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Oncogenic point mutations in the Myb DNA-binding domain alter the DNA-binding properties of Myb at a physiological target gene.

Ivanova O, Braas D, Klempnauer KH - Nucleic Acids Res. (2007)

Bottom Line: Interestingly, the activation of the enhancer was abolished by the oncogenic amino acid substitutions.We demonstrated that a single Myb-binding site is responsible for the activation of the lysozyme enhancer by Myb and showed that the v-Myb protein of AMV was unable to bind to this site.Our data demonstrate for the first time that oncogenic activation of Myb alters its DNA-binding specificity at a physiological Myb target gene.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie, Westfälische-Wilhelms-Universität Münster, Wilhelm-Klemm-Strasse 2, D-48149 Münster, Germany.

ABSTRACT
The oncoprotein v-Myb of avian myeloblastosis virus (AMV) transforms myelomonocytic cells by deregulating specific target genes. Previous work has shown that the oncogenic potential of v-Myb was activated by truncation of N- and C-terminal sequences of c-Myb and was further increased by amino acid substitutions in the DNA-binding domain and other parts of the protein. We have analyzed the activation of the chicken lysozyme gene which is strongly activated by c-Myb but not by its oncogenic counterpart v-Myb. We report that Myb acts on two different cis-regulatory elements, the promoter and an enhancer located upstream of the gene. Interestingly, the activation of the enhancer was abolished by the oncogenic amino acid substitutions. We demonstrated that a single Myb-binding site is responsible for the activation of the lysozyme enhancer by Myb and showed that the v-Myb protein of AMV was unable to bind to this site. Our data demonstrate for the first time that oncogenic activation of Myb alters its DNA-binding specificity at a physiological Myb target gene.

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Synergistic activation of the −2.7 kb enhancer by Myb, C/EBP and PU.1. (A) QT6 cells were transfected with the reporter gene pTATA or pTATA-2.7 and different combinations of expression vectors for v-MybREV, C/EBPα, C/EBPβ and PU.1, as indicated at the bottom. To control the transfection efficiencies, cells were additionally transfected with the β-galactosidase plasmid pCMVβ. Cells were harvested 24 h after transfection and analyzed for luciferase and β-galactosidase activities. The columns show the average luciferase activity normalized to the β-galactosidase activity. Thin lines show standard deviations. The numbers above the columns indicate the extent of synergy between Myb and C/EBPα, C/EBPβ and PU.1, respectively. These numbers were determined by dividing the luciferase activity observed in the presence of both factors together by the sum of the luciferase activities observed for each factor alone. (B) QT6 cells were transfected with pTATA-2.7 and different combinations of expression vectors, as indicated at the bottom. Cells were analyzed as described in A. (C) C/EBPα, C/EBPβ and recombinant protein constructs are shown schematically at the top. QT6 cells were transfected with pTATA-2.7 and the expression vectors indicated below the columns. To control the transfection efficiencies, cells were additionally transfected with the β-galactosidase plasmid pCMVβ. Cells were analyzed as in (A).
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Figure 3: Synergistic activation of the −2.7 kb enhancer by Myb, C/EBP and PU.1. (A) QT6 cells were transfected with the reporter gene pTATA or pTATA-2.7 and different combinations of expression vectors for v-MybREV, C/EBPα, C/EBPβ and PU.1, as indicated at the bottom. To control the transfection efficiencies, cells were additionally transfected with the β-galactosidase plasmid pCMVβ. Cells were harvested 24 h after transfection and analyzed for luciferase and β-galactosidase activities. The columns show the average luciferase activity normalized to the β-galactosidase activity. Thin lines show standard deviations. The numbers above the columns indicate the extent of synergy between Myb and C/EBPα, C/EBPβ and PU.1, respectively. These numbers were determined by dividing the luciferase activity observed in the presence of both factors together by the sum of the luciferase activities observed for each factor alone. (B) QT6 cells were transfected with pTATA-2.7 and different combinations of expression vectors, as indicated at the bottom. Cells were analyzed as described in A. (C) C/EBPα, C/EBPβ and recombinant protein constructs are shown schematically at the top. QT6 cells were transfected with pTATA-2.7 and the expression vectors indicated below the columns. To control the transfection efficiencies, cells were additionally transfected with the β-galactosidase plasmid pCMVβ. Cells were analyzed as in (A).

Mentions: The differential activity of v-MybREV and v-MybAMV at the −2.7 kb enhancer suggested that further analysis of the enhancer might provide a clue about why the AMV version of v-Myb does not activate the lysozyme gene. Several proteins have previously been implicated in binding to the −2.7 kb lysozyme enhancer, including Ets and C/EBP family members as well as an unknown protein binding to an ‘AP-1-like’ site (26,32,33). The protein binding to the Ets-binding site has been tentatively identified as PU.1 (26,33). To explore whether Myb cooperates with one of the known proteins that bind to the enhancer we performed co-transfection experiments with different combinations of expression vectors for Myb, PU.1, C/EBPα and C/EBPβ (Figure 3A). Herschlag and Johnson (34) defined synergism in transcriptional activation as existing when the effects of two factors are more than additive. To determine whether Myb is synergistic with any of the other factors we therefore compared the activity of the reporter gene, in the presence of each factor alone, to the activity observed when the same factors were expressed together. When transfected on its own, each of the factors was able to stimulate the activity of the enhancer to some extent. When expressed together, more than additive stimulation was observed when Myb was combined with C/EBPα or with PU.1, whereas merely additive stimulation was observed when Myb was combined with C/EBPβ. The degree of synergy is expressed in a quantitative manner by the numbers on top of the columns in Figure 3A, where a factor larger than one indicates more than additive effects. From these numbers it is apparent that C/EBPα and PU.1 synergize with Myb according to the definition of Herschlag and Johnson (34) whereas Myb and C/EBPβ are not synergistic. We also assessed the combined effects of C/EBPα and PU.1, and of Myb, C/EBPα and PU.1 on the enhancer (Figure 3B). Interestingly, C/EBPα and PU.1 did not act synergistically in the absence of Myb whereas the presence of Myb resulted in a very strong stimulation of the enhancer activity. Thus, it appears that Myb, C/EBPα and PU.1 act in concert to activate the lysozyme enhancer.Figure 3.


Oncogenic point mutations in the Myb DNA-binding domain alter the DNA-binding properties of Myb at a physiological target gene.

Ivanova O, Braas D, Klempnauer KH - Nucleic Acids Res. (2007)

Synergistic activation of the −2.7 kb enhancer by Myb, C/EBP and PU.1. (A) QT6 cells were transfected with the reporter gene pTATA or pTATA-2.7 and different combinations of expression vectors for v-MybREV, C/EBPα, C/EBPβ and PU.1, as indicated at the bottom. To control the transfection efficiencies, cells were additionally transfected with the β-galactosidase plasmid pCMVβ. Cells were harvested 24 h after transfection and analyzed for luciferase and β-galactosidase activities. The columns show the average luciferase activity normalized to the β-galactosidase activity. Thin lines show standard deviations. The numbers above the columns indicate the extent of synergy between Myb and C/EBPα, C/EBPβ and PU.1, respectively. These numbers were determined by dividing the luciferase activity observed in the presence of both factors together by the sum of the luciferase activities observed for each factor alone. (B) QT6 cells were transfected with pTATA-2.7 and different combinations of expression vectors, as indicated at the bottom. Cells were analyzed as described in A. (C) C/EBPα, C/EBPβ and recombinant protein constructs are shown schematically at the top. QT6 cells were transfected with pTATA-2.7 and the expression vectors indicated below the columns. To control the transfection efficiencies, cells were additionally transfected with the β-galactosidase plasmid pCMVβ. Cells were analyzed as in (A).
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Figure 3: Synergistic activation of the −2.7 kb enhancer by Myb, C/EBP and PU.1. (A) QT6 cells were transfected with the reporter gene pTATA or pTATA-2.7 and different combinations of expression vectors for v-MybREV, C/EBPα, C/EBPβ and PU.1, as indicated at the bottom. To control the transfection efficiencies, cells were additionally transfected with the β-galactosidase plasmid pCMVβ. Cells were harvested 24 h after transfection and analyzed for luciferase and β-galactosidase activities. The columns show the average luciferase activity normalized to the β-galactosidase activity. Thin lines show standard deviations. The numbers above the columns indicate the extent of synergy between Myb and C/EBPα, C/EBPβ and PU.1, respectively. These numbers were determined by dividing the luciferase activity observed in the presence of both factors together by the sum of the luciferase activities observed for each factor alone. (B) QT6 cells were transfected with pTATA-2.7 and different combinations of expression vectors, as indicated at the bottom. Cells were analyzed as described in A. (C) C/EBPα, C/EBPβ and recombinant protein constructs are shown schematically at the top. QT6 cells were transfected with pTATA-2.7 and the expression vectors indicated below the columns. To control the transfection efficiencies, cells were additionally transfected with the β-galactosidase plasmid pCMVβ. Cells were analyzed as in (A).
Mentions: The differential activity of v-MybREV and v-MybAMV at the −2.7 kb enhancer suggested that further analysis of the enhancer might provide a clue about why the AMV version of v-Myb does not activate the lysozyme gene. Several proteins have previously been implicated in binding to the −2.7 kb lysozyme enhancer, including Ets and C/EBP family members as well as an unknown protein binding to an ‘AP-1-like’ site (26,32,33). The protein binding to the Ets-binding site has been tentatively identified as PU.1 (26,33). To explore whether Myb cooperates with one of the known proteins that bind to the enhancer we performed co-transfection experiments with different combinations of expression vectors for Myb, PU.1, C/EBPα and C/EBPβ (Figure 3A). Herschlag and Johnson (34) defined synergism in transcriptional activation as existing when the effects of two factors are more than additive. To determine whether Myb is synergistic with any of the other factors we therefore compared the activity of the reporter gene, in the presence of each factor alone, to the activity observed when the same factors were expressed together. When transfected on its own, each of the factors was able to stimulate the activity of the enhancer to some extent. When expressed together, more than additive stimulation was observed when Myb was combined with C/EBPα or with PU.1, whereas merely additive stimulation was observed when Myb was combined with C/EBPβ. The degree of synergy is expressed in a quantitative manner by the numbers on top of the columns in Figure 3A, where a factor larger than one indicates more than additive effects. From these numbers it is apparent that C/EBPα and PU.1 synergize with Myb according to the definition of Herschlag and Johnson (34) whereas Myb and C/EBPβ are not synergistic. We also assessed the combined effects of C/EBPα and PU.1, and of Myb, C/EBPα and PU.1 on the enhancer (Figure 3B). Interestingly, C/EBPα and PU.1 did not act synergistically in the absence of Myb whereas the presence of Myb resulted in a very strong stimulation of the enhancer activity. Thus, it appears that Myb, C/EBPα and PU.1 act in concert to activate the lysozyme enhancer.Figure 3.

Bottom Line: Interestingly, the activation of the enhancer was abolished by the oncogenic amino acid substitutions.We demonstrated that a single Myb-binding site is responsible for the activation of the lysozyme enhancer by Myb and showed that the v-Myb protein of AMV was unable to bind to this site.Our data demonstrate for the first time that oncogenic activation of Myb alters its DNA-binding specificity at a physiological Myb target gene.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie, Westfälische-Wilhelms-Universität Münster, Wilhelm-Klemm-Strasse 2, D-48149 Münster, Germany.

ABSTRACT
The oncoprotein v-Myb of avian myeloblastosis virus (AMV) transforms myelomonocytic cells by deregulating specific target genes. Previous work has shown that the oncogenic potential of v-Myb was activated by truncation of N- and C-terminal sequences of c-Myb and was further increased by amino acid substitutions in the DNA-binding domain and other parts of the protein. We have analyzed the activation of the chicken lysozyme gene which is strongly activated by c-Myb but not by its oncogenic counterpart v-Myb. We report that Myb acts on two different cis-regulatory elements, the promoter and an enhancer located upstream of the gene. Interestingly, the activation of the enhancer was abolished by the oncogenic amino acid substitutions. We demonstrated that a single Myb-binding site is responsible for the activation of the lysozyme enhancer by Myb and showed that the v-Myb protein of AMV was unable to bind to this site. Our data demonstrate for the first time that oncogenic activation of Myb alters its DNA-binding specificity at a physiological Myb target gene.

Show MeSH
Related in: MedlinePlus