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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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c-Myb occupancy on the MYC promoter is SUMO-independent. ChIP were performed with K562 cells to assess occupancy of c-Myb wild-type and SUMO-negative 2KR-mutant on the human MYC promoter. K562 cells were stably transfected with 3×FLAG- and HA-tagged c-Myb wild-type and 2KR, and clones were picked based on immunoblotting (inserted panel) showing similar expression of the integrated and endogenous c-Myb. ChIP was performed using anti-FLAG antibody, while an isotype IgG antibody was used as negative control. Occupancy was analysed by amplifying the MYC promoter by real-time PCR after reversal of the cross-linking. An unrelated DNA region (UDR) was used as negative control. The UDR was chosen from the gene desert region (53), the exact location is: chr2: 22153688+22153788. The results are calculated from triplicates of real-time PCR reactions and are expressed as percentage of recovery ± SD relative to the input. v, stable cell lines with integrated empty vector pEF1neo; asterisk, endogenous c-Myb; double asterisk, stably integrated, double-tagged c-Myb.
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Figure 4: c-Myb occupancy on the MYC promoter is SUMO-independent. ChIP were performed with K562 cells to assess occupancy of c-Myb wild-type and SUMO-negative 2KR-mutant on the human MYC promoter. K562 cells were stably transfected with 3×FLAG- and HA-tagged c-Myb wild-type and 2KR, and clones were picked based on immunoblotting (inserted panel) showing similar expression of the integrated and endogenous c-Myb. ChIP was performed using anti-FLAG antibody, while an isotype IgG antibody was used as negative control. Occupancy was analysed by amplifying the MYC promoter by real-time PCR after reversal of the cross-linking. An unrelated DNA region (UDR) was used as negative control. The UDR was chosen from the gene desert region (53), the exact location is: chr2: 22153688+22153788. The results are calculated from triplicates of real-time PCR reactions and are expressed as percentage of recovery ± SD relative to the input. v, stable cell lines with integrated empty vector pEF1neo; asterisk, endogenous c-Myb; double asterisk, stably integrated, double-tagged c-Myb.

Mentions: An EMSA analysis showed that the specific DNA binding observed was similar for c-Myb wild-type and 2KR as well as for the SUMO-fusion protein (Supplementary Figure S1), indicating that SUMO moieties conjugated or fused to the C-terminal of c-Myb have no significant influence on the activity of the DNA binding domain localized in the N-terminal of the protein. To monitor the DNA binding in vivo, we analysed c-Myb occupancy on the endogenous MYC promoter, an established target gene of c-Myb, harbouring multiple MREs [(39) and references therein]. For this analysis, we generated stable cell lines derived from K562 with integrated double-tagged c-Myb constructs in both wild-type and 2KR mutant versions. The levels of expressions of c-Myb in these cells are very close to the endogenous gene product and thus represent physiological levels of c-Myb (see western-insert in Figure 4). The ChIP signals were highly similar for the two versions of c-Myb, suggesting no significant difference in chromatin occupancy between wild-type and 2KR c-Myb (Figure 4). We also tested the same MYC promoter region in a reporter assay and found c-Myb 2KR to activate this promoter significantly stronger than wild-type c-Myb did, confirming the relevance of this promoter for SC (Supplementary Figure S2). Taken together, these data do not support an explanation of the SUMO-induced change in synergizing properties of c-Myb based on changes in DNA binding properties, neither in vitro nor in vivo.Figure 4.


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)

c-Myb occupancy on the MYC promoter is SUMO-independent. ChIP were performed with K562 cells to assess occupancy of c-Myb wild-type and SUMO-negative 2KR-mutant on the human MYC promoter. K562 cells were stably transfected with 3×FLAG- and HA-tagged c-Myb wild-type and 2KR, and clones were picked based on immunoblotting (inserted panel) showing similar expression of the integrated and endogenous c-Myb. ChIP was performed using anti-FLAG antibody, while an isotype IgG antibody was used as negative control. Occupancy was analysed by amplifying the MYC promoter by real-time PCR after reversal of the cross-linking. An unrelated DNA region (UDR) was used as negative control. The UDR was chosen from the gene desert region (53), the exact location is: chr2: 22153688+22153788. The results are calculated from triplicates of real-time PCR reactions and are expressed as percentage of recovery ± SD relative to the input. v, stable cell lines with integrated empty vector pEF1neo; asterisk, endogenous c-Myb; double asterisk, stably integrated, double-tagged c-Myb.
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Figure 4: c-Myb occupancy on the MYC promoter is SUMO-independent. ChIP were performed with K562 cells to assess occupancy of c-Myb wild-type and SUMO-negative 2KR-mutant on the human MYC promoter. K562 cells were stably transfected with 3×FLAG- and HA-tagged c-Myb wild-type and 2KR, and clones were picked based on immunoblotting (inserted panel) showing similar expression of the integrated and endogenous c-Myb. ChIP was performed using anti-FLAG antibody, while an isotype IgG antibody was used as negative control. Occupancy was analysed by amplifying the MYC promoter by real-time PCR after reversal of the cross-linking. An unrelated DNA region (UDR) was used as negative control. The UDR was chosen from the gene desert region (53), the exact location is: chr2: 22153688+22153788. The results are calculated from triplicates of real-time PCR reactions and are expressed as percentage of recovery ± SD relative to the input. v, stable cell lines with integrated empty vector pEF1neo; asterisk, endogenous c-Myb; double asterisk, stably integrated, double-tagged c-Myb.
Mentions: An EMSA analysis showed that the specific DNA binding observed was similar for c-Myb wild-type and 2KR as well as for the SUMO-fusion protein (Supplementary Figure S1), indicating that SUMO moieties conjugated or fused to the C-terminal of c-Myb have no significant influence on the activity of the DNA binding domain localized in the N-terminal of the protein. To monitor the DNA binding in vivo, we analysed c-Myb occupancy on the endogenous MYC promoter, an established target gene of c-Myb, harbouring multiple MREs [(39) and references therein]. For this analysis, we generated stable cell lines derived from K562 with integrated double-tagged c-Myb constructs in both wild-type and 2KR mutant versions. The levels of expressions of c-Myb in these cells are very close to the endogenous gene product and thus represent physiological levels of c-Myb (see western-insert in Figure 4). The ChIP signals were highly similar for the two versions of c-Myb, suggesting no significant difference in chromatin occupancy between wild-type and 2KR c-Myb (Figure 4). We also tested the same MYC promoter region in a reporter assay and found c-Myb 2KR to activate this promoter significantly stronger than wild-type c-Myb did, confirming the relevance of this promoter for SC (Supplementary Figure S2). Taken together, these data do not support an explanation of the SUMO-induced change in synergizing properties of c-Myb based on changes in DNA binding properties, neither in vitro nor in vivo.Figure 4.

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