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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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The NRD region of c-Myb harbours a SRAF that can switch from being repressive to be activating. (A) CV-1 cells were transfected with 0.2 or 0.4 µg of plasmids expressing Gal4p-DBD (GBD) fused to c-Myb NRD (amino acid residues 410–640) wild-type, 2KR, WT-SUMO-1, 2KR-SUMO-1 or 2EA. The reporter output from the E1b-driven Gal4p-responsive reporter plasmid (5×GRE, 0.2 µg) was normalized to the effect of Gal4p-DBD (0.2 or 0.4 µg), which was set to 100. The results are presented as RLU ± SEM. (B) Based on parallel transfections, using an 1×GRE-E1b-Luc reporter plasmid (0.2 µg), the SFs of the constructs assayed in (A) were calculated. The results are presented as SF ± SEM. (C) CV-1 cells were transfected with plasmids expressing GBD, GBD-NRD or GBD-NRD-2KR together with the SUMO-protease SENP1 or a protease-dead SENP1 mutant. The results are presented as fold change ± SEM, relative to the activity levels of the GBD constructs in the absence of the protease.
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Figure 6: The NRD region of c-Myb harbours a SRAF that can switch from being repressive to be activating. (A) CV-1 cells were transfected with 0.2 or 0.4 µg of plasmids expressing Gal4p-DBD (GBD) fused to c-Myb NRD (amino acid residues 410–640) wild-type, 2KR, WT-SUMO-1, 2KR-SUMO-1 or 2EA. The reporter output from the E1b-driven Gal4p-responsive reporter plasmid (5×GRE, 0.2 µg) was normalized to the effect of Gal4p-DBD (0.2 or 0.4 µg), which was set to 100. The results are presented as RLU ± SEM. (B) Based on parallel transfections, using an 1×GRE-E1b-Luc reporter plasmid (0.2 µg), the SFs of the constructs assayed in (A) were calculated. The results are presented as SF ± SEM. (C) CV-1 cells were transfected with plasmids expressing GBD, GBD-NRD or GBD-NRD-2KR together with the SUMO-protease SENP1 or a protease-dead SENP1 mutant. The results are presented as fold change ± SEM, relative to the activity levels of the GBD constructs in the absence of the protease.

Mentions: The classical way of identifying an AF is by fusing the domain under investigation to the Gal4p DBD and monitoring the induced activation of a Gal4p-responsive reporter. When we tested the NRD region of c-Myb (amino acids 410–640) in such a system, we saw that the wild-type version of NRD had a weak repressive effect (Figure 6A). However, the SUMO-negative 2KR version of NRD was no longer acting negatively, but had switched into behaving as an activator (Figure 6A). To make sure that this notable observation was not some peculiarity related to the minimal E1B promoter in the pG5E1bLuc reporter used, we repeated the experiment with an SNRPN-driven Gal4p-responsive luciferase reporter and observed an equal effect (data not shown). We also tested out the E→A mutants instead of the K→R mutants and observed the same changes in the properties of the NRD becoming an activating domain upon reduced sumoylation (Figure 6A). The fusion of SUMO-1 to the active 2KR-version of the NRD abolished its AF totally. All Gal4p-DBD fusion constructs showed equal expression levels as evaluated by western (data not shown) analysis. It is noteworthy that this gain of AF paralleled a strong increase in synergy properties as observed when SF-values were measured for all the Gal4p-fusions using a 1×GRE reporter in addition to the 5×GRE reporter (Figure 6B).Figure 6.


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)

The NRD region of c-Myb harbours a SRAF that can switch from being repressive to be activating. (A) CV-1 cells were transfected with 0.2 or 0.4 µg of plasmids expressing Gal4p-DBD (GBD) fused to c-Myb NRD (amino acid residues 410–640) wild-type, 2KR, WT-SUMO-1, 2KR-SUMO-1 or 2EA. The reporter output from the E1b-driven Gal4p-responsive reporter plasmid (5×GRE, 0.2 µg) was normalized to the effect of Gal4p-DBD (0.2 or 0.4 µg), which was set to 100. The results are presented as RLU ± SEM. (B) Based on parallel transfections, using an 1×GRE-E1b-Luc reporter plasmid (0.2 µg), the SFs of the constructs assayed in (A) were calculated. The results are presented as SF ± SEM. (C) CV-1 cells were transfected with plasmids expressing GBD, GBD-NRD or GBD-NRD-2KR together with the SUMO-protease SENP1 or a protease-dead SENP1 mutant. The results are presented as fold change ± SEM, relative to the activity levels of the GBD constructs in the absence of the protease.
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Related In: Results  -  Collection

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Figure 6: The NRD region of c-Myb harbours a SRAF that can switch from being repressive to be activating. (A) CV-1 cells were transfected with 0.2 or 0.4 µg of plasmids expressing Gal4p-DBD (GBD) fused to c-Myb NRD (amino acid residues 410–640) wild-type, 2KR, WT-SUMO-1, 2KR-SUMO-1 or 2EA. The reporter output from the E1b-driven Gal4p-responsive reporter plasmid (5×GRE, 0.2 µg) was normalized to the effect of Gal4p-DBD (0.2 or 0.4 µg), which was set to 100. The results are presented as RLU ± SEM. (B) Based on parallel transfections, using an 1×GRE-E1b-Luc reporter plasmid (0.2 µg), the SFs of the constructs assayed in (A) were calculated. The results are presented as SF ± SEM. (C) CV-1 cells were transfected with plasmids expressing GBD, GBD-NRD or GBD-NRD-2KR together with the SUMO-protease SENP1 or a protease-dead SENP1 mutant. The results are presented as fold change ± SEM, relative to the activity levels of the GBD constructs in the absence of the protease.
Mentions: The classical way of identifying an AF is by fusing the domain under investigation to the Gal4p DBD and monitoring the induced activation of a Gal4p-responsive reporter. When we tested the NRD region of c-Myb (amino acids 410–640) in such a system, we saw that the wild-type version of NRD had a weak repressive effect (Figure 6A). However, the SUMO-negative 2KR version of NRD was no longer acting negatively, but had switched into behaving as an activator (Figure 6A). To make sure that this notable observation was not some peculiarity related to the minimal E1B promoter in the pG5E1bLuc reporter used, we repeated the experiment with an SNRPN-driven Gal4p-responsive luciferase reporter and observed an equal effect (data not shown). We also tested out the E→A mutants instead of the K→R mutants and observed the same changes in the properties of the NRD becoming an activating domain upon reduced sumoylation (Figure 6A). The fusion of SUMO-1 to the active 2KR-version of the NRD abolished its AF totally. All Gal4p-DBD fusion constructs showed equal expression levels as evaluated by western (data not shown) analysis. It is noteworthy that this gain of AF paralleled a strong increase in synergy properties as observed when SF-values were measured for all the Gal4p-fusions using a 1×GRE reporter in addition to the 5×GRE reporter (Figure 6B).Figure 6.

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