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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 ID in Sp3 harbours a SRAF. CV-1 cells were transfected with 0.2 µg of plasmids expressing Gal4p-DBD fused to (A) Sp3 (amino acid residues 81–613) wild-type or the kee SUMO-negative mutant, or (C) Sp3 ID (amino acid residues 534–612) wild-type and kee mutant. 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 µg) and set to 100. The results are presented as RLU ± SEM. (B and D) Based on parallel transfections, using an 1×GRE-E1b-Luc reporter plasmid (0.2 µg), the SFs for the GBD-Sp3 and GBD-Sp3-ID constructs assayed in (A) and (C) were calculated. The results are presented as SF ± SEM.
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Figure 7: The ID in Sp3 harbours a SRAF. CV-1 cells were transfected with 0.2 µg of plasmids expressing Gal4p-DBD fused to (A) Sp3 (amino acid residues 81–613) wild-type or the kee SUMO-negative mutant, or (C) Sp3 ID (amino acid residues 534–612) wild-type and kee mutant. 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 µg) and set to 100. The results are presented as RLU ± SEM. (B and D) Based on parallel transfections, using an 1×GRE-E1b-Luc reporter plasmid (0.2 µg), the SFs for the GBD-Sp3 and GBD-Sp3-ID constructs assayed in (A) and (C) were calculated. The results are presented as SF ± SEM.

Mentions: Activators with two AFs are known from the literature, the steroid hormone receptor family being one prominent example. In fact, SC was first found in members of this family (9). To test whether the SUMO-dependent repressor-to-activator switch is a more general phenomenon, we chose to study the Sp3 transcription factor because this factor has been reported to activate or repress transcription depending on the promoter context (42). Moreover, the repressive function of Sp3 is mediated by SUMO-conjugation of K551 located in an ID. First, we confirmed using our Gal4-responsive reporter that mutation of its SUMO-conjugation site turned a Gal4p-Sp3 fusion protein into an activator (Figure 7A) as reported (42). This was accompanied by a large increase in SF value indicating a parallel change in synergy properties (Figure 7B). Next, we asked whether the small ID region harbouring the sumoylation site when removed from its two glutamine-rich activation domains would show SRAF behaviour. As seen for c-Myb NRD, wild-type Sp3-ID had a weak repressive effect by itself, while the SUMO-negative version was clearly activating (Figure 7C), showing the presence of a SRAF in Sp3. In parallel, we measured a large increase in SF value for SUMO-negative Sp3-ID (Figure 7D), linking the functional switch to changes in synergy behaviour. We also extended our analysis to the sumoylated CRD in p53. Again, we observed a similar change, but with more modest alterations (Supplementary Figure S3).Figure 7.


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 ID in Sp3 harbours a SRAF. CV-1 cells were transfected with 0.2 µg of plasmids expressing Gal4p-DBD fused to (A) Sp3 (amino acid residues 81–613) wild-type or the kee SUMO-negative mutant, or (C) Sp3 ID (amino acid residues 534–612) wild-type and kee mutant. 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 µg) and set to 100. The results are presented as RLU ± SEM. (B and D) Based on parallel transfections, using an 1×GRE-E1b-Luc reporter plasmid (0.2 µg), the SFs for the GBD-Sp3 and GBD-Sp3-ID constructs assayed in (A) and (C) were calculated. The results are presented as SF ± SEM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 7: The ID in Sp3 harbours a SRAF. CV-1 cells were transfected with 0.2 µg of plasmids expressing Gal4p-DBD fused to (A) Sp3 (amino acid residues 81–613) wild-type or the kee SUMO-negative mutant, or (C) Sp3 ID (amino acid residues 534–612) wild-type and kee mutant. 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 µg) and set to 100. The results are presented as RLU ± SEM. (B and D) Based on parallel transfections, using an 1×GRE-E1b-Luc reporter plasmid (0.2 µg), the SFs for the GBD-Sp3 and GBD-Sp3-ID constructs assayed in (A) and (C) were calculated. The results are presented as SF ± SEM.
Mentions: Activators with two AFs are known from the literature, the steroid hormone receptor family being one prominent example. In fact, SC was first found in members of this family (9). To test whether the SUMO-dependent repressor-to-activator switch is a more general phenomenon, we chose to study the Sp3 transcription factor because this factor has been reported to activate or repress transcription depending on the promoter context (42). Moreover, the repressive function of Sp3 is mediated by SUMO-conjugation of K551 located in an ID. First, we confirmed using our Gal4-responsive reporter that mutation of its SUMO-conjugation site turned a Gal4p-Sp3 fusion protein into an activator (Figure 7A) as reported (42). This was accompanied by a large increase in SF value indicating a parallel change in synergy properties (Figure 7B). Next, we asked whether the small ID region harbouring the sumoylation site when removed from its two glutamine-rich activation domains would show SRAF behaviour. As seen for c-Myb NRD, wild-type Sp3-ID had a weak repressive effect by itself, while the SUMO-negative version was clearly activating (Figure 7C), showing the presence of a SRAF in Sp3. In parallel, we measured a large increase in SF value for SUMO-negative Sp3-ID (Figure 7D), linking the functional switch to changes in synergy behaviour. We also extended our analysis to the sumoylated CRD in p53. Again, we observed a similar change, but with more modest alterations (Supplementary Figure S3).Figure 7.

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