some cases induces protein stability (6, 41). For exam-
ple, SUMOylation of the inhibitor of NF-?B (I?B?) antago-
nizes its ubiquitin-dependent degradation (6). SUMOylation
has been shown to affect the integrity of nuclear bodies and
polycomb group bodies, possibly by regulating the localization
of promyelocytic leukemia/SP100 and polycomb proteins to
these nuclear domains (21, 35, 41, 42, 44). SUMOylation has
also been reported to control the activity of a number of
transcription factors through mechanisms not dependent on
nuclear localization (10, 12, 34, 46).
There are many reports of SUMOylation negatively regulat-
ing the activity of transcriptional activators (34). For example,
SUMOylation confers repression activity on Elk-1 by promot-
ing its interactions with HDAC-2 (51). ERK-mediated phos-
phorylation of Elk-1 induces its deSUMOylation and causes its
switch to a transcriptional activator (50). In a fashion similar to
that of Elk-1, SUMOylation of the coactivator p300 promotes
recruitment of HDAC-6 and induces transcriptional repression
* Corresponding author. Mailing address: Cutaneous Biology Re-
search Center, Massachusetts General Hospital, Harvard Medical
School, Charlestown, MA 02129. Phone: (617) 724-8279. Fax: (617)
726-4453. E-mail: firstname.lastname@example.org.
of some promoters (11). The components of the SUMO path-
way were also reported to negatively regulate transcription.
The SUMO moiety or the E2-conjugating enzyme Ubc9, when
tethered to the Gal4 DNA-binding domain, represses tran-
scription (39, 43). Nonetheless, SUMOylation was also re-
ported to increase the transcriptional activity of p53 and Tcf4
(14, 38, 49).
In this report, we show that Ikaros protein is SUMOylated in
vivo and that this modification negatively impacts its properties
as a transcriptional repressor. Two SUMOylation sites were
identified within the Ikaros N-terminal repression domain, at
positions K58 and K240. Mutation of these residues prevents
Ikaros SUMOylation and unexpectedly increases Ikaros activ-
ity as a repressor. In line with this finding, deSUMOylases
increase Ikaros-mediated repression, whereas E3 SUMO li-
gases reverse this effect. Ikaros SUMOylation interferes with
its ability to repress transcription not by changing its nuclear
localization to pericentromeric heterochromatin but by dis-
rupting its interactions with HDAC-dependent and -indepen-
dent corepressors, but not with other nuclear regulators. These
studies provide us with a new example of how gene repression
by Ikaros and its corepressors can be regulated by SUMO.
MATERIALS AND METHODS
Plasmids and reagents. The CDM8-Ik-1, CMV2-FlagIk-1, and pMX-GFP-
IRES-Ik-1 expression and reporter plasmids used for repression and activation
studies were previously described (13, 26). The reagents used in the yeast two-
hybrid screen were described previously (24). SUMO1GG fused to green fluo-
rescent protein (GFP-SUMO1), GFP-SUMO1GA, and Flag-tagged PIASx? and
PIASx? were generously provided by J. Palvimo. Flag-tagged PIAS1 and -3 were
obtained from K. Shuai. Myc-tagged Senp1 was provided by E. Yeh, and GFP-
Axam and a mutant form of Axam were provided by A. Kikuchi. Hemagglutinin
(HA)-SUMO1, SUMO2, and SUMO3 were provided by R. Hay. Flag-SUMO1
and Flag-SUMO2 were provided by Gregory David and Rudolf Grosschedl.
pcDNA3 HA-ubiquitin and mutant forms of ubiquitin were provided by T.
Kamitani. Ikaros antibodies 4E9, 8H2, and IKD14 have been previously de-
scribed (24, 45). Anti-SUMO2/3 was provided by H Saitoh. Anti-GMP1 (anti-
SUMO1) was purchased from Zymed, anti-GFP was from Clontech and Santa
Cruz Biotechnology, anti-HA was from Santa Cruz Biotechnology, and anti-
Flag(M2) antibody and Flag(M2) peptide were from Sigma.
Yeast two-hybrid screen. A thymocyte library generated in the pJG vector was
screened with Aiolos (amino acids 60 to 554), full-length Ikaros (amino acids 1
to 518), and their subdomains Y4, Y6, and Y9 (24) fused to the LexA DNA-
binding domain (expressed from vector pEG202). The screen was carried out as
described in reference 24, with an EGY48 yeast strain expressing the fusion
Cell lines, transfections, and infections. 293T, NIH 3T3, and U2 OS cells were
maintained in Dulbecco’s modified Eagle’s medium supplemented with 10%
fetal bovine serum (HyClone). Ikaros-null T-cell line NA1 was derived from the
thymuses of Ikaros-null mice (8) and maintained in RPMI medium supple-
mented with 10% fetal bovine serum. Transfections of 293T cells were carried
out by the HEPES-buffered saline–-CaPO4precipitation method. NIH 3T3 and
U2 OS cells were transfected with the Polyfect transfection reagent (Gibco BRL)
at the manufacturer’s recommendation. For infections, retroviruses were gener-
ated after transfecting the packaging cell line Phoenix with the pMX-GFP-IRES
plasmid or the Ikaros-expressing derivatives (13). At 48 h after transfection, viral
supernatants were recovered and used to infect NA1 cells by spinning them at
1,800 rpm for 45 min in a Beckman GS-6KR centrifuge (as described in infection
protocols at http://www.stanford.edu/group/nolan). At 24 h after infection, GFP-
positive cells were sorted and extracts were prepared and subjected to Western
Immunofluorescence studies. NIH 3T3 cells were transfected with Ikaros with
or without GFP-SUMO1 or with GFP-SUMO1 and PIASx?. At 24 h after
transfection, cells were split into chamber slides and left to grow for an additional
12 h. Cells were then fixed and permeabilized with 70% ethanol for 30 min at
room temperature; this was followed by extensive washing and by blocking of
unspecific staining. Cells were then stained with Ikaros antibodies as described
Extract preparation, immunoprecipitation, and Western analysis. For thymo-
cyte extracts, thymuses were surgically removed from wild-type mice; cell sus-
pensions were prepared and split in two. One half was lysed with buffer C (20
mM HEPES [pH 7.9], 25% glycerol, 0.42 M NaCl, 0.2 mM EDTA, 1.5 mM
MgCl2, protease inhibitors) containing 1% NP-40, and the other half was lysed
with buffer C containing 1% sodium dodecyl sulfate (SDS) and 1 mM N-ethyl-
maleimide (NEM). The latter buffer was also used to generate NA1 cell extracts.
Other whole-cell extracts and nuclear extracts from transfected or untransfected
cells were prepared as previously described (24, 29) in the presence or absence
of NEM. For immunoprecipitations, the extracts were precleared with protein
G-agarose beads (Roche). The precleared extracts were incubated with the
antibody of interest or the relevant isotype control in the presence of 30 ?l of
protein G-agarose beads and rotated for 4 h to overnight. Beads were then
collected, washed at least four times with lysis buffer, and resuspended in SDS
sample buffer. For the anti-Flag immunoprecipitations, anti-Flag(M2) agarose
beads and M2 peptide were used to immunoprecipitate and elute complexes
from beads, respectively. Eluents were subjected to SDS-polyacrylamide gel
electrophoresis (PAGE) and transferred to polyvinylidene difluoride mem-
branes, probed with the relevant antibody, and examined by autoradiography by
Enhance Chemical Luminescence.
Transcriptional studies. For the repression studies, Gal4-Ikaros fusions (0.5 to
1 ?g), the 5XGal4 thymidine kinase (tk) promoter driving chloramphenicol
acetyltransferase (CAT) (10 ?g), and the growth hormone (GH) control
(pXGH5; 0.5 ?g) were transfected into NIH 3T3 or U2 OS cells as indicated.
Cells were harvested 36 h after transfection and split into two samples. One half
was processed for the CAT assay as previously described (45), and the other half
was used to generate proteins and test their level by Western blotting. GH assays
were performed as recommended by the manufacturer (Nichols Institute). CAT
values were normalized to the GH levels.
For the activation assays, 1 ?g of the reporter 4XIKBS2 (Ikaros binding site)
tkCAT, 1 ?g of Ikaros expression vector, and 0.5 ?g of the GH plasmid were
used. Cells were harvested after 36 h and processed as described above for the
repression experiments. All of the transcription assays were performed at least
three times in duplicate or triplicate.
Ikaros is SUMOylated in lymphocytes. To identify Ikaros
family interactors, a thymocyte library was used in a yeast
two-hybrid screen with the Aiolos and Ikaros proteins as baits
as described previously (24). Three different types of cDNA
were isolated multiple times, which encoded Aiolos and Ikaros
interactors belonging to the SUMO pathway (Fig. 1A). One
was SUMO1, the second was the component of the E1-SUMO
activating enzyme Uba2, and the third was the E2-conjugating
enzyme Ubc9. These proteins exhibited stronger interactions
with Ikaros in the yeast two-hybrid system (Fig. 1A). Delinea-
tion of the Ikaros-Ubc9 interaction interface revealed their
association through the N-terminal (Y6, exons 2 to 7 [amino
acids 1 to 282], and Y9, exons 2 to 4 [amino acids 1 to 141]) but
not the C-terminal region (Y4, amino acids 364 to 518 of exon
8) of the Ikaros protein (Fig. 1A and B).
Protein SUMOylation occurs on the lysine of a ?KxE pro-
tein motif, where ? represents the hydrophobic residue isoleu-
cine (I), leucine (L), or valine (V) and x is any amino acid.
Analysis of the Ikaros protein sequence revealed four putative
SUMOylation sites, at positions K58 and K240, located in the
N-terminal half of the protein, and K425 and K459, located in
the C-terminal region (Fig. 1B). Of these, the first three rep-
resent perfect consensus motifs for SUMOylation. The Ikaros
SUMOylation motifs containing K58 and K240 are highly con-
served across species from skates to humans (16). These two
motifs were also found to be conserved in Aiolos at positions
K63 and K256. The other two Ikaros family members, Helios
VOL. 25, 2005 Ikaros SUMOylation DISRUPTS BINDING TO COREPRESSORS2689
and Eos, had only one SUMOylation motif at their N termini,
corresponding to Ikaros K58.
Given the potential of Ikaros for SUMOylation, we tested
whether the protein was modified in vivo in primary T cells.
Nuclear extracts, prepared from murine thymocytes in the
presence or absence of 1% SDS and the SUMO-isopeptidase
inhibitor NEM, were examined for Ikaros protein expression.
Three slower-migrating Ikaros protein species were detected
only in the presence of NEM, indicating that a fraction of the
protein is SUMOylated (Fig. 1C, ?NEM/SDS, bands M1, M2,
and B1; see next section). Other Ikaros-interacting proteins,
i.e., mSin3A and HDAC2, were also tested and found not to be
SUMOylated in thymocytes (data not shown).
Mapping of the Ikaros SUMOylation sites. We next inves-
tigated which of the Ikaros SUMOylation motifs were used in
vivo. For this study, the putative lysine-SUMO acceptors were
mutated to arginine singly or in combination. Vectors express-
ing an untagged version of Ikaros (Ik-1) or the SUMOylation
motif mutant forms and GFP-SUMO1 were cotransfected in
the epithelial 293T cell line. Ikaros proteins were immunopre-
cipitated with anti-Ikaros antibody and immunoblotted with
Ikaros and GFP antibodies to better reveal the modified forms
(Fig. 2A, ?-Ikaros and ?-GFP). In addition to the main Ikaros
protein band, three slower-migrating species were observed
with Ikaros antibodies (Fig. 2A, lanes 3 and 5), similar to those
observed in thymocytes (Fig. 1C). Antibodies to GFP con-
firmed that these protein species were the result of Ikaros-
GFP-SUMO1 conjugation (Fig. 2A, right half).
The first of the modified bands contains Ikaros protein that
is monoSUMOylated at K58 as this disappears upon the K58
mutation (Fig. 2A, band M1, lane 6; also Fig. 1C). The second
contains Ikaros that is monoSUMOylated at K240 as this is
also affected by the corresponding mutation (Fig. 2A, band
M2, lane 7; also Fig. 1C). Ikaros protein that is biSUMOylated
at both the K58 and K240 sites runs at the third position, and
this is equally affected by either of the single mutations (Fig.
2A, band B1, lane 12; also Fig. 1C). In sharp contrast to the
Ikaros mutations at K58 and K240, mutations at K425 and
K459 had no effect on the slower-migrating forms (Fig. 2A,
lanes 8 and 9). Similar findings on SUMOylation of Ikaros and
its mutant forms were obtained with the human cell line U2 OS
(data not shown). Other members of the SUMO family,
SUMO2 and -3, were also tested for the ability to be conju-
gated to Ikaros. A series of expression and immunoprecipita-
tion studies demonstrated that all three of the SUMO proteins
could be conjugated to Ikaros at positions K58 and K240 (data
The Ikaros sites of SUMOylation were confirmed in lym-
phoid cells, where Ikaros is normally expressed. The T-cell line
NA1, derived from Ikaros-null mice, was infected with pMX-
GFP retroviruses harboring either Ik-1, the Ik-1 K58 and 240R
SUMOylation mutant form, or the parental vector as a control
(13). pMX-GFP-Ik-1 and pMX-GFP-Ik-1 K58 and 240R, but
not the parental control vector, reconstituted Ikaros protein
expression, which was detected as a major band at ?65 kDa
(Fig. 2B). Reconstitution with Ik-1, but not with the SUMOy-
lation mutant form of Ikaros, also provided three Ikaros-re-
lated slower-migrating protein species (Fig. 2B). These were
similar in size to those observed in primary thymocytes that
express Ikaros (Fig. 1C). This study verifies that the higher-
molecular-weight Ikaros species observed in thymocytes and in
an Ikaros-reconstituted T-cell line represent SUMOylated
forms of the Ikaros protein. Furthermore, it shows that Ikaros
lysines 58 and 240 are major sites for SUMOylation in vivo in
both lymphoid and nonlymphoid cells.
Ikaros SUMOylation is actively regulated by SUMO iso-
peptidases and E3 ligases. SUMOylation is a dynamic process
that is controlled by opposing enzymatic activities. In line
with previous observations, Ikaros SUMOylation was exam-
ined in the presence of differing amounts of SUMOylase and
FIG. 1. Ikaros interacts with components of the SUMO pathway
and is SUMOylated in primary lymphocytes. (A) SUMO1 and the
SUMO enzymes E1 (Uba2 component) and E2 (Ubc9) were isolated
in a yeast two-hybrid screen with Aiolos and Ikaros. These were found
to interact specifically with both Ikaros and Aiolos but not with control
baits like Myc and Mxi-1. The Ikaros-Ubc9 interacting domain was
located at the N-terminal half of Ikaros (Y6), which harbors two
putative Ikaros SUMOylation sites. nd, not determined. (B) Schematic
representation of full-length Ikaros isoform 1 (Ik-1) showing the loca-
tion of potential SUMOylation sites (I/V/L)KXE (asterisks). The mod-
ifiable lysine in each putative SUMOylation site is indicated by num-
ber. Horizontal rectangles represent translated exons (Ex), while
vertical rectangles identify Ikaros zinc fingers (F1 to -6). The Ikaros
N-terminal (Y6 and Y9) and C-terminal (Y4) subdomains used in the
yeast two-hybrid assay are also shown. (C) Whole-cell extracts from
murine thymocytes were generated in the presence (?) or absence (?)
of 1% SDS and the SUMOylation inhibitor NEM (NEM/SDS). Pro-
teins were separated by SDS–7% PAGE and subjected to Western blot
analysis for Ikaros proteins. Ikaros isoforms 1, 2, 3, and 7 are shown.
The three presumed SUMOylated Ikaros protein species are shown by
arrows (M1, M2, and B1; see nomenclature explanation in Results).
Positions of approximate molecular weight markers are shown on the
2690 GO ´MEZ-DEL ARCO ET AL.MOL. CELL. BIOL.
GFP-SUMO1-Ikaros proteins were detected upon coexpres-
sion of Ik-1 and GFP-SUMO1 in 293T cells (Fig. 3A, Contr.).
Ikaros SUMOylation was progressively reduced when the level
of the isopeptidase Senp1 was increased (Fig. 3A, MT-Senp1).
A similar effect on Ikaros deSUMOylation was also observed
when the expression of another isopeptidase, Axam, was in-
creased (data not shown).
In contrast to the effect of isopeptidases, an increase in
SUMO E3 ligase (PIASx?) increased Ikaros SUMOylation
(Fig. 3B; see also Fig. 5B and 7B). Immunoprecipitation stud-
ies revealed a strong interaction between Ikaros and the E3
ligase PIASx? and a weaker interaction with PIAS3 (Fig. 3C).
The PIASx? and PIAS1 family members were not detected
within the Ikaros immunoprecipitates (Fig. 3C). Thus, Ikaros
SUMOylation is regulated by the PIAS family of ligases, prob-
ably through Ikaros’ specific interactions with two members of
Ikaros SUMOylation mutant forms are more potent tran-
scriptional repressors. We next examined whether SUMOyla-
tion of Ikaros can influence its activity as a repressor or acti-
vator of transcription. Ikaros’ function as a transcriptional
repressor can be revealed upon its tethering to the Gal4 DNA-
binding domain (29). Gal4–Ik-1 or the Gal4–Ik-1–SUMO mu-
tant form was coexpressed with the 5XGal4-tk-CAT reporter
in U2 OS or NIH 3T3 cells (Fig. 4A and data not shown). As
previously shown (29), Gal4–Ik-1 repressed transcription (Fig.
FIG. 2. Ikaros SUMOylation sites established in nonlymphoid and lymphoid cells. (A) GFP-tagged SUMO1 was coexpressed with wild-type
Ik-1 (WT) or the Ikaros mutant forms Ik-1K58R, Ik-1K240R, Ik-1K425R, Ik-1K459R, Ik-1K240,425,459R, and Ik-1K58,240,459R into 293T cells.
A mock transfection is also shown in lane 1 (vertical hyphen). Whole-cell extracts were immunoprecipitated with Ikaros antibody (I) or control
immunoglobulin G (G). Immunoprecipitates were separated by SDS–8% PAGE and analyzed by Western blotting with Ikaros and GFP antibodies
consecutively. The unmodified (uIk-1) and SUMOylated forms of Ikaros (M1 [monoSUMOylated at K58], M2 [monoSUMOylated at K240], and
B1 [biSUMOylated at K58 and K240]; see Results for an explanation) are indicated (GFP-SUMO1 Ik-1). In the GFP blot, the asterisk indicates
an unspecific cross-reactive band. (B) An Ikaros-null T-cell line, NA1, was infected with pMX-GFP-IRES Ik-1 (Ik-1, lane 2), the SUMO
double-mutant form (K58,240R, lane 3), or the pMX-GFP-IRES control (pMX-GFP, lane 1). At 24 h after infection, cells expressing GFP were
sorted and whole-cell extracts were prepared in the presence of 1% SDS and 1 mM NEM. Proteins were separated and analyzed by Western
blotting with Ikaros antibodies. The unmodified (uIk-1) and SUMOylated (M1, M2, and B1) Ikaros forms are indicated (SUMOy Ik-1). A loading
control immunoblot (IB) is also shown (Sin3A). The positions of approximate molecular weight markers are shown at the left of each panel.
VOL. 25, 2005 Ikaros SUMOylation DISRUPTS BINDING TO COREPRESSORS2691
4A, WT). Significantly, the Ikaros K58R (data not shown),
K240R, and K58 and 240R SUMOylation mutant forms were
stronger repressors of transcription than the wild-type Ikaros
protein, a fraction of which is likely SUMOylated (Fig. 4A).
Interestingly, a similar increase in repression was detected with
either of the single-mutant forms or the double-SUMOylation
mutant form of Ikaros, suggesting that co-occupancy of these
SUMOylation sites is required to relieve repression. As shown
by Western blot analysis, the effect of the mutations in Ikaros
FIG. 3. SUMOylation of Ikaros is controlled by the relative activ-
ities of SUMOylases and deSUMOylases. (A) Ik-1 and GFP-SUMO1
were coexpressed in 293T cells with increasing amounts of a Myc-
tagged version of the deSUMOylase Senp1 (MT-Senp1) or empty
vector as a control (Contr.). Ikaros proteins were immunoprecipitated
and analyzed by Western blotting. SUMOylated (SUMOy. Ik-1) and
unmodified (uIk-1) Ikaros proteins are shown. The level of expression
of Senp1 is shown in the lower part. (B) FlagIk-1 was coexpressed with
GFP-SUMO1 with or without PIASx? in 293T cells. Whole-cell ex-
tracts were subjected to Western blot analysis with Ikaros. SUMOy-
lated (SUMOy. Ik-1) and unmodified (uIk-1) Ikaros proteins are de-
marcated. A loading control for the immunoblots (IB) is shown
(Sin3B). Positions of approximate molecular weight markers are also
indicated. (C) Ikaros interactions with SUMO E3 ligases. Ik-1 was
coexpressed with the Flag-tagged SUMO E3 ligases PIASx?, PIASx?,
PIAS1, and PIAS3 in 293T cells. Whole-cell extracts were subjected to
immunoprecipitation with Ikaros antibodies (?-Ik) or control mouse
immunoglobulin G (IgG) and probed with Flag (IB:Flag) and Ikaros
(IB: Ik) antibodies. The expression levels of the different E3 ligases are
shown at the left (Input).
FIG. 4. SUMOylation of Ikaros specifically interferes with its abil-
ity to repress transcription. (A) Gal4 (?) or Gal4–Ik-1 fusion proteins
(wild type [WT], K240R, and K58,240R) were cotransfected with the
5XGal4-tk-CAT reporter and GH control into U2 OS cells (described
in Materials and Methods). Cell extracts were analyzed for CAT ac-
tivity and protein expression. Reporter activity was normalized to GH
levels, and fold repression relative to the Gal4 vector was calculated
(29). Schematic diagrams of the Gal4-Ikaros fusion protein and the
reporter used are depicted. Results of two representative experiments
(Exp.), out of seven with similar findings, are displayed. Western blots
(immunoblots [IB]) assessing protein expression are shown. YY1 pro-
tein expression was used as a loading control. (B) Ikaros’ activity as a
potentiator of gene expression is not affected by SUMOylation. Wild-
type Ik-1 (WT), Ik-1 K58R (K58), Ik-1 K240R (K240), Ik-1 K58,240R
(K58,240), or an expression vector control was cotransfected together
with the 4XIk-tk-CAT reporter and the GH control plasmid into U2
OS cells. Reporter activity was normalized to GH levels, and fold
activation relative to that obtained with the empty vector was calcu-
lated (28). One representative experiment, out of three, is shown.
Protein expression is shown by immunoblots (IB), which used Sin3A as
a loading control. In both panels, schematic diagrams of the Ikaros
protein and the reporters are provided. In both panels, each experi-
mental point was performed in duplicate with less than 5% variability.
2692 GO ´MEZ-DEL ARCO ET AL.MOL. CELL. BIOL.
on its repression activity was not due to a change in protein
expression or stability (Fig. 4A, bottom, and data not shown).
The effect of SUMOylation on Ikaros’ ability to potentiate
gene expression was also examined in a transient transcription
assay (28). Ikaros and its SUMO mutant forms were cotrans-
fected with a 4XIk-tk-CAT reporter (32) in U2 OS cells (Fig.
4B). Expression of either the wild-type Ikaros protein or its
SUMOylation mutant forms provided a similar strong poten-
tiation of reporter activity (Fig. 4B).
Taken together, these data support the model in which
SUMOylation of Ikaros at both the K58 and K240 sites spe-
cifically negates its ability to repress gene expression.
The SUMO pathway directly regulates Ikaros’ activity as a
repressor. Importantly, Ikaros mutations that prevent its
SUMOylation enhance its potential as a transcriptional repres-
sor (Fig. 4A). The role of the SUMO pathway in regulating
Ikaros’ repressor function was examined by varying the level of
enzymes that have disparate effects on SUMOylation.
Gal4–Ik-1 and the 5XGal4-tk-CAT reporter were coex-
pressed with or without GFP-SUMO1 and the E3 ligase
PIASx? in U2 OS cells (Fig. 5A). Cell lysates were prepared
for CAT assays and for Western blot analysis. As previously
shown (29), expression of Gal-4–Ikaros represses the Gal-4
reporter (Fig. 5A, lane 3). However, when Gal-4–Ikaros was
coexpressed with PIASx? and GFP-SUMO1, high levels of
reporter activity were observed and very little repression was
obtained (Fig. 5A, lane 4). Importantly, coexpression of
PIASx? resulted in a significant increase in both the level and
complexity of Ikaros SUMOylation, whereas the overall Ikaros
protein expressed in these two experimental points was not
significantly different (Fig. 5A, Western blot assays, compare
lanes 3 and 4; also data not shown). As expression of the E3
SUMOylase did not alter Gal4-dependent transcription, the
observed effect is Ikaros dependent (Fig. 5A, lane 2).
Given that enzymes that promote SUMOylation attenuate
Ikaros’ repression activity, we set out to test whether the con-
verse was also true; that is, whether deSUMOylases enhance
Ikaros-mediated repression. To demonstrate an increase in an
already strong repression potential, we adapted the experimen-
tal design to achieve lower levels of Ikaros protein. As shown
in Fig. 5B, the amount of protein generated by 0.5 ?g of
Gal4–Ik-1 gave moderate repression (Fig. 5B, lane 3). Coex-
pression of the deSUMOylase Senp1 increased Ikaros-medi-
ated repression and the level of unmodified Ikaros protein,
possibly by antagonizing its SUMOylation (Fig. 5B, compare
lanes 3 and 4). It is important to note that the level of repres-
sion provided by wild-type Ikaros and deSUMOylases was sim-
ilar to that generated by the Ikaros SUMO mutant forms when
expressed at similar levels (Fig. 5B, compare lanes 4 and 8).
As shown in Fig. 5A, coexpression of PIASx? and GFP-
SUMO1 increased Ikaros SUMOylation, decreased the level
of unmodified protein, and reduced its repression activity (Fig.
5B, lane 7). In contrast, coexpression of PIASx? with the
Ikaros K58 and K240R SUMOylation mutant form had no
such effect on its strong repression activity (Fig. 5B, lane 8).
Taken together, these studies demonstrate that SUMOyla-
tion can directly and inversely regulate transcriptional repres-
sion by Ikaros. The more SUMOylated Ikaros protein there is,
the less repression it can provide.
Ikaros SUMOylation does not interfere with its nuclear
localization to pericentromeric heterochromatin. To deter-
mine the mechanism by which SUMOylation regulates Ikaros-
mediated repression, we examined the effects on the protein’s
FIG. 5. Functional interactions of Ikaros with the SUMO pathway. (A) One microgram of Gal4 (lanes 1 and 2) or Gal4–Ik-1 (lanes 3 and 4)
was cotransfected, together with the 5XGal4-tk-CAT reporter, Flag-PIASx? (3 ?g), and GFP-SUMO1 (2 ?g) where indicated, into U2 OS cells.
Total extracts were prepared in the absence of NEM for CAT assay or in the presence of the deSUMOylase inhibitor for Western blotting. Western
blot assays of the indicated proteins were performed to assess their expression and the levels of Ikaros SUMOylation. (B) Gal4, Gal4–Ik-1, and
Gal4–Ik-1 K58,240R expression plasmids (0.5 ?g of each) were cotransfected together with Gal4-tk-CAT reporter and Flag-PIASx? plus
GFP-SUMO1 or MT-Senp1 (3 ?g) into U2 OS cells. Samples were processed as described for panel A. Two independently performed experiments
(Exp), out of four with similar findings, are displayed. Unmodified (uIk-1) and SUMOylated (SUMOy. Ik-1) Ikaros proteins are shown. The bar
and asterisk indicate Ikaros modified by endogenous SUMO protein. In both panels, each experimental point was performed in duplicate with less
than 5% variability. WT, wild type.
VOL. 25, 2005 Ikaros SUMOylation DISRUPTS BINDING TO COREPRESSORS 2693
nuclear localization. In proliferating cells, Ikaros undergoes a
dynamic redistribution into pericentromeric heterochromatin,
an event that has been correlated in the past with its function
as a repressor and silencer of gene expression (1, 24).
Untagged Ikaros and its SUMOylation mutant form (K58
and K240R) were cotransfected together with GFP-SUMO1
and Senp1 or PIASx? into NIH 3T3 fibroblasts where, as in
cycling lymphocytes, Ikaros localizes into pericentromeric het-
erochromatin (4, 28). Immunofluorescence analysis with
Ikaros antibodies indicated that most of both wild-type Ikaros
and its SUMOylation mutant form localized into pericentro-
meric heterochromatin (Fig. 6A). The localization of Ikaros
(and its single- or double-SUMOylation mutant form) re-
mained unaffected by the presence or absence of GFP-
FIG. 6. SUMOylation of Ikaros does not interfere with its localization into pericentromeric heterochromatin. (A) Wild-type Ik-1 (WT) and the
SUMO double-mutant form (Ik-1 K58,240R) were coexpressed together with GFP-SUMO1 and the isopeptidase Senp1 or GFP-SUMO1 and the
E3 ligase PIASx? into NIH 3T3 cells as indicated. Cells were fixed and analyzed by immunofluorescence microscopy for Ikaros proteins.
Condensed DNA in centromeric heterochromatin was revealed with 4?,6?-diamidino-2-phenylindole (DAPI) (DNA). The percentage of cells with
Ikaros localizing in heterochromatin and the number of cells counted for each experimental condition (n) are shown. (B) Wild-type Ik-1 (WT) and
the indicated SUMO mutant forms were coexpressed together with GFP-SUMO1 in NIH 3T3 cells. Cells were fixed and analyzed by fluorescence
microscopy for GFP and Ikaros proteins. Condensed DNA in centromeric heterochromatin was revealed with DAPI (DNA).
2694 GO ´MEZ-DEL ARCO ET AL.MOL. CELL. BIOL.
SUMO1 (Fig. 6 and data not shown) or by the presence of the
isopeptidase Senp1 or the E3 ligase PIASx? (Fig. 6A and data
The distribution of GFP-SUMO1 was also revealed by flu-
orescence. In the absence of Ikaros or in the presence of the
K58 and 240R double-SUMOylation mutant form, GFP-
SUMO1 was widely distributed throughout the nucleus with
only a small selective presence in pericentromeric heterochro-
matin. However, in the presence of wild-type Ikaros or a sin-
gle-SUMOylation mutant (K58 or K240) form of Ikaros, most
of the GFP-SUMO1 protein was detected in pericentromeric
heterochromatin together with Ikaros (Fig. 6B). This is likely
the result of interaction and covalent association between
Ikaros and GFP-SUMO1 that dictates the overall nuclear dis-
tribution of GFP-SUMO1.
Taken together, these results indicate that Ikaros SUMOy-
lation does not influence its activity as a repressor by altering
its nuclear localization. SUMOylated Ikaros, which has lost its
function as a repressor, can still localize into pericentromeric
SUMOylation of Ikaros inhibits its interactions with tran-
scriptional corepressors. Ikaros has been proposed to regulate
transcription by associating with chromatin remodeling and
other transcription regulators (24, 26, 27, 29). SUMOylation of
Ikaros may specifically interfere with its ability to associate
with some of these factors, which are engaged in repression.
To examine this possibility, Flag-tagged Ik-1 was expressed
in 293T cells without and with GFP-SUMO1, and its associa-
tion with its previously reported interactors Mi-2? (NuRD
complex), Sin3A, Sin3B, CtBP, and Brg-1 (Swi/Snf complex)
was tested by Ikaros immunoprecipitation, followed by West-
ern blotting (Fig. 7A). In the absence of GFP-SUMO1, a small
fraction of Ikaros was SUMOylated and strong interactions
with endogenously expressed Sin3A, Sin3B, CtBP, Mi-2?, and
Brg-1 were detected (Fig. 7A, ? GFP-SUMO1). In sharp con-
trast, when GFP-SUMO1 was expressed, a major fraction of
Ikaros was SUMOylated and its interaction with Sin3A, Sin3B,
CtBP, and Mi-2? was greatly reduced (Fig. 7A, ? GFP-
SUMO1). The effect was most pronounced with CtBP, which
was almost absent in the Ikaros immunoprecipitate. The inter-
actions of Ikaros with its corepressors were also evaluated
upon coexpression of GFP-SUMO1 and the SUMO E3 ligase
PIASx?. As shown in Fig. 7B, a dramatic increase in Ikaros
SUMOylation disrupted Sin3A interactions.
In sharp contrast to Ikaros interactions with Sin3A, Sin3B,
Mi-2?, and CtBP, which were greatly reduced upon its
SUMOylation, Ikaros association with the Swi/Snf ATPase
Brg-1 was mildly affected (Fig. 7). A previous established in-
FIG. 7. SUMOylation interferes with Ikaros’ ability to associate with corepressors of transcription. (A) CMV2 Flag-tagged Ikaros (Flag–Ik-1)
was cotransfected together with (?) or without (?) GFP-SUMO1 into 293T cells. Ikaros proteins were immunoprecipitated (IP) with a mouse
immunoglobulin G-protein G-agarose (IgG) or anti-Flag(M2)-agarose (Flag) beads. Proteins were eluted from the precipitated immunocomplexes
with an excess of Flag(M2) peptide. Eluted proteins were separated by SDS–7% PAGE and analyzed by Western blotting for Brg-1, CtBP, Mi-2?,
Sin3A, Sin3B, and Ikaros proteins. Unmodified (uIk-1) and GFP-SUMO1-modified (GFP-SUMO1 Ik-1) Ikaros proteins are indicated. The
asterisk indicates an Ikaros species likely monomodified by endogenous SUMO. (B) Flag–Ik-1 was coexpressed with GFP-SUMO1 (?) and
PIASx? (?) into 293T cells. Ikaros proteins were immunoprecipitated (IP) and analyzed as described for panel A. Western blot assays for GFP,
Ikaros, Sin3A, and SV40 TAg are shown. Unmodified and SUMOylated Ikaros species are demarcated (uIk-1 and GFP-SUMO1 Ik-1, respec-
tively). In both panels, the positions of approximate molecular weight markers are shown at the left.
VOL. 25, 2005 Ikaros SUMOylation DISRUPTS BINDING TO COREPRESSORS2695
teraction between Ikaros and the simian virus 40 (SV40) T
antigen (TAg) in 293T cells was also not influenced by
SUMOylation (Fig. 7B).
These data strongly support a scenario in which SUMOyla-
tion of Ikaros specifically inhibits its interactions with HDAC-
dependent (Sin3 and Mi-2?), as well as HDAC-independent
(CtBP), corepressors of transcription. It does not, however,
significantly affect its interactions with components of the Swi/
Snf complex or with the viral protein SV40 TAg.
Here we provide the first evidence that Ikaros interacts with
components of the SUMO pathway and is SUMOylated in
vivo. We identify two SUMOylation sites on Ikaros and show
that their simultaneous modification supports the loss of
Ikaros’ repression function. We demonstrate that SUMOyla-
tion of Ikaros disrupts its interactions with HDAC-dependent
and HDAC-independent corepressors of transcription but
does not affect its localization into pericentromeric hetero-
Different lines of evidence have provided support for the
idea that Ikaros directly interacts with components of the
SUMO pathway. Yeast two-hybrid studies demonstrated its
potential interaction with SUMO1, the component of the E1-
activating enzyme heterodimer Uba2, and the E2-conjugating
enzyme Ubc9. Immunofluorescence studies with mammalian
cells have shown an Ikaros-dependent localization of SUMO1
into pericentromeric heterochromatin that is disrupted by mu-
tations in the Ikaros SUMOylation motifs (data not shown).
Finally, Ikaros immunoprecipitations from these cells revealed
a preferential association with two of the four members of the
SUMO E3 ligase family, PIASx? and PIAS3.
Ikaros is predominantly SUMOylated in vivo at lysines 58
and 240, which are located at the N-terminal half of the pro-
tein. These two Ikaros SUMOylation motifs are highly con-
served across species (16), suggesting that this type of regula-
tion of the Ikaros protein is conserved through evolution. In
addition, sites corresponding to Ikaros K58 and K240 are also
found in Aiolos, suggesting a similar type of control in the
second Ikaros family member.
Mutations in either of the SUMOylation sites of Ikaros
drastically elevated its activity as a repressor of transcription,
whereas mutations in both gave no added effect. Consistent
with these findings, Ikaros’ repressive potential was reduced
when the protein was SUMOylated at both K58 and K240 but
remained unaffected when it was monoSUMOylated at either
of these sites (Fig. 4). Taken together, these studies indicate
that Ikaros’ activity as a repressor is dependent on the com-
plexity of SUMOylation, as both of its SUMOylation sites must
be modified to relieve repression.
The level of Ikaros SUMOylation and its potential for re-
pression can be regulated by the opposing activities of
SUMOylases and deSUMOylases. Whereas SUMOylases de-
crease repression mediated by Ikaros, deSUMOylases like
Senp1 increase its repression activity to a level that is similar to
that obtained with the SUMOylation mutant forms of Ikaros.
Relative expression of these enzymes, as well as accessibility of
the Ikaros protein to these factors, may dictate its overall
SUMOylation. For example, the Ikaros-interacting PIAS E3
ligases are distributed in promyelocytic leukemia bodies, chro-
matin, and nuclear matrix (19, 40), where Ikaros is likely
SUMOylated. Of the deSUMOylating enzymes, Senp1 and
Axam are present in the nucleoplasm whereas others are in the
cytoplasm and nucleolus (34).
The mechanism by which SUMOylation controls Ikaros re-
pression function is central to our understanding of its dual
function as an activator and repressor of gene expression.
Ikaros’ ability to localize into pericentromeric heterochroma-
tin has been correlated with its ability to repress and silence
gene expression (2, 7). However, although SUMOylation alle-
viates Ikaros’ repression it does not do so by affecting its
pericentromeric heterochromatin localization. This argues that
Ikaros’ localization into this nuclear compartment is not cen-
tral to the protein’s ability to repress transcription. However,
SUMOylation disrupts the well-established associations be-
tween Ikaros and corepressors of transcription like CtBP, Sin3,
and Mi-2? of the NURD complex (24, 26, 29). The inability of
Ikaros to be integrated into HDAC-dependent and HDAC-
independent pathways of repression is likely to inadvertently
affect its function as a repressor. SUMOylation, however, has
no significant effect on Ikaros’ interactions with components of
the Swi/Snf complex, which can provide chromatin fluidity and,
like Ikaros, can positively regulate developmentally important
loci like the gene for CD8 (3, 17).
How does SUMOylation of Ikaros interfere with its interac-
tions with HDAC-dependent and -independent corepressors?
SUMOylation of Ikaros may simply block access to their bind-
ing sites. For example, the Ikaros K58 SUMOylation site lies
next to the CtBP interaction motif (amino acids 34 to 38) and
may be responsible for the more severe disruption of Ikaros-
CtBP interactions relative to the other corepressors. The pro-
teins Mi-2? and Sin3 share binding domains located at the N-
and C-terminal regions of the Ikaros proteins, and accessibility
to these common interaction domains is likely to be similarly
affected by SUMOylation. SUMOylation may affect Ikaros-
corepressor associations by inducing conformational changes
that alter their interaction interface.
Studies with primary thymocytes and cycling T cells show
that a small but significant fraction of the total Ikaros protein
is SUMOylated. Nonetheless, mutation of the SUMOylation
sites of Ikaros has a strong effect on its activity as a repressor.
Given the dynamic and transient nature of SUMOylation, it
may be required to initiate the disassembly of Ikaros-repressor
complexes but not to maintain them in a separate state. De-
SUMOylated Ikaros may then be preferentially retained in a
different type of protein complex, i.e., Swi/Snf, that is not
greatly affected by SUMOylation. SUMO modifications regu-
lating the disassembly of septin ring structures during mitosis
have been reported in yeast (20).
In conclusion, SUMO conjugation may play a pivotal role in
dictating Ikaros’ ability to interact with distinct transcriptional
cofactors. This type of posttranslational modification may un-
derlie the protein’s ability to be integrated into a pool of
potentiators versus repressors of gene expression. In the fu-
ture, it will be important to determine how signaling pathways,
for example, those that control Ikaros phosphorylation, impact
SUMOylation. It is likely that coordinating these distinct post-
translational modifications is critical for effecting key events in
gene expression during lymphocyte development.
2696 GO ´MEZ-DEL ARCO ET AL.MOL. CELL. BIOL.
We thank G. David, R. Grosschedl, R. Hay, T. Kamitani, A. Kiku-
chi, J. Palvimo, H. Saitoh, K. Shuai, and E. Yeh for the generous gift
of reagents used in this work. We are very grateful to J. Kim and N.
Avitahl for the two-hybrid screen and the generation of the NA1 cell
line, respectively. We also thank E. Heller, S. Ng, J. Seavitt, C. Wil-
liams, and T. Yoshida for careful reading of and valuable comments on
P.G.-D. was supported by Fellowships from the Leukemia and Lym-
phoma Society of America and the Ministry of Science and Technology
of Spain and is a recipient of a Lady Tata Memorial Trust Award. This
research was supported by NIH grant RO1-AI380342-09 to K.G.
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