Limits...
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.

Show MeSH

Related in: MedlinePlus

Different c-Myb-mediated synergy on natural chromatin-embedded promoters. (A) Schematic presentation of the regulatory elements of the mim-1 and lysozyme genes according to (35–37). Both genes contain functional MREs in enhancer elements as well as in promoters. Question mark indicates functionality uncertain. HD11 cells were transfected with plasmids expressing c-Myb-HA or the sumoylation-negative mutant c-Myb-2KR-HA. (B) A western blot performed with anti-HA antibody, using lysates from the same cultures used for activity measurements. The α-tubulin was used as loading control. Activation of the endogenous Myb-target genes (C) mim-1 and (D) lysozyme was measured by quantitative real-time PCR. Target gene expression data were normalized by the relative expression of the housekeeping gene HPRT1 and represented as relative to empty vector-transfected cells, which were set to 100. The results represent the mean ± SEM of three independent biological assays, each analysed in duplicate for expression levels.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2926607&req=5

Figure 3: Different c-Myb-mediated synergy on natural chromatin-embedded promoters. (A) Schematic presentation of the regulatory elements of the mim-1 and lysozyme genes according to (35–37). Both genes contain functional MREs in enhancer elements as well as in promoters. Question mark indicates functionality uncertain. HD11 cells were transfected with plasmids expressing c-Myb-HA or the sumoylation-negative mutant c-Myb-2KR-HA. (B) A western blot performed with anti-HA antibody, using lysates from the same cultures used for activity measurements. The α-tubulin was used as loading control. Activation of the endogenous Myb-target genes (C) mim-1 and (D) lysozyme was measured by quantitative real-time PCR. Target gene expression data were normalized by the relative expression of the housekeeping gene HPRT1 and represented as relative to empty vector-transfected cells, which were set to 100. The results represent the mean ± SEM of three independent biological assays, each analysed in duplicate for expression levels.

Mentions: Most natural promoters are complex and often activated by a combination of transcription factors, several of which may be SUMO-conjugated. The activation of a natural promoter through desumoylation would be expected to involve removal of SUMO from various transcription factors. Unfortunately, this situation is not easy to mimic in an experimental setup. However, two Myb-responsive genes have been characterized in detail with respect to chromosomal organization of their Myb-responsive regulatory regions (Figure 3A). The mim-1 gene is activated by c-Myb through interaction with two distinct regions, a promoter and an enhancer region, both of which contain several MREs (35,36). The lysozyme gene has a similar organization but with fewer active MREs involved (36,37). Hence, we addressed the physiological relevance of the SUMO-mediated SC of c-Myb by comparing the activation of these two endogenous target genes by wild-type c-Myb and c-Myb 2KR, both expressed to similar levels (Figure 3B). We used c-Myb negative chicken macrophage HD11 cells since these cells express C/EBPβ known to cooperate with c-Myb in transcriptional activation of both mim-1 and lysozyme (24,38). As shown in Figure 3C, co-transfection of c-Myb wild-type or 2KR mutant resulted in a clear differential activation of mim-1, as expected if several MREs contribute to the activity of the chromatin embedded promoter. For the lysozyme gene (Figure 3D), the difference between c-Myb wild-type and 2KR was much less pronounced, consistent with fewer MREs being involved. We conclude that natural promoters differ significantly in their synergy behaviour when c-Myb is compared in the two states. We expect, however, that a larger difference would have been observed if we had been able to compare two or more cooperating factors in both SUMO-conjugated and non-conjugated states.Figure 3.


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)

Different c-Myb-mediated synergy on natural chromatin-embedded promoters. (A) Schematic presentation of the regulatory elements of the mim-1 and lysozyme genes according to (35–37). Both genes contain functional MREs in enhancer elements as well as in promoters. Question mark indicates functionality uncertain. HD11 cells were transfected with plasmids expressing c-Myb-HA or the sumoylation-negative mutant c-Myb-2KR-HA. (B) A western blot performed with anti-HA antibody, using lysates from the same cultures used for activity measurements. The α-tubulin was used as loading control. Activation of the endogenous Myb-target genes (C) mim-1 and (D) lysozyme was measured by quantitative real-time PCR. Target gene expression data were normalized by the relative expression of the housekeeping gene HPRT1 and represented as relative to empty vector-transfected cells, which were set to 100. The results represent the mean ± SEM of three independent biological assays, each analysed in duplicate for expression levels.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2926607&req=5

Figure 3: Different c-Myb-mediated synergy on natural chromatin-embedded promoters. (A) Schematic presentation of the regulatory elements of the mim-1 and lysozyme genes according to (35–37). Both genes contain functional MREs in enhancer elements as well as in promoters. Question mark indicates functionality uncertain. HD11 cells were transfected with plasmids expressing c-Myb-HA or the sumoylation-negative mutant c-Myb-2KR-HA. (B) A western blot performed with anti-HA antibody, using lysates from the same cultures used for activity measurements. The α-tubulin was used as loading control. Activation of the endogenous Myb-target genes (C) mim-1 and (D) lysozyme was measured by quantitative real-time PCR. Target gene expression data were normalized by the relative expression of the housekeeping gene HPRT1 and represented as relative to empty vector-transfected cells, which were set to 100. The results represent the mean ± SEM of three independent biological assays, each analysed in duplicate for expression levels.
Mentions: Most natural promoters are complex and often activated by a combination of transcription factors, several of which may be SUMO-conjugated. The activation of a natural promoter through desumoylation would be expected to involve removal of SUMO from various transcription factors. Unfortunately, this situation is not easy to mimic in an experimental setup. However, two Myb-responsive genes have been characterized in detail with respect to chromosomal organization of their Myb-responsive regulatory regions (Figure 3A). The mim-1 gene is activated by c-Myb through interaction with two distinct regions, a promoter and an enhancer region, both of which contain several MREs (35,36). The lysozyme gene has a similar organization but with fewer active MREs involved (36,37). Hence, we addressed the physiological relevance of the SUMO-mediated SC of c-Myb by comparing the activation of these two endogenous target genes by wild-type c-Myb and c-Myb 2KR, both expressed to similar levels (Figure 3B). We used c-Myb negative chicken macrophage HD11 cells since these cells express C/EBPβ known to cooperate with c-Myb in transcriptional activation of both mim-1 and lysozyme (24,38). As shown in Figure 3C, co-transfection of c-Myb wild-type or 2KR mutant resulted in a clear differential activation of mim-1, as expected if several MREs contribute to the activity of the chromatin embedded promoter. For the lysozyme gene (Figure 3D), the difference between c-Myb wild-type and 2KR was much less pronounced, consistent with fewer MREs being involved. We conclude that natural promoters differ significantly in their synergy behaviour when c-Myb is compared in the two states. We expect, however, that a larger difference would have been observed if we had been able to compare two or more cooperating factors in both SUMO-conjugated and non-conjugated states.Figure 3.

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