Eur J Biochem 270, 1338–1348 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03504.x Transactivation properties of c-Myb are critically dependent on two SUMO-1 acceptor sites that are conjugated in a PIASy enhanced manner ˚ Øyvind Dahle1, Tor Ø Andersen1, Oddmund Nordgard1, Vilborg Matre1, Giannino Del Sal2,3 and Odd S Gabrielsen1 Department of Biochemistry, University of Oslo, Norway; 2Laboratorio Nazionale CIB, Area Science Park, Trieste, Italy; 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 cotransfected with FLAG-tagged SUMO-1 Here we provide independent evidence that human c-Myb is also subject to SUMO-1 conjugation under more physiological conditions 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 twohybrid 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 sumoylation 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 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]) Homozygous null c-Myb/Rag1 chimerical mice are blocked in early T-cell development, while mice with a c-mybnull 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 transactivation domain (TAD) and a C-terminal negative regulatory domain (NRD) The DBD of c-Myb is comprised of the three imperfect repeats: R1, R2 and R3, 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 phosphorylation 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: + 47 22 85 44 43, Tel.: + 47 22 85 73 46, E-mail: o.s.gabrielsen@biokjemi.uio.no 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 (Received December 2002, revised 31 January 2003, accepted February 2003) Keywords: c-Myb; transcription; SUMO-1; Ubc9; PIASy Ó FEBS 2003 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 sumoylation 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] Furthermore, 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 Sumoylation of c-Myb (Eur J Biochem 270) 1339 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 performed 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 [35S]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 Plasmids Antibodies 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 fulllength 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 SalI in 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 pGEX6P-2 between SalI and NotI 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] 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) 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 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 RNATM RT-PCR Miniprep kit (Stratagene) RNA (1–2 lg) was reverse transcribed with Superscript 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 Diagnostics) using the LightCycler FastStart DNA Master SYBR Green I mix for amplification (Roche Diagnostics) Reactions were performed in 20 lL with 0.5 lM primers and mM MgCl2 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 1340 Ø Dahle et al (Eur J Biochem 270) Ó FEBS 2003 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/Pi 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 experiment 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 sumoylation 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 and 5) The same doublets of bands were seen in the control lanes and 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 conjugation enzymes were sufficient to cause sumoylation This experiment was similar to what is shown in the control lanes 2, and 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 Fig 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 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 Ó FEBS 2003 verify that the observed modifications were indeed due to SUMO-1 conjugation we performed at coimmunoprecipitation experiment While the lysate from CV-1 cells transfected 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 modification 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 immunoprecipitation 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, GSTUBC9 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, respectively (+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 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 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 Sumoylation of c-Myb (Eur J Biochem 270) 1341 bis-sumoylated wild type protein (modified in K503 and K527) is formed as efficiently as mono-sumoylated (presumably 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 twohybrid 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 lowcopy CEN vector Screening of · 106 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 reporterselective 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 NRDregion 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 million independent 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 1342 Ø Dahle et al (Eur J Biochem 270) Ó FEBS 2003 Fig Human c-Myb is sumoylated by UBC9 in vitro (A) In vitro translated 35S-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 35S-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 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 Ó FEBS 2003 Sumoylation of c-Myb (Eur J Biochem 270) 1343 Fig 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-chlorindol3-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 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 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- Fig Mutation of SUMO-1 conjugation sites enhances c-Myb activity (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 methods Abbreviations are as above and also ÔAMV vMÕ, AMV v-Myb; ÔSÕ, cotransfected with a SUMO-1 expressing plasmid pCDNA3-HASUMO-1 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 Ó FEBS 2003 1344 Ø Dahle et al (Eur J Biochem 270) Fig 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 lg and 1.0 lg) as indicated As negative control 2KR c-Myb was cotransfected 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 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 PIASyenhanced 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 experiment 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 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 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 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 preparation of M, which probably is due to sumoylation being a Fig 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 with one third used for preparation of the total fraction (T) and the 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 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, probably as a consequence of altered protein–protein interactions 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 transactivation 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 distribution 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 modification 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 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) immunoprecipitations 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 sumoylation 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 conjugation 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 activation Sumoylation appears to have an effect on the activity of several transcription factors, such as p53 [26,32], c-Jun Sumoylation of c-Myb (Eur J Biochem 270) 1345 [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 transcriptional 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-Myb both in transfection assays and when activation of an 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 factors 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 modulation 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 Gal4two hybrid system, is rather weak and technically not suitable to investigate whether it is modulated by sumoylation We did test the other possibility of altered posttranslational 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 protein able to interact with other proteins than the nonsumoylated 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 1346 Ø Dahle et al (Eur J Biochem 270) 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 suggested 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 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38037–38044  ... insoluble part after subnuclear fractionation, which indicates that sumoylation might be involved in modulating the protein–protein interactions of c-Myb Materials and methods Sumoylation of c-Myb. .. 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... 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