Transactivation properties of c-Myb are critically dependent
on two SUMO-1 acceptor sites that are conjugated
in a PIASy enhanced manner
Øyvind Dahle
1
, Tor Ø. Andersen
1
, Oddmund Nordga
˚
rd
1
, Vilborg Matre
1
, Giannino Del Sal
2,3
and Odd S. Gabrielsen
1
1
Department of Biochemistry, University of Oslo, Norway;
2
Laboratorio Nazionale CIB, Area Science Park, Trieste, Italy;
3
Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Universita
`
degli Studi di Trieste, Italy
The transcription factor v-Myb is a potent inducer of
myeloid leukemias, and its cellular homologue c-Myb
plays a crucial role in the regulation of hematopoiesis.
Recently, Bies and coworkers (Bies, J., Markus, J. &
Wolff, L. (2002) J. Biol. Chem, 277, 8999–9009) presented
evidence that murine c-Myb can be sumoylated under

T-cell development, while mice with a c-myb
null
mutation
display severe hematopoietic defects leading to in utero
death at E15 [2,3]. The c-Myb protein consists of an
N-terminal DNA-binding domain (DBD), a central trans-
activation domain (TAD) and a C-terminal negative
regulatory domain (NRD). The DBD of c-Myb is com-
prised of the three imperfect repeats: R
1
,R
2
and R
3
,each
related to the helix-turn-helix motif [4–7].
Oncogenic alterations, as found in AMV v-Myb, include
both N- and C-terminal deletions as well as point mutations
[8]. AMV v-myb is a potent and cell-type specific oncogene
that transforms target cells in the macrophage lineage and
induces monocytic leukemia [8,9]. Several studies have
attempted to define oncogenic determinants of v-myb.
N- and C-terminal deletions remove several sites of protein
modification, including an N-terminal CK2 phosphoryla-
tion site (S11 and S12) [10], and a putative MAPK-site
(S528) [11–13] as well as acetylation sites [14,15] located in
the deleted portion of the C-terminal NRD. In addition,
specific point mutations in v-Myb abolish protein–protein
interactions [5], as well as phosphorylation as in the case of
V117D [16]. c-Myb has recently also been reported to be

found. A more general proposition is that sumoylation plays
a role in the stabilization of higher order protein complexes
and modification of protein–protein interactions [18]. This is
consistent with the role of sumoylation in PML nuclear
bodies where it is important for PML nuclear body dynamics
and for recruiting other nuclear body components [28].
In the present study we have extended the findings of Bies
et al. [17] by providing several lines of independent evidence
for this novel modification of c-Myb. We show that c-Myb
interacts strongly with Ubc9 causing sumoylation at two
specific sites in the NRD region of the protein, K527 being a
dominant site and K503 being secondary. When sumoyla-
tion was blocked by mutation of the two modification sites,
this caused a large increase in transcriptional activity of
c-Myb, both when assayed with a transiently transfected
reporter gene and when measuring a resident Myb-target
gene. SUMO-1 conjugation was significantly enhanced by
cotransfection with PIASy, which is the E3 like factor
reported to enhance sumoylation of LEF1 [29]. Further-
more, PIASy seems to increase the fraction of Myb species
in the insoluble part after subnuclear fractionation, which
indicates that sumoylation might be involved in modulating
the protein–protein interactions of c-Myb.
Materials and methods
Plasmids
The yeast bait plasmid pDBT-hcM encoding full-length
human c-Myb fused to the Gal4p DBD was generated
from a cDNA clone [30] and the vector pDBT [31]. The
mammalian expression plasmid pCIneo-hcM contains full-
length human c-Myb cDNA with an optimized ATG

(Promega). Data from three independent transfection
experiments were normalized for protein concentration in
the samples. Equal transfection efficiency was verified by
Western analysis of the transfected species.
In vitro
conjugation assay
The various forms of human c-Myb were generated in
the TNT rabbit reticulocyte lysate system (Promega) in the
presence of [
35
S]methionine. Templates used were either the
appropriate plasmid (pCIneo-hcM) or a PCR product with
T7 promoter added during amplification (ÔTpC-fragmentÕ:
amino acids 410–639). GST-SUMO-1 [37] and GST-UBC9
were expressed and affinity-purified using standard methods
(Amersham Pharmacia Biotech). SUMO-activating enzyme
(E1 fraction) was prepared from CV1 cells as described [37].
SUMO-1 conjugation assays were performed as described
in [32] with purified GST-UBC9 included and incubation
for two hours at 30 °C. Reaction mixtures were analysed on
10% polyacrylamide gels revealed by fluorography.
Antibodies
For Myb detection, we used the polyclonal antibody H141
(Santa Cruz) and the monoclonal antibody 5e11 [38].
SUMO-1 was detected with monoclonal antibodies from
Zymed. PIASy-T7 was detected using anti-T7 Ig (Novagen).
Immunoprecipitation and Western blot
CV-1 cells were transfected with the indicated plasmids to
analyse sumoylation of c-Myb. After transfection, cells were
lysed and subjected to coimmunoprecipitation as described

same primer sets on a positive control template. The mRNA
levels of GAPDH were thus set at 100%.
Nuclear matrix preparation
CV-1 cells seeded out in 10 cm Petri dishes were transfected
with the indicated plasmids. Cells were harvested 24 h after
transfection in NaCl/P
i
and 30% were lysed directly in
loading buffer as a control for transfection. Nuclear matrix
samples and soluble fractions were prepared essentially as
described in [39].
Results
Bies et al. [17] have shown that murine c-Myb can be
sumoylated under overexpression conditions in COS7 cells
when cotransfected with FLAG-tagged SUMO-1. The
conjugation sites were mapped to the NRD region of the
protein. This work raised several questions that we have
addressed in a parallel study focusing on human c-Myb.
Several lines of independent evidence for sumoylation
of c-Myb
Our first interest was to find independent evidence for this
novel type of post-translational modification of c-Myb to
better establish its physiological relevance. In particular, we
were concerned by the overexpression conditions exclusively
used in the previous work on sumoylation of c-Myb [17].
We therefore initially performed a cotransfection experi-
ment similar to those reported by Bies et al.[17]but
replacing the COS cells with CV-1 cells, known to cause less
amplification of transfected plasmids than COS cells [40].
When CV-1 cells were cotransfected with constructs

migrating forms (Fig. 1B, lane 4). In this mutant, only a
single additional band is seen, probably due to a less efficient
sumoylation of the remaining K503 site. Again, the 2KR
mutant showed no retarded bands (Fig. 1B, lane 5). To
Fig. 1. Human c-Myb is sumoylated in residues 503 and 527. (A) CV-1
cells transfected with the Myb-expressing plasmids as indicated, and in
addition with (+) or without (–) pGFP-SUMO-1. The Myb proteins
expressed were full-length human c-Myb (hcM) and c-Myb mutated in
lysine 503 (K503R) or 527 (K527R) or both (2KR). Cells were lysed
directly in loading buffer before separation on SDS/PAGE and
immunoblotting revealed by a monoclonal anti-Myb Ig (5E11). (B)
CV-1 cells transfected with empty pCIneo vector (v) or plasmids
expressing indicated Myb proteins as in (A). Cell lysates were subjected
to direct immunoblot with monoclonal anti-(c-Myb) Ig. (C) CV-1 cells
were transfected as in (B). Immunoprecipitation was performed with
monoclonal anti-SUMO-1 Ig (upper panel) and polyclonal anti-
(c-Myb) Ig (lower panel). After SDS/PAGE the blot was revealed by
mAb 5E11. (D) Cell lysates from Jurkat cells expressing endogenous
c-Myb, was subjected to immunoprecipitation with polyclonal
anti-HA Ig, polyclonal anti-(c-Myb) Ig and polyclonal anti-(SUMO-1)
Ig. After SDS/PAGE of the immunoprecipitates, immunoblot analysis
was performed using monoclonal anti-(c-Myb) Ig. The arrow indicates
the migration of unmodified c-Myb.
1340 Ø. Dahle et al.(Eur. J. Biochem. 270) Ó FEBS 2003
verify that the observed modifications were indeed due to
SUMO-1 conjugation we performed at coimmunoprecipi-
tation experiment. While the lysate from CV-1 cells trans-
fected with wild type c-Myb contained modified Myb-forms
that became immunoprecipitated with the anti-SUMO-1 Ig
(Fig. 1C, lane 2), this was not the case with the 2KR mutant

forms were generated with sizes corresponding to the
addition of one or two moieties of GST-SUMO-1, respect-
ively (+39 kDa and +78 kDa) (Fig. 2, lane 4). These
modified forms disappeared when either GST-UBC9 or
GST-SUMO-1 was omitted from the reaction mixture
(Fig. 2, lanes 3 and 5), strongly suggesting that they
correspond to c-Myb conjugated to SUMO-1 peptides.
Both retarded bands observed with the wild type protein
disappeared when the double mutant (2KR) was subjected
to in vitro sumoylation, demonstrating their function as
conjugation sites (Fig. 2, lanes 7 and 9). Consistent with the
location of K503 and K527 in a region that is deleted in
AMV v-Myb, an AMV v-Myb protein did not generate
retarded modified forms in this system (results not shown).
The two single mutants, K503R and K527R, and the 2KR
mutant were also subjected to in vitro sumoylation in the
context of a c-Myb fragment (amino acids 410–566, more
efficiently translated in vitro). When the conjugated forms of
the single mutants were compared, it was evident that the
two sites were not equivalent. While the K527R mutation
caused a sharp drop in sumoylation efficiency, requiring a
high input of UBC9 to become sumoylated on the
remaining site, the K503R protein was still efficiently
sumoylated at low inputs of UBC9 similar to wild type. This
strongly suggests that K527 is a much more efficiently
conjugated site than K503. It is also noteworthy that
bis-sumoylated wild type protein (modified in K503 and
K527) is formed as efficiently as mono-sumoylated (pre-
sumably mainly modified in K527), while mono-sumoylated
K527R protein (presumably modified in K503) is formed

involved in the UBC9 interaction (results not shown). These
two-hybrid results show that Ubc9 is amongst the strongest
interaction partners of c-Myb as judged by a low-copy bait
screening in a cDNA library containing 2 million inde-
pendent clones, lending further support to the importance
of the c-Myb–Ubc9 interaction.
We conclude that SUMO-1 conjugation of c-Myb is not
only a phenomenon induced under favourable conditions of
overexpression of c-Myb and SUMO-1, but a robust
modification caused by a strong interaction between
c-Myb and Ubc9. This leads to modification at two residues
in the NRD part of the protein with K527 being the major
sumoylation site. The conjugation of SUMO-1 to c-Myb
raises the question of the role of this modification with
respect to the transcriptional activity of c-Myb.
Disruption of the SUMO-1 acceptor sites in c-Myb
causes a superactivation phenotype
Bies et al. [17] observed that c-Myb mutated in one of the
sumoylation sites was more active than wild type Myb in an
effector-reporter assay under overexpression conditions in
COS7 cells. To confirm this observation in CV-1 cells and
to extend the analysis to clarify the relative functional
importance of the two conjugation sites, we compared
reporter activation induced by the individual mutants (K503
and K527), the double mutant (2KR) and wild type c-Myb
using a reporter with multimerized Myb response elements
(Fig. 4A). While full-length c-Myb caused a modest level of
reporter activation (1.3-fold relative to empty effector), the
K503R mutant was slightly more active (3.6-fold), the
K527R mutant significantly more active (9.6-fold) and

wild type TpC c-Myb, respectively; ÔK503R mono-SÕ, single sumoylated K503R TpC c-Myb; ÔK527R mono-SÕ, single sumoylated K527R TpC
c-Myb.
1342 Ø. Dahle et al.(Eur. J. Biochem. 270) Ó FEBS 2003
cooperate with C/EBPb/NF-M, which is constitutively
expressed in these cells [5,41]. When assayed by real-time
PCR, the AMV version caused only a marginal mim-1
activation, while c-Myb induced a significant level of mim-1
expression (Fig. 4B, lanes 1 and 7). In this assay the 2KR
double mutant (lane 3) induced mim-1 expression to a
fourfold higher level than did wild type c-Myb (lane 1).
Cotransfection of SUMO-1 did not significantly change this
difference in behaviour, probably as the endogenous level of
SUMO-1 was already high and did not increase much after
SUMO-1 transfection (data not shown). Taken together,
these data clearly demonstrate that the conjugation sites in
K503 and K527 are critical for the potency of c-Myb to
activate the expression of a resident chromosomal c-Myb
target gene.
PIASy enhances sumoylation of c-Myb and its
association with the nuclear matrix
Conjugation of SUMO-1 to target proteins has recently
been found to involve E3 enzymes in the PIAS family [22–
25]. PIASy has been reported to enhance conjugation of
SUMO-1 to LEF1 [29]. Based on this observation, we tested
whether PIASy also enhanced sumoylation of c-Myb, as
both LEF1 and c-Myb have been reported to be important
for differentiation in the hematopoietic system [42]. As
shown in Fig. 5, increasing amounts of transfected PIASy
caused a parallel increase in the intensity of the retarded
c-Myb species corresponding to single and double sumoyl-

bait plasmid expressing lamin (pLam) and full-length human c-Myb
(pDBT-hcM-FL) were transformed into the a-mating type of the same
strain. Mating was performed to create the diploid combinations
indicated in the figure. These were subjected to 5-bromo-4-chlorindol-
3-yl b-
D
-galactoside overlay assay to reveal activation of the LacZ
reporter gene as blue colour. Left panel: PJ69–4a cells transformed
with plasmids encoding UBC9 fused to GAL4-AD (pACT2-UBC9),
c-Myb fused to GAL4-DBD (pDBT-hcM) and the two corresponding
empty vectors in the indicated combinations. LacZ reporter activity
was measured by a liquid b-galactosidase assay. The results are shown
as mean values ± SEM of four independent experiments, each carried
out in triplicate.
Ó FEBS 2003 Sumoylation of c-Myb (Eur. J. Biochem. 270) 1343
parallel decrease in the basal activity of the reporter in the
Myb-negative controls (data not shown). This general
negative effect precluded any definitive conclusions from
this experiment as to whether SUMO-1 conjugated Myb is
transcriptionally less active than nonconjugated.
To investigate additional consequences of PIASy-
enhanced sumoylation of c-Myb, we examined whether
the partitioning of c-Myb within the nucleus was altered.
This hypothesis was based on the fact that sumoylation
modulates the protein–protein interactions between PML
and its protein partners [28,43] and that PIASy is localized
to the nuclear matrix [29]. Therefore, we examined whether
PIASy-enhanced sumoylation of c-Myb had a general effect
on the interactions of c–Myb with other proteins in the
nucleus by doing a nuclear matrix (M) preparation experi-

differences might also be implicated in the increased activity
of 2KR compared to wild type c-Myb, but the mechanisms
for this remain unidentified.
Discussion
In the present study we have shown that the human
transcription factor c-Myb is subject to conjugation by the
small ubiquitin-related modifier, SUMO-1, at two sites in
the NRD region of the protein, K527 being a principal
sumoylation site and K503 a secondary one. Both sites are
important for transcriptional activity and their mutation
causes a large enhancement of Myb-dependent transactiva-
tion. Sumoylation of c-Myb was strongly enhanced by
coexpression of PIASy, which is the E3-like factor reported
to enhance sumoylation of LEF1 [29]. This E3-induced
increase in sumoylation also caused a shift in the distribu-
tion of Myb species towards the insoluble fraction after
subnuclear fractionation.
Bies et al. [17] recently reported that sumoylation of
murine c-Myb can be induced by overexpression in COS7
cells of both c-Myb and FLAG-tagged SUMO-1. Here, we
have reported a related study on human c-Myb that not
only confirms the findings of Bies and coworkers, but also
addresses several questions not answered by the previous
study. In particular, we were concerned that the modifica-
tion had only been strictly demonstrated by cotransfections
in COS cells. This cell line is well known to cause
amplification of effector plasmids containing an SV40
Fig. 6. PIASy recruits wild type c-Myb to the nuclear matrix. CV-1
cells were transfected with plasmids expressing wild type c-Myb alone
or in combination with PIASy as indicated. The same number of cells

two-hybrid screening shows that the SUMO-1 conjugase
Ubc9 is one of a few major Myb-interacting proteins
expressed in bone marrow or erythroleukemia cell lines.
We believe these independent data are important to be
confident that this novel type of modification of c-Myb is a
relevant one.
The two sites in c-Myb became conjugated with unequal
efficiency, K527 being a principal sumoylation site and
K503 a secondary one, despite both having identical core
sequence motifs IKQE. It is possible that the presence of
prolines close to K527 creates a more favourable context at
this site [44]. The difference is clearly seen by the dissimilar
effects of mutations in the two sites. The K527R mutant was
severely reduced in sumoylation in vivo (Fig. 1A,B) and a
poor substrate in vitro compared to the K503R mutant
(Fig. 2C), despite both harbouring one remaining conjuga-
tion site. The large difference in efficiency could mean that
the K527 site is the only physiologically relevant site, as
indicated by the observation that endogenously expressed
c-Myb in Jurkat cells was detected with only one SUMO-1
peptide conjugated (Fig. 1D). We cannot exclude, however,
that the two sites have distinct properties and that the
sumoylation of them depends on the biological context or is
controlled by specific E3 enzymes. It has recently been
reported that PML harbours two independent sumoylation
sites with distinct properties [45]. It is also possible that a
stepwise addition occurs. The UBC9-titration experiments
in vitro suggested that K503-sumoylation occurred more
efficiently if K527 was already modified. As SUMO-1 seems
to bind E3-type proteins [22], a possible scenario is that the

endogenous target gene is monitored. That the relative
enhancements were different in the two systems tested
certainly relates to the many differences between the two
cellular assays, including the use of a synthetic promoter
(multimerized Myb response elements) vs. a chromatin
embedded target gene, different cooperation between fac-
tors on the two promoters, and cofactors present in the
hematopoietic cell line not present in CV-1 cell line.
When single and double mutants were compared we
observed a clear correlation between the increase in
transcriptional activity of the individual mutants (Fig. 4)
and the reduction in their degree of sumoylation (Fig.
1A,B). Most probably these differences are caused by
abolished sumoylation, which alters the transactivation
properties. Such changes could occur directly, by modula-
tion of Myb’s intrinsic activation potential, or indirectly
through changes in subnuclear associations.
A direct transcriptional effect could result from changes
in intramolecular interactions or altered post-translational
modifications. The first would fit with the finding of a main
conjugation site (K527) within the previously identified
EVES region of c-Myb [51]. However, in our hands the
reported EVES–DBD interaction, when assayed in a Gal4-
two hybrid system, is rather weak and technically not
suitable to investigate whether it is modulated by sumoy-
lation. We did test the other possibility of altered post-
translational modifications in experiments where we
compared c-Myb wild type and the 2KR mutant with
respect to CBP interaction (CoIP experiments) and level of
acetylation, but did not observe any differences related to

that it is not simply PIASy that sequesters c-Myb into the
nuclear matrix. Whatever the mechanism, it is possible that
the trafficking of 2KR changes compared to wild type
c-Myb, and this leads to subtle changes in localization or
subnuclear associations. This might cause secondary effects
resulting in the observed increased activity of 2KR. The
emerging picture of the functional nuclear architecture
consisting of specialized domains with distinct biological
functions implies that most nuclear proteins are regulated
by and exert their functions from higher order protein
complexes at specific locations [55,56]. If, as suggested here,
sumoylation is involved in regulating the association of
c-Myb with higher order complexes, it would be important
to study the effects of sumoylation of c-Myb in a more
biological context than transfected reporter assays provide.
Deletion of the carboxy-terminal region of c-Myb
augments its transcriptional and transformation properties
(reviewed in [1,8,57]). For this reason the carboxy-terminal
part of the protein has been referred to as a negative
regulatory domain (NRD). More detailed mapping sugges-
ted the presence of two subdomains each contributing to the
NRD effect, the first of which harbours a putative leucine
zipper domain [58]. The second subdomain spans the amino
acid residues 495–640 in chicken c-Myb [59,60], and thus
encompasses both sumoylation sites. It has been proposed
that an additional cellular protein is required for negative
regulation of transcriptional activation by the NRD [8].
NRD regions in several transcription factors, including
c-Myb, share a common motif called the SC motif (synergy
control) [44], which appears to limit the transcriptional

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