Tài liệu Báo cáo khoa học: Transactivation properties of c-Myb are critically dependent on two SUMO-1 acceptor sites that are conjugated in a PIASy enhanced manner pptx

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

overexpression conditions in COS7 cells when cotrans-
fected with FLAG-tagged SUMO-1. Here we provide
independent evidence that human c-Myb is also subject to
SUMO-1 conjugation under more physiological condi-
tions as revealed by coimmunoprecipitation analysis of
Jurkat cells and transfected CV-1 cells. Analysis in an
in vitro conjugation system showed that modification of
the two sites K503 and K527 is interdependent. A two-
hybrid screening revealed that the SUMO-1 conjugase
Ubc9 is one of a few major Myb-interacting proteins. The
moderate basal level of sumoylation was greatly enhanced
by cotransfection of PIASy, an E3 ligase for SUMO-1.
The functional consequence of abolishing sumoylation
was enhanced activation both of a transiently transfected
reporter gene and of a resident Myb-target gene. When
single and double mutants were compared, we found a
clear correlation between reduction in sumoylation and
increase in transcriptional activation. Enhancing sumoy-
lation by contransfection of PIASy had a negative effect
on both Myb-induced and basal level reporter activation.
Furthermore, PIASy caused a shift in nuclear distribution
of c-Myb towards the insoluble matrix fraction. We
propose that the negative influence on transactivation
properties by the negative regulatory domain region of
c-Myb depends on the sumoylation sites located here.
Keywords: c-Myb; transcription; SUMO-1; Ubc9; PIASy.
The c-Myb transcription factor plays a central role in the
regulation of cell growth and differentiation, in particular in
hematopoietic progenitor cells (reviewed in [1]). Homozy-
gous null c-Myb/Rag1 chimerical mice are blocked in early

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

subjected to SUMO-1 (small ubiquitin-related modifier)
conjugation [17].
The SUMO-1 protein is related to ubiquitin, but its
function, although presently unclear, seem to be other
than proteasomal degradation (reviewed in [18,19]). The
sequence homology between ubiquitin and SUMO-1 is
low, but the structures are highly similar [20], and they
use related conjugation mechanisms [21], including the
use of E3-like factors, which was recently identified for
sumoylation as the PIAS proteins (protein inhibitor of
activated STATs) [22–25]. The sequence YKXE has
been proposed as a consensus sequence for SUMO-1
conjugation [26]. The process of sumoylation is conserved
from yeast to man and is a dynamic and reversible
Correspondence to O. S. Gabrielsen, Department of Biochemistry,
University of Oslo, PO Box 1041 Blindern, N-0316 Oslo, Norway.
Fax:+4722854443,Tel.:+4722857346,
E-mail:
Abbreviations: DBD, DNA-binding domain; MRE, Myb recognition
element; NRD, negative regulatory domain; PIAS, protein inhibitior
of activated STATs; SUMO-1, small ubiquitin-related modifier;
TAD, transactivation domain; Ubc9, ubiquitin conjugation enzyme 9.
(Received 5 December 2002, revised 31 January 2003,
accepted 6 February 2003)
Eur. J. Biochem. 270, 1338–1348 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03504.x
process. Both sumoylation and desumoylation are needed
for viability in yeast [27].
Because several different classes of proteins are targets for
SUMO-1 conjugation, it is rather unlikely that a single
explanation for the biological role of sumoylation will 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

context. A c-Myb-HA fusion cDNA was generated by
cloning oligos encoding the C-terminal part of c-Myb in
fusion with an HA-tag, between PshAI and SalIin
pCIneo-hcM, to give the plasmid pCIneo-hcM-HA. The
cDNAs encoding c-Myb mutants K503R, K527R and
K503/527R (abbreviated 2KR) were generated using the
Quick Change Site-Directed Mutagenesis Kit (Stratagene)
on a subfragment of human c-MYB. Plasmids expressing
full-length SUMO-1 with an HA epitope (HA-SUMO-1)
or in fusion with GFP (GFP-SUMO-1) have been
described [32]. The expression plasmid pGEX-UBC9 was
constructed from a human UBC9 cDNA (isolated in the
two-hybrid screening), and cloned in-frame into pGEX-
6P-2 between SalIandNotI. All cloned fragments
generated by PCR were verified by sequencing. The
c-Myb-responsive luciferase reporter construct pGL2/tk/
3xGG contains multimerized Myb response elements and
its construction is described in [33].
Yeast two-hybrid screen
The yeast two-hybrid screen was performed in the yeast
strain PJ69-4a [34,35] with pDBT-hcM as bait and using
two Matchmaker cDNA libraries (Clontech): from human
bone marrow (HL4053AH) and from the erythroleukemia
cell line K562 (HL4032AH).
Cell culture, transfections and luciferase assays
CV-1 and HD11 cells were grown as described [33,36].
Transient transfections were performed by lipofection
(Lipofectamine-Plus, Gibco Life Technologies) or using
Fugene (Roche Diagnostics). Luciferase assays were per-
formed in triplicate using the Luciferase Assay Reagent

(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

[32] using standard methods.
RNA isolation and real time PCR
Total RNA was extracted from transfected HD11 cells
using Absolutely RNA
TM
RT-PCR Miniprep kit (Strata-
gene). RNA (1–2 lg) was reverse transcribed with Super-
script II reverse transcriptase (Life Technologies). The
cDNA was diluted fivefold prior to PCR amplification
using primers specific for chicken mim-1 and chicken
GAPDH, respectively. Real-time PCR was performed on
a LightCycler rapid thermal cycler system (Roche Diag-
nostics) using the LightCycler FastStart DNA Master
SYBR Green I mix for amplification (Roche Diagnostics).
Reactions were performed in 20 lL with 0.5 l
M
primers
and 3 m
M
MgCl
2
. The amplification specificity of the PCR
products was confirmed by using melting curve analysis and
gel electrophoresis. We calculated the relative level of mim-1
mRNA as 100/E
(CP1–CP2)
, where CP1 and CP2 are crossing
Ó FEBS 2003 Sumoylation of c-Myb (Eur. J. Biochem. 270) 1339
points for mim-1 and GAPDH mRNAs, respectively, and E
is the average efficiency of amplification obtained with the

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

expressing human c-Myb and GFP-tagged SUMO-1, two
retarded doublet bands were observed (Fig. 1A, lane 3).
These totally disappeared when the two putative sumoyla-
tion sites (in human c-Myb K503 and K527) were both
mutated (Ô2KR-MybÕ, Fig. 1A, lane 9). Single mutations
K503R and K527R had intermediary effects, with a strong
reduction with K527R and less effect with K503R where
only the upper doublet disappeared (Fig. 1A, lanes 7 and 5).
The same doublets of bands were seen in the control lanes 2
and 4 due to endogenous SUMO-1. Sumoylation at two
sites in c-Myb would be expected to generate two retarded
simple bands. Hence, the doublets probably represent
c-Myb with one and two conjugated SUMO-1 moieties,
respectively, combined with or without a second type of
modification (such as phosphorylation) affecting migration.
This confirms the observations of Bies et al. [17] under more
moderate conditions of overexpression.
To increase the stringency further we performed a similar
experiment in the absence of transfected SUMO-1 to see
whether endogenous levels of the peptide and its conjuga-
tion enzymes were sufficient to cause sumoylation. This
experiment was similar to what is shown in the control lanes
2, 4 and 6 in Fig. 1A but the use of a higher exposure allows
the effects of the mutants to be more evident. Again shifted
Myb-bands were observed in addition to the main 75 kDa
band (Fig. 1B, lane 2), although the mobility shifts now
were more modest, consistent with conjugation of untagged
SUMO-1. The K503R mutant caused the upper doublet to
disappear (Fig. 1B, lane 3). The K527R mutant caused a
much more important reduction in intensity of the slower

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

(Fig. 1C, lane 3). This supports that wild type c-Myb
becomes conjugated with SUMO-1, and that this modifi-
cation is abolished in the 2KR mutant. Both variants of
c-Myb were equally expressed (Fig. 1C).
Having shown that c-Myb is sumoylated in CV-1 cells by
endogenous levels of the conjugation machinery, we finally
addressed whether the same was true for endogenous c-Myb
proteins in myb-positive cells. Therefore we carried out a
similar analysis in Jurkat cells. Immunoprecipitation of
c-Myb with polyclonal anti-(c-Myb) Ig, and detection with
monoclonal anti(-c-Myb) Ig revealed the main c-Myb band
at 75 kDa and several c-Myb species with higher molecular
mass (Fig. 1D, lane 2). Immunoprecipitation of sumoylated
proteins with polyclonal anti-(SUMO-1) Ig in the same
experiment revealed that at least one of these bands are
sumoylated c-Myb. This was also confirmed by immuno-
precipitation of c-Myb with polyclonal anti-(c-Myb) Ig and
detection with monoclonal anti-(SUMO-1) Ig, which
revealed one band with a size corresponding to c-Myb
conjugated with one SUMO-1 molecule (results not shown).
We conclude that the SUMO-1 conjugation c-Myb
observed by Bies et al. [17] under overexpression conditions
seems to be a robust phenomenon that also occurs under
more physiological conditions.
A second line of experiments further supported that
c-Myb is a good substrate for SUMO-1 conjugation. An
in vitro system for sumoylation was set up to investigate
sumoylation of c-Myb (Fig. 2). When in vitro translated
human c-Myb was incubated with an E1 fraction, GST-
UBC9 and GST-SUMO-1, two more slowly migrating

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

with low efficiency. This suggests that K527-conjugation
enhances the efficiency of sumoylation at the other site.
A third line of independent evidence for sumoylation of
c–Myb is the interaction between c-Myb and Ubc9, the
latter acting as an E2-type SUMO-1 conjugase. Assuming
such an interaction, Bies et al. [17] performed a direct two-
hybrid test for this interaction between Ubc9 fused to
Gal4p-DBD and c-Myb domains fused to Gal4p-TAD.
Both fusion proteins were expressed from high-copy yeast
vectors. In an independent series of experiments we set up a
two-hybrid screen using full-length human c-Myb as bait
fused to Gal4p-DBD, but in our case expressed from a low-
copy CEN vector. Screening of 4 · 10
6
transformants from
two mixed cDNA Matchmaker libraries (human bone
marrow and human erythroleukemia K562 cell line)
resulted in the isolation of 23 triple-positive independent
clones. Three of these were identical to mRNA for
human ubiquitin-conjugating enzyme UBC9 (Accession
no. AJ002385). Retransformation and growth on reporter-
selective media (not shown) verified the Myb–UBC9
interaction, and by determination of reporter activation
using both a 5-bromo-4-chlorindol-3-yl b-
D
-galactoside
overlay and a liquid b-galactosidase assay (Fig. 3). Similar
analysis of several subdomains of c-Myb revealed strongest
subdomain interaction with the EVES-domain in the NRD-
region of c-Myb, suggesting that this region might be

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

Ó FEBS 2003 Sumoylation of c-Myb (Eur. J. Biochem. 270) 1341
finally the double mutant c-Myb-2KR gave rise to a 23-fold
increase in reporter activity, which is 17-fold higher than
wild type c-Myb. A Western blot confirmed that all Myb
variants were equally expressed (Fig. 4A). It is noteworthy
that a clear correlation seems to exist between the increase in
transcriptional activity of the individual mutants (Fig. 4A)
and the reduction in their degree of sumoylation (Fig.
1A,B).
Because effector-reporter assays in transfected cells is a
method with recognized limitations, we wanted to see
whether the conjugation sites influenced transcriptional
activity in a more physiological setting and therefore tested
activation of the resident mim-1 gene using the HD11 cell
line, an established Myb-model [36]. The mim-1 target gene
is only activated by c-Myb, not by v-Myb, when residing in
its chromosomal locus because v-Myb has lost the ability to
Fig. 2. Human c-Myb is sumoylated by UBC9 in vitro. (A) In vitro translated
35
S-labelled full-length c-Myb was incubated in the presence (+) or in
the absence (–) of the indicated components described in Materials and methods (lanes 1–5). Single sumoylated (1· Sumo) and bis-sumoylated (2·
Sumo) Myb, respectively. In lanes 6–9 full-length c-Myb (hcM) and c-Myb mutated at K503 and K527 (2KR) were compared in the presence (+)
or absence (–) of the full set of sumoylation components. (B) Wild type or mutant (K503R and K527R) subdomains of human c-Myb (TpC
fragments, amino acids 410–639) were
35
S-labelled in vitro and subjected to sumoylation as in 2A, but with variable limiting amounts of GST-UBC9
as indicated (given as ng of UBC9 only). The amount of sumoylated Myb species were quantified by the
NIH IMAGE
1.62 software (upper panel) and
the different sumoylated Myb species measured are shown in the lower panel. ÔWt bis-SÕ and Ôwt mono-SÕ represent double and single sumoylated

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-

ated c-Myb (Fig. 5, lanes 1–4). No corresponding enhanced
bands were observed with the 2KR mutant (Fig. 5, lane 5).
Thus, PIASy enhances conjugation of SUMO-1 to c-Myb,
and probably functions as an E3 enzyme for this process.
Now being able to greatly enhance the fraction of
conjugated Myb molecules, we asked how this affected
Myb-dependent transactivation, using a luciferase reporter
in transfected CV-1 cells. As expected, the input of PIASy
down-regulated Myb-dependent reporter activation was
increased, but it turned out that PIASy expression caused a
Fig. 4. Mutation of SUMO-1 conjugation sites enhances c-Myb acti-
vity. (A) Luciferase assays were performed on lysates from CV-1 cells
transfected with plasmids encoding the indicated proteins, abbreviated
as in the legend to Fig. 1, and a Myb-responsive reporter plasmid. An
aliquot of the lysates was analysed by Western blotting with anti-Myb
Ig to confirm expression of transfected Myb (lower panel). Note that
standard lysis was used without precaution to avoid desumoylation
upon cell lysis, hence less shifts are seen than in Fig. 1B. (B) The
indicated plasmids were transfected into HD11 cells and total RNA
was isolated. Activation of the endogenous Myb-target gene, mim-1,
was measured by real time PCR as described in Materials and meth-
ods. Abbreviations are as above and also ÔAMV vMÕ, AMV v-Myb; ÔSÕ,
cotransfected with a SUMO-1 expressing plasmid pCDNA3-HA-
SUMO-1.
Fig. 3. UBC9 is a major Myb-interacting protein. UBC9 was found
three times among 23 triple-positive independent clones isolated in a
yeast two-hybrid screening with full-length human c-Myb as bait. The
UBC9/c–Myb interaction was verified as shown. Right panel: Empty
library vector (pACT2) and pACT2-UBC9 were transformed into the
yeast two-hybrid strain PJ69-4a. Similarly, empty bait vector (pDBT),

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-

ment as described in [39]. This experiment was done by
transfecting CV-1 cells with either c-Myb alone or c-Myb
together with PIASy. When c-Myb was expressed alone, a
large portion of the c-Myb species was in the soluble
fraction (S) compared to the M-fraction (Fig. 6, lanes 5 and
4). Sumoylated c-Myb was only visible here when a
comparable sample of the cells was lysed directly in the
loading buffer (lane 6). In contrast, coexpression with
PIASy resulted in a distinct change in the distribution of
c-Myb, with more c-Myb retained in M than found in S
(lanes 1 and 2). The sumoylated c-Myb species appeared to
accumulate preferentially in the M-fraction. However,
because unsumoylated c-Myb species were also detected in
M (Fig. 6, lanes 1 and 4), as was a significant fraction of the
2KR mutant (not shown), sumoylation cannot solely be
responsible for recruiting c-Myb to the nuclear matrix
fraction. It is equally likely that PIASy somehow causes
accumulation of c-Myb in the nuclear matrix and enhances
its sumoylation there, which then stabilizes its association
with the M-fraction. We also noticed that the amount of
sumoylated c-Myb was not maintained during the prepar-
ation of M, which probably is due to sumoylation being a
reversible process (Fig. 6, T, M and S). This might give a
larger fraction of unsumoylated c-Myb in M than is actually
the case in vivo.
We conclude that PIASy enhances sumoylation of c-Myb
significantly, and that sumoylated and unsumoylated
c-Myb show differences in intranuclear distribution, prob-
ably as a consequence of altered protein–protein inter-
actions between c-Myb and its protein partners. Such

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

from both transfections was used for nuclear matrix preparation as
described in Materials and methods. The cell suspension was divided
withonethirdusedforpreparationofthetotalfraction(T)andthe
remaining two-thirds used to make the soluble and nuclear matrix
fraction. Total protein concentration of the soluble fraction was used
to normalize between the preparations. After separation of the proteins
on 10% SDS/PAGE, c-Myb species in the soluble (S), insoluble (M),
and total (T) fractions were detected with immunoblotting using anti-
(c-Myb) Ig as in Fig. 1.
Fig. 5. PIASy enhances sumoylation of c-Myb. A plasmid expressing
wild type c-Myb was cotransfected into CV-1 cells with increasing
amounts of plasmid expressing PIASy (0 lg, 0.25 lg, 0.5 lgand
1.0 lg) as indicated. As negative control 2KR c-Myb was cotrans-
fected with 1.0 lg PIASy plasmid. The upper panel shows immunoblot
(IB) detection with anti-(c-Myb) Ig, and the lower panel shows PIASy
expression revealed with anti-T7 Ig and IB detection.
1344 Ø. Dahle et al.(Eur. J. Biochem. 270) Ó FEBS 2003
origin of replication (here pcDNA3-derived) due to the
presence of SV40 large T-antigen [40], which will lead to
significantly increased levels of expression. In the present
study, we have tried to overcome these limitations and
provide three lines of independent evidence that human
c-Myb is indeed subject to SUMO-1 conjugation: (a) immu-
noprecipitations and Western analysis of Jurkat cells and
transfected CV-1 cells confirms that the modification
occurs at K503 and K527 under more physiological
conditions than previously reported, (b) analysis of sumoy-
lation in an in vitro conjugation system shows that c-Myb is
a good substrate for SUMO-1 conjugation and that
modification of the two sites are interdependent, and (c) a

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

strong K527 is modified first, followed by enhanced
recruitment of an E3 activity through binding to SUMO-1
causing more efficient modification of the remaining weaker
site (although E3 was not added in vitro, a rather crude
source of E1 was used).
An important novel finding is that the modest level of
modification observed in continuously growing cells is not
constitutive but can be enhanced significantly upon a
change in the level of a specific E3 enzyme (Fig. 5). PIASy,
the E3-like factor reported to enhance sumoylation of LEF1
[29], was found to drastically enhance sumoylation of
c-Myb on both sites. This E3-induced increase also caused a
shift in distribution of Myb species towards the insoluble
fraction after subnuclear fractionation, as discussed below.
The functional implications of c-Myb being prone to
SUMO-1 conjugation are not yet fully understood. One
obvious possibility is regulation of transcriptional activa-
tion. Sumoylation appears to have an effect on the activity
of several transcription factors, such as p53 [26,32], c-Jun
[46], Lef1 [29], AR [47], Sp3 [48], IRF-1 [49] and HDAC4
[50]. Sp3 is a particularly illustrative example of a factor
where SUMO modification, as with c-Myb, silences tran-
scriptional activity. Analysis of several mutations in Sp3
showed that those that prevented SUMO modification all
strongly enhanced the transcriptional activity of the factor
[48]. Similarly, SUMO-1 conjugation in c-Myb occurs at
sites that are very important for the activity of the factor.
Even a conservative mutation (KfiR) keeping the charge
unchanged, causes a large enhancement of the activity of
c-Mybbothintransfectionassaysandwhenactivationofan

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

the mutation (data not shown). Other possible direct
mechanisms exist: sumoylation could affect transcription
as an intrinsic part of the transcriptional activation process
through interference with ubiquitylation. Recent reports
have shown an unexpected involvement of ubiquitylation in
transcriptional activation [52–54]. Bies et al. [17] propose
that sumoylation stabilizes the c-Myb protein. This cannot,
however, explain the increased activity of 2KR, as it was
reported that mutating the sumoylation sites has no effect
on the stability of the protein [17].
Another possibility is that SUMO-1 could mediate
protein–protein interactions, making the sumoylated pro-
tein able to interact with other proteins than the nonsumoy-
lated protein, which is the case for PML [28,43]. This
concept that SUMO-1 conjugation stabilizes higher order
protein complexes was recently suggested as a common
theme for sumoylated proteins [18]. We therefore examined
whether PIASy-enhanced sumoylation of c-Myb would
alter its distribution within the nucleus. We observed that
the portion of c-Myb in the insoluble part of the nucleus was
increased after cotransfection with PIASy in a nuclear
Ó FEBS 2003 Sumoylation of c-Myb (Eur. J. Biochem. 270) 1345
matrix preparation experiment (Fig. 6). This experiment
also showed that there is an accumulation of sumoylated
Myb in the insoluble fraction of the nucleus, indicating that
sumoylation stabilizes the association of c-Myb with
insoluble structures in the nucleus. The mechanism for this
altered distribution remains to be elucidated. We were not
able to detect direct interactions between c-Myb and PIASy
in cotransfection experiments (results not shown) suggesting

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

synergy of these regulators through a mechanism involving
altered higher-order protein–protein interactions. It is
intriguing that this motif matches exactly the consensus
sequence for SUMO-1 conjugation, and several of the
proteins previously identified to contain the SC motif have
later been shown to be sumoylated in these sites, for
example c-Myb [17], C/EBP [61], AR [47] and Sp3 [48]. We
therefore propose that sumoylation of the NRD region of
c-Myb makes an important contribution to its negative
influence on transactivation properties. This effect may
rely on alterations in higher-order interactions with cooper-
ating proteins or subnuclear structures. Sumoylation as an
effector of NRD function may thus be a working model
linking effects on transcriptional activation with effects on
subnuclear associations.
Acknowledgements
This work was supported by The Norwegian Research Council (ØD,
TØA, ON, OB, OSG), The Norwegian Cancer Society (OB, OSG), and
the Anders Jahres Foundation (OSG). We thank Tone Berge for
construction of plasmids expressing C-terminal tagged c-Myb. We are
grateful to A. Leutz for providing the HD11 cell line, and to Dr
Jonathan P. Sleeman for the source of the 5E11 monoclonal antibody.
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