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A SUMO-regulated activation function controls synergy of c-Myb through a repressor-activator switch leading to differential p300 recruitment.

Molvaersmyr AK, Saether T, Gilfillan S, Lorenzo PI, Kvaløy H, Matre V, Gabrielsen OS - Nucleic Acids Res. (2010)

Bottom Line: Focusing on the haematopoietic transcription factor c-Myb, we found evidence for a strong SC linked to SUMO-conjugation in its negative regulatory domain (NRD), while AMV v-Myb has escaped this control.When NRD is sumoylated, the activity of c-Myb is reduced.We therefore propose a general model for SUMO-mediated SC, where SUMO controls synergy by determining the number and strength of AFs associated with a promoter leading to differential chromatin signatures.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Oslo, Oslo, Norway.

ABSTRACT
Synergy between transcription factors operating together on complex promoters is a key aspect of gene activation. The ability of specific factors to synergize is restricted by sumoylation (synergy control, SC). Focusing on the haematopoietic transcription factor c-Myb, we found evidence for a strong SC linked to SUMO-conjugation in its negative regulatory domain (NRD), while AMV v-Myb has escaped this control. Mechanistic studies revealed a SUMO-dependent switch in the function of NRD. When NRD is sumoylated, the activity of c-Myb is reduced. When sumoylation is abolished, NRD switches into being activating, providing the factor with a second activation function (AF). Thus, c-Myb harbours two AFs, one that is constitutively active and one in the NRD being SUMO-regulated (SRAF). This double AF augments c-Myb synergy at compound natural promoters. A similar SUMO-dependent switch was observed in the regulatory domains of Sp3 and p53. We show that the change in synergy behaviour correlates with a SUMO-dependent differential recruitment of p300 and a corresponding local change in histone H3 and H4 acetylation. We therefore propose a general model for SUMO-mediated SC, where SUMO controls synergy by determining the number and strength of AFs associated with a promoter leading to differential chromatin signatures.

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A SUMO-mediated SC is operating on c-Myb. (A) Schematic picture of the different EMSA probes used to identify the minimal phasing between the MREs, and the luciferase reporters used in this study. The reporter plasmids contained increasing numbers of MREs (1× to 5×, all with 10 bp phasing) upstream of a core promoter from human MYC driving the luciferase reporter gene. (B) Recombinant c-Myb DBD (R123: 0–25 fmol) was bound to 2×MRE(GG) oligonucleotides (20 fmol each) with different phasing (6, 10, 15 or 20 bp) and complexes were separated on EMSA gels. F, free probe; */**, one or two R123 molecule(s) in complex with the probe, respectively. (C) CV-1 cells were transfected with reporter plasmids containing increasing numbers of MREs (1× to 5×) as indicated and a plasmid encoding c-Myb wild-type (inverted filled triangle) or a SUMO-negative c-Myb 2KR (filled diamond). Reporter activation is presented as relative luciferase units (RLU) ± SEM. (D) The definition of the SF. (E) CV-1 cells were transfected with reporters containing one or four MREs [(1×MRE(GG)-MYC or 4×MRE(GG)-MYC] and plasmids encoding c-Myb wild-type, c-Myb with both SUMO-conjugation sites mutated from lysine to arginine (2KR) or single SUMO-conjugation sites mutated (K503R/K527R, upper panel). CV-1 cells were co-transfected with plasmids encoding the different c-Myb SUMO-mutants and PIASy (ratio 4:1) to visualize the sumoylation pattern. The cells were lysed directly in SDS–PAGE loading buffer to maintain the modifications (lower panel). c-Myb-S and c-Myb-2S: c-Myb modified with one or two SUMO moieties, respectively. (F) SFs were measured using wild-type c-Myb and c-Myb 2KR expression plasmids together with two variants of a luciferase reporter activated by four MREs, differing only in their helical phasing. In the cis reporter, pGL4-4×MRE(GG)-MYC, the MRE phasing is 10 bp, while in the trans reporter, pGL4-4×MRE(GG)-MYC abab, the MRE phasing is 15 bp. The results are presented as SF ± SEM.
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Figure 1: A SUMO-mediated SC is operating on c-Myb. (A) Schematic picture of the different EMSA probes used to identify the minimal phasing between the MREs, and the luciferase reporters used in this study. The reporter plasmids contained increasing numbers of MREs (1× to 5×, all with 10 bp phasing) upstream of a core promoter from human MYC driving the luciferase reporter gene. (B) Recombinant c-Myb DBD (R123: 0–25 fmol) was bound to 2×MRE(GG) oligonucleotides (20 fmol each) with different phasing (6, 10, 15 or 20 bp) and complexes were separated on EMSA gels. F, free probe; */**, one or two R123 molecule(s) in complex with the probe, respectively. (C) CV-1 cells were transfected with reporter plasmids containing increasing numbers of MREs (1× to 5×) as indicated and a plasmid encoding c-Myb wild-type (inverted filled triangle) or a SUMO-negative c-Myb 2KR (filled diamond). Reporter activation is presented as relative luciferase units (RLU) ± SEM. (D) The definition of the SF. (E) CV-1 cells were transfected with reporters containing one or four MREs [(1×MRE(GG)-MYC or 4×MRE(GG)-MYC] and plasmids encoding c-Myb wild-type, c-Myb with both SUMO-conjugation sites mutated from lysine to arginine (2KR) or single SUMO-conjugation sites mutated (K503R/K527R, upper panel). CV-1 cells were co-transfected with plasmids encoding the different c-Myb SUMO-mutants and PIASy (ratio 4:1) to visualize the sumoylation pattern. The cells were lysed directly in SDS–PAGE loading buffer to maintain the modifications (lower panel). c-Myb-S and c-Myb-2S: c-Myb modified with one or two SUMO moieties, respectively. (F) SFs were measured using wild-type c-Myb and c-Myb 2KR expression plasmids together with two variants of a luciferase reporter activated by four MREs, differing only in their helical phasing. In the cis reporter, pGL4-4×MRE(GG)-MYC, the MRE phasing is 10 bp, while in the trans reporter, pGL4-4×MRE(GG)-MYC abab, the MRE phasing is 15 bp. The results are presented as SF ± SEM.

Mentions: Previous studies have reported that the activity of c-Myb is significantly enhanced by removal of its two SUMO-conjugation sites (19,20). To better understand the mechanism of this enhancement, we asked whether c-Myb was subject to the phenomenon of SC. Systematic analysis of this type of behaviour requires a dedicated set of reporter constructs with defined changes in the multiplicity of response elements. Hence, we constructed a set of reporter plasmids based on the pGL3 backbone with identical core promoters (from MYC P2), activated by one to five copies of an optimal Myb-response element (MRE) each with a phasing of 10 bp (Figure 1A). Binding to differently spaced MREs was examined in a systematic EMSA study using recombinant c-Myb. Here, we observed that a phasing of 6 bp caused interference from one bound factor on the binding of a second, while a phasing of 10 bp did not (Figure 1B). When reporters with one to five MREs were compared, wild-type c-Myb activated the various reporters with only minor differences (all about 3-fold) suggesting a modest response to increasing multiplicity (Figure 1C). In contrast, the SUMO-conjugation negative mutant, c-Myb 2KR, showed a remarkable increase in activity when the number of MREs was augmented (Figure 1C). In order to quantify this difference in synergistic behaviour, we defined a relative ‘synergy factor’ (SF) as the ratio of the reporter activity with four MREs to the activity with one MRE, divided by four (the latter to obtain ratios independent on the number of MREs). A simple proportional increase in reporter activity with the number of MREs should then give an SF = 1, while a positive synergy will result in SF > 1. An SF < 1 would then imply a less than additive effect, which might be termed ‘negative synergy’ (illustrated in Figure 1D). We calculated the SF based on four MREs in this work, but very similar SFs were obtained when we calculated ratios based on three or more MREs (data not shown). It should be noticed that SF is a relative quantity showing little dependence on the expression level of the factor tested. Using this simple quantification scheme, we compared wild-type and mutant c-Myb (Figure 1E). In fact, wild-type c-Myb displayed a negative synergy (SF = 0.26), while the SUMO-negative c-Myb 2KR showed a strong positive synergy (SF = 3.9). Single mutants, retaining one of the two SUMO-conjugation sites, showed intermediate levels (K503R: SF = 0.98, K527R: SF = 0.72). By comparing the SF data for the SUMO mutants (Figure 1E, upper panel) with their modification pattern (Figure 1E, lower panel), it seems clear that SF is inversely related to the level of c-Myb sumoylation. It may be noticed that the multiple bands representing one (c-Myb 1S) or two (c-Myb 2S) conjugated SUMO-moieties appear to be caused by various states of phosphorylation (data not shown). Taken together, these results suggest that the synergistic behaviour of c-Myb is highly dependent on its ability to become sumoylated.Figure 1.


A SUMO-regulated activation function controls synergy of c-Myb through a repressor-activator switch leading to differential p300 recruitment.

Molvaersmyr AK, Saether T, Gilfillan S, Lorenzo PI, Kvaløy H, Matre V, Gabrielsen OS - Nucleic Acids Res. (2010)

A SUMO-mediated SC is operating on c-Myb. (A) Schematic picture of the different EMSA probes used to identify the minimal phasing between the MREs, and the luciferase reporters used in this study. The reporter plasmids contained increasing numbers of MREs (1× to 5×, all with 10 bp phasing) upstream of a core promoter from human MYC driving the luciferase reporter gene. (B) Recombinant c-Myb DBD (R123: 0–25 fmol) was bound to 2×MRE(GG) oligonucleotides (20 fmol each) with different phasing (6, 10, 15 or 20 bp) and complexes were separated on EMSA gels. F, free probe; */**, one or two R123 molecule(s) in complex with the probe, respectively. (C) CV-1 cells were transfected with reporter plasmids containing increasing numbers of MREs (1× to 5×) as indicated and a plasmid encoding c-Myb wild-type (inverted filled triangle) or a SUMO-negative c-Myb 2KR (filled diamond). Reporter activation is presented as relative luciferase units (RLU) ± SEM. (D) The definition of the SF. (E) CV-1 cells were transfected with reporters containing one or four MREs [(1×MRE(GG)-MYC or 4×MRE(GG)-MYC] and plasmids encoding c-Myb wild-type, c-Myb with both SUMO-conjugation sites mutated from lysine to arginine (2KR) or single SUMO-conjugation sites mutated (K503R/K527R, upper panel). CV-1 cells were co-transfected with plasmids encoding the different c-Myb SUMO-mutants and PIASy (ratio 4:1) to visualize the sumoylation pattern. The cells were lysed directly in SDS–PAGE loading buffer to maintain the modifications (lower panel). c-Myb-S and c-Myb-2S: c-Myb modified with one or two SUMO moieties, respectively. (F) SFs were measured using wild-type c-Myb and c-Myb 2KR expression plasmids together with two variants of a luciferase reporter activated by four MREs, differing only in their helical phasing. In the cis reporter, pGL4-4×MRE(GG)-MYC, the MRE phasing is 10 bp, while in the trans reporter, pGL4-4×MRE(GG)-MYC abab, the MRE phasing is 15 bp. The results are presented as SF ± SEM.
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Figure 1: A SUMO-mediated SC is operating on c-Myb. (A) Schematic picture of the different EMSA probes used to identify the minimal phasing between the MREs, and the luciferase reporters used in this study. The reporter plasmids contained increasing numbers of MREs (1× to 5×, all with 10 bp phasing) upstream of a core promoter from human MYC driving the luciferase reporter gene. (B) Recombinant c-Myb DBD (R123: 0–25 fmol) was bound to 2×MRE(GG) oligonucleotides (20 fmol each) with different phasing (6, 10, 15 or 20 bp) and complexes were separated on EMSA gels. F, free probe; */**, one or two R123 molecule(s) in complex with the probe, respectively. (C) CV-1 cells were transfected with reporter plasmids containing increasing numbers of MREs (1× to 5×) as indicated and a plasmid encoding c-Myb wild-type (inverted filled triangle) or a SUMO-negative c-Myb 2KR (filled diamond). Reporter activation is presented as relative luciferase units (RLU) ± SEM. (D) The definition of the SF. (E) CV-1 cells were transfected with reporters containing one or four MREs [(1×MRE(GG)-MYC or 4×MRE(GG)-MYC] and plasmids encoding c-Myb wild-type, c-Myb with both SUMO-conjugation sites mutated from lysine to arginine (2KR) or single SUMO-conjugation sites mutated (K503R/K527R, upper panel). CV-1 cells were co-transfected with plasmids encoding the different c-Myb SUMO-mutants and PIASy (ratio 4:1) to visualize the sumoylation pattern. The cells were lysed directly in SDS–PAGE loading buffer to maintain the modifications (lower panel). c-Myb-S and c-Myb-2S: c-Myb modified with one or two SUMO moieties, respectively. (F) SFs were measured using wild-type c-Myb and c-Myb 2KR expression plasmids together with two variants of a luciferase reporter activated by four MREs, differing only in their helical phasing. In the cis reporter, pGL4-4×MRE(GG)-MYC, the MRE phasing is 10 bp, while in the trans reporter, pGL4-4×MRE(GG)-MYC abab, the MRE phasing is 15 bp. The results are presented as SF ± SEM.
Mentions: Previous studies have reported that the activity of c-Myb is significantly enhanced by removal of its two SUMO-conjugation sites (19,20). To better understand the mechanism of this enhancement, we asked whether c-Myb was subject to the phenomenon of SC. Systematic analysis of this type of behaviour requires a dedicated set of reporter constructs with defined changes in the multiplicity of response elements. Hence, we constructed a set of reporter plasmids based on the pGL3 backbone with identical core promoters (from MYC P2), activated by one to five copies of an optimal Myb-response element (MRE) each with a phasing of 10 bp (Figure 1A). Binding to differently spaced MREs was examined in a systematic EMSA study using recombinant c-Myb. Here, we observed that a phasing of 6 bp caused interference from one bound factor on the binding of a second, while a phasing of 10 bp did not (Figure 1B). When reporters with one to five MREs were compared, wild-type c-Myb activated the various reporters with only minor differences (all about 3-fold) suggesting a modest response to increasing multiplicity (Figure 1C). In contrast, the SUMO-conjugation negative mutant, c-Myb 2KR, showed a remarkable increase in activity when the number of MREs was augmented (Figure 1C). In order to quantify this difference in synergistic behaviour, we defined a relative ‘synergy factor’ (SF) as the ratio of the reporter activity with four MREs to the activity with one MRE, divided by four (the latter to obtain ratios independent on the number of MREs). A simple proportional increase in reporter activity with the number of MREs should then give an SF = 1, while a positive synergy will result in SF > 1. An SF < 1 would then imply a less than additive effect, which might be termed ‘negative synergy’ (illustrated in Figure 1D). We calculated the SF based on four MREs in this work, but very similar SFs were obtained when we calculated ratios based on three or more MREs (data not shown). It should be noticed that SF is a relative quantity showing little dependence on the expression level of the factor tested. Using this simple quantification scheme, we compared wild-type and mutant c-Myb (Figure 1E). In fact, wild-type c-Myb displayed a negative synergy (SF = 0.26), while the SUMO-negative c-Myb 2KR showed a strong positive synergy (SF = 3.9). Single mutants, retaining one of the two SUMO-conjugation sites, showed intermediate levels (K503R: SF = 0.98, K527R: SF = 0.72). By comparing the SF data for the SUMO mutants (Figure 1E, upper panel) with their modification pattern (Figure 1E, lower panel), it seems clear that SF is inversely related to the level of c-Myb sumoylation. It may be noticed that the multiple bands representing one (c-Myb 1S) or two (c-Myb 2S) conjugated SUMO-moieties appear to be caused by various states of phosphorylation (data not shown). Taken together, these results suggest that the synergistic behaviour of c-Myb is highly dependent on its ability to become sumoylated.Figure 1.

Bottom Line: Focusing on the haematopoietic transcription factor c-Myb, we found evidence for a strong SC linked to SUMO-conjugation in its negative regulatory domain (NRD), while AMV v-Myb has escaped this control.When NRD is sumoylated, the activity of c-Myb is reduced.We therefore propose a general model for SUMO-mediated SC, where SUMO controls synergy by determining the number and strength of AFs associated with a promoter leading to differential chromatin signatures.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Oslo, Oslo, Norway.

ABSTRACT
Synergy between transcription factors operating together on complex promoters is a key aspect of gene activation. The ability of specific factors to synergize is restricted by sumoylation (synergy control, SC). Focusing on the haematopoietic transcription factor c-Myb, we found evidence for a strong SC linked to SUMO-conjugation in its negative regulatory domain (NRD), while AMV v-Myb has escaped this control. Mechanistic studies revealed a SUMO-dependent switch in the function of NRD. When NRD is sumoylated, the activity of c-Myb is reduced. When sumoylation is abolished, NRD switches into being activating, providing the factor with a second activation function (AF). Thus, c-Myb harbours two AFs, one that is constitutively active and one in the NRD being SUMO-regulated (SRAF). This double AF augments c-Myb synergy at compound natural promoters. A similar SUMO-dependent switch was observed in the regulatory domains of Sp3 and p53. We show that the change in synergy behaviour correlates with a SUMO-dependent differential recruitment of p300 and a corresponding local change in histone H3 and H4 acetylation. We therefore propose a general model for SUMO-mediated SC, where SUMO controls synergy by determining the number and strength of AFs associated with a promoter leading to differential chromatin signatures.

Show MeSH
Related in: MedlinePlus