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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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SUMO-dependent recruitment to chromatin. (A) ChIPs were performed in a HEK 293 reporter cell line (33) harbouring an integrated transgene as illustrated [drawing based on Fig.2 in ref. (33)]. The reporter cell line was transfected with Gal-fusion derivatives of c-Myb as illustrated. Occupancy of factors and presence of chromatin marks were assessed on the luciferase promoter 5×Gal4 responsive elements (5×GRE) and on an intronic element in the neighbouring NCOA5 gene. Arrows indicate the regions amplified during analysis of ChIP samples by quantitative real-time PCR (ChIP–qPCR). (B) HEK 293 cells were transfected with plasmids expressing Gal4p-DBD (GBD) fused to c-Myb (amino acid residues 233–640) wild-type or 2KR. The output from the integrated luciferase was normalized to the effect of GBD alone, which was set to 100. The results are presented as RLU ± SEM. Immunoblotting was performed to control protein expression. Cells were lysed directly in SDS–PAGE loading buffer to maintain the modifications, and analysed using anti-HA antibody. GBD-Myb 1S and 2S: GBD-c-Myb modified with one or two SUMO proteins, respectively. (C) HEK 293 cells were transfected with plasmids expressing Gal4p-DBD fused to c-Myb wild-type, Sp3 wild-type or the kee SUMO-negative mutant of Sp3. The luciferase output was treated and presented as in (B). Occupancies of (D) Gal-Myb, (E) Mi-2α, (F) p300, (G) acetylated histone H3 and (H) acetylated histone H4 on the 5×GRE promoter and on the NCOA5 intron were analysed using ChIP–qPCR with cells transfected with GBD-Myb wild-type, 2KR or empty vector. In (E) Gal-Myb was co-transfected with FLAG-tagged Mi-2α.
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Figure 9: SUMO-dependent recruitment to chromatin. (A) ChIPs were performed in a HEK 293 reporter cell line (33) harbouring an integrated transgene as illustrated [drawing based on Fig.2 in ref. (33)]. The reporter cell line was transfected with Gal-fusion derivatives of c-Myb as illustrated. Occupancy of factors and presence of chromatin marks were assessed on the luciferase promoter 5×Gal4 responsive elements (5×GRE) and on an intronic element in the neighbouring NCOA5 gene. Arrows indicate the regions amplified during analysis of ChIP samples by quantitative real-time PCR (ChIP–qPCR). (B) HEK 293 cells were transfected with plasmids expressing Gal4p-DBD (GBD) fused to c-Myb (amino acid residues 233–640) wild-type or 2KR. The output from the integrated luciferase was normalized to the effect of GBD alone, which was set to 100. The results are presented as RLU ± SEM. Immunoblotting was performed to control protein expression. Cells were lysed directly in SDS–PAGE loading buffer to maintain the modifications, and analysed using anti-HA antibody. GBD-Myb 1S and 2S: GBD-c-Myb modified with one or two SUMO proteins, respectively. (C) HEK 293 cells were transfected with plasmids expressing Gal4p-DBD fused to c-Myb wild-type, Sp3 wild-type or the kee SUMO-negative mutant of Sp3. The luciferase output was treated and presented as in (B). Occupancies of (D) Gal-Myb, (E) Mi-2α, (F) p300, (G) acetylated histone H3 and (H) acetylated histone H4 on the 5×GRE promoter and on the NCOA5 intron were analysed using ChIP–qPCR with cells transfected with GBD-Myb wild-type, 2KR or empty vector. In (E) Gal-Myb was co-transfected with FLAG-tagged Mi-2α.

Mentions: Having explored how SUMO-conjugation affects intrinsic properties of c-Myb, we next investigated SUMO-modulated interactions. To assess interactions in the context of multiple SUMO-conjugated c-Myb proteins bound to a chromatinized promoter, we took advantage of the model system developed by Suske and co-workers (33), in which an array of Gal4-responsive elements (5×GRE) in front of a luciferase reporter is integrated in the genome of HEK 293 cells (Figure 9A). First, we made Gal4-c-Myb fusion constructs of the same design as used previously for Sp3 (33), with the c-Myb DBD replaced by Gal4-DBD (Figure 9A). Reporter assays showed a dramatic SUMO-dependent switch in activation in this system, with wild-type c-Myb showing quite modest activation and the SUMO-negative c-Myb 2KR behaving as a potent activator of the integrated reporter, being about 900-fold more active than wild-type c-Myb (Figure 9B, lower panel). Both forms of c-Myb were equally expressed (Figure 9B, upper panel). For comparison the sumoylation-deficient Gal4-Sp3-KEE mutant versus wild-type Gal4-Sp3 also showed a SUMO-dependent shift, but only about 10-fold (Figure 9C). Notably, the level of activation observed with sumoylation-competent Gal-Myb was comparable to that seen with sumoylation-deficient Gal4-Sp3-KEE (Figure 9C). ChIP analysis of c-Myb occupancy confirmed equal binding of both forms of c-Myb to the promoter region (Figure 9D). This is consistent with what we observed in the EMSA studies (Figure 1B and Supplementary Figure S1) and on the MYC promoter (Figure 4), confirming that SUMO-status does not affect the promoter recruitment of c-Myb.Figure 9.


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)

SUMO-dependent recruitment to chromatin. (A) ChIPs were performed in a HEK 293 reporter cell line (33) harbouring an integrated transgene as illustrated [drawing based on Fig.2 in ref. (33)]. The reporter cell line was transfected with Gal-fusion derivatives of c-Myb as illustrated. Occupancy of factors and presence of chromatin marks were assessed on the luciferase promoter 5×Gal4 responsive elements (5×GRE) and on an intronic element in the neighbouring NCOA5 gene. Arrows indicate the regions amplified during analysis of ChIP samples by quantitative real-time PCR (ChIP–qPCR). (B) HEK 293 cells were transfected with plasmids expressing Gal4p-DBD (GBD) fused to c-Myb (amino acid residues 233–640) wild-type or 2KR. The output from the integrated luciferase was normalized to the effect of GBD alone, which was set to 100. The results are presented as RLU ± SEM. Immunoblotting was performed to control protein expression. Cells were lysed directly in SDS–PAGE loading buffer to maintain the modifications, and analysed using anti-HA antibody. GBD-Myb 1S and 2S: GBD-c-Myb modified with one or two SUMO proteins, respectively. (C) HEK 293 cells were transfected with plasmids expressing Gal4p-DBD fused to c-Myb wild-type, Sp3 wild-type or the kee SUMO-negative mutant of Sp3. The luciferase output was treated and presented as in (B). Occupancies of (D) Gal-Myb, (E) Mi-2α, (F) p300, (G) acetylated histone H3 and (H) acetylated histone H4 on the 5×GRE promoter and on the NCOA5 intron were analysed using ChIP–qPCR with cells transfected with GBD-Myb wild-type, 2KR or empty vector. In (E) Gal-Myb was co-transfected with FLAG-tagged Mi-2α.
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Figure 9: SUMO-dependent recruitment to chromatin. (A) ChIPs were performed in a HEK 293 reporter cell line (33) harbouring an integrated transgene as illustrated [drawing based on Fig.2 in ref. (33)]. The reporter cell line was transfected with Gal-fusion derivatives of c-Myb as illustrated. Occupancy of factors and presence of chromatin marks were assessed on the luciferase promoter 5×Gal4 responsive elements (5×GRE) and on an intronic element in the neighbouring NCOA5 gene. Arrows indicate the regions amplified during analysis of ChIP samples by quantitative real-time PCR (ChIP–qPCR). (B) HEK 293 cells were transfected with plasmids expressing Gal4p-DBD (GBD) fused to c-Myb (amino acid residues 233–640) wild-type or 2KR. The output from the integrated luciferase was normalized to the effect of GBD alone, which was set to 100. The results are presented as RLU ± SEM. Immunoblotting was performed to control protein expression. Cells were lysed directly in SDS–PAGE loading buffer to maintain the modifications, and analysed using anti-HA antibody. GBD-Myb 1S and 2S: GBD-c-Myb modified with one or two SUMO proteins, respectively. (C) HEK 293 cells were transfected with plasmids expressing Gal4p-DBD fused to c-Myb wild-type, Sp3 wild-type or the kee SUMO-negative mutant of Sp3. The luciferase output was treated and presented as in (B). Occupancies of (D) Gal-Myb, (E) Mi-2α, (F) p300, (G) acetylated histone H3 and (H) acetylated histone H4 on the 5×GRE promoter and on the NCOA5 intron were analysed using ChIP–qPCR with cells transfected with GBD-Myb wild-type, 2KR or empty vector. In (E) Gal-Myb was co-transfected with FLAG-tagged Mi-2α.
Mentions: Having explored how SUMO-conjugation affects intrinsic properties of c-Myb, we next investigated SUMO-modulated interactions. To assess interactions in the context of multiple SUMO-conjugated c-Myb proteins bound to a chromatinized promoter, we took advantage of the model system developed by Suske and co-workers (33), in which an array of Gal4-responsive elements (5×GRE) in front of a luciferase reporter is integrated in the genome of HEK 293 cells (Figure 9A). First, we made Gal4-c-Myb fusion constructs of the same design as used previously for Sp3 (33), with the c-Myb DBD replaced by Gal4-DBD (Figure 9A). Reporter assays showed a dramatic SUMO-dependent switch in activation in this system, with wild-type c-Myb showing quite modest activation and the SUMO-negative c-Myb 2KR behaving as a potent activator of the integrated reporter, being about 900-fold more active than wild-type c-Myb (Figure 9B, lower panel). Both forms of c-Myb were equally expressed (Figure 9B, upper panel). For comparison the sumoylation-deficient Gal4-Sp3-KEE mutant versus wild-type Gal4-Sp3 also showed a SUMO-dependent shift, but only about 10-fold (Figure 9C). Notably, the level of activation observed with sumoylation-competent Gal-Myb was comparable to that seen with sumoylation-deficient Gal4-Sp3-KEE (Figure 9C). ChIP analysis of c-Myb occupancy confirmed equal binding of both forms of c-Myb to the promoter region (Figure 9D). This is consistent with what we observed in the EMSA studies (Figure 1B and Supplementary Figure S1) and on the MYC promoter (Figure 4), confirming that SUMO-status does not affect the promoter recruitment of c-Myb.Figure 9.

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