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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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Electrophoretic mobility shift experiments. (A) Western blot analysis of the bacterially expressed v-MybREV and v-MybAMV proteins. The amounts (in μl) loaded onto the gel are shown at the bottom. Full-length Myb protein is marked by an arrow. (B) Binding reactions contained the radiolabeled double-stranded oligonucleotides shown and bacterially expressed Myb protein, as indicated above and below the lanes. The numbers below the lanes refer to the amounts (in μl) of protein added. Control reactions contained no Myb protein. Protein–DNA complexes were visualized by electrophoresis on native polyacrylamide gels followed by autoradiography. The black and white arrowheads mark complexes of full-length v-Myb and a proteolytic degradation product, respectively.
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Figure 6: Electrophoretic mobility shift experiments. (A) Western blot analysis of the bacterially expressed v-MybREV and v-MybAMV proteins. The amounts (in μl) loaded onto the gel are shown at the bottom. Full-length Myb protein is marked by an arrow. (B) Binding reactions contained the radiolabeled double-stranded oligonucleotides shown and bacterially expressed Myb protein, as indicated above and below the lanes. The numbers below the lanes refer to the amounts (in μl) of protein added. Control reactions contained no Myb protein. Protein–DNA complexes were visualized by electrophoresis on native polyacrylamide gels followed by autoradiography. The black and white arrowheads mark complexes of full-length v-Myb and a proteolytic degradation product, respectively.

Mentions: The data illustrated in Figure 1 demonstrated that v-MybREV, in which most of the amino acid substitutions found in the v-Myb protein of AMV had been reverted, activated the −2.7 kb lysozyme enhancer, while v-MybAMV was unable to do so. Since our work had shown Myb-binding site 3 to play a key role in the activation of the enhancer, we wondered whether or not v-MybAMV was able to bind to this site. In the light of the current model of the Myb DNA-binding domain (37) a direct effect of the substitutions on DNA binding seemed unlikely because the amino acid residues that have been substituted in the DNA-binding domain of AMV v-Myb point away from the bound DNA. On the other hand, by using artificial sites that were systematically altered in certain positions of the Myb consensus binding motif Brendeford et al. (38) have shown that the AMV-specific amino acid substitutions indeed can have some effect on the DNA-binding activity of the Myb DNA-binding domain. To investigate whether the amino acid substitutions in the DNA-binding domain of AMV-v-Myb affect the ability to recognize binding site 3 of the −2.7 kb enhancer, we performed electrophoretic mobility shift assays using bacterially expressed v-Myb proteins containing or lacking AMV-specific amino acid substitutions within the DNA-binding domain. The experiment shown in Figure 6A demonstrated that bacterially expressed v-Myb, lacking AMV-specific amino acid substitutions within the DNA-binding domain, was indeed able to recognize Myb-binding site 3 in vitro. Binding to this site was weaker than to the Myb binding site A of the mim-1 promoter, which was expected because binding site 3 of the lysozyme enhancer differs significantly from the Myb consensus binding motif. Mutation of binding site 3 abolished binding of Myb, consistent with the observation that mutation of this site abolished the activation of the enhancer by Myb. In the experiment illustrated in Figure 6B we compared the ability of both versions of Myb to bind to the mim-1 A site and to Myb-binding site 3. Both proteins recognized the mim-1 A site equally well; however, the v-Myb protein containing the AMV-specific amino acid substitutions was virtually unable to bind to Myb-binding site 3. This observation clearly demonstrated that the amino acid substitutions in the DNA-binding domain of AMV v-Myb have affected the sequence specificity of the protein in such a way that it no longer recognizes Myb-binding site 3 of the lysozyme enhancer. This lack of binding provides a straightforward explanation for the inability of v-MybAMV to activate the lysozyme enhancer.Figure 6.


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)

Electrophoretic mobility shift experiments. (A) Western blot analysis of the bacterially expressed v-MybREV and v-MybAMV proteins. The amounts (in μl) loaded onto the gel are shown at the bottom. Full-length Myb protein is marked by an arrow. (B) Binding reactions contained the radiolabeled double-stranded oligonucleotides shown and bacterially expressed Myb protein, as indicated above and below the lanes. The numbers below the lanes refer to the amounts (in μl) of protein added. Control reactions contained no Myb protein. Protein–DNA complexes were visualized by electrophoresis on native polyacrylamide gels followed by autoradiography. The black and white arrowheads mark complexes of full-length v-Myb and a proteolytic degradation product, respectively.
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Figure 6: Electrophoretic mobility shift experiments. (A) Western blot analysis of the bacterially expressed v-MybREV and v-MybAMV proteins. The amounts (in μl) loaded onto the gel are shown at the bottom. Full-length Myb protein is marked by an arrow. (B) Binding reactions contained the radiolabeled double-stranded oligonucleotides shown and bacterially expressed Myb protein, as indicated above and below the lanes. The numbers below the lanes refer to the amounts (in μl) of protein added. Control reactions contained no Myb protein. Protein–DNA complexes were visualized by electrophoresis on native polyacrylamide gels followed by autoradiography. The black and white arrowheads mark complexes of full-length v-Myb and a proteolytic degradation product, respectively.
Mentions: The data illustrated in Figure 1 demonstrated that v-MybREV, in which most of the amino acid substitutions found in the v-Myb protein of AMV had been reverted, activated the −2.7 kb lysozyme enhancer, while v-MybAMV was unable to do so. Since our work had shown Myb-binding site 3 to play a key role in the activation of the enhancer, we wondered whether or not v-MybAMV was able to bind to this site. In the light of the current model of the Myb DNA-binding domain (37) a direct effect of the substitutions on DNA binding seemed unlikely because the amino acid residues that have been substituted in the DNA-binding domain of AMV v-Myb point away from the bound DNA. On the other hand, by using artificial sites that were systematically altered in certain positions of the Myb consensus binding motif Brendeford et al. (38) have shown that the AMV-specific amino acid substitutions indeed can have some effect on the DNA-binding activity of the Myb DNA-binding domain. To investigate whether the amino acid substitutions in the DNA-binding domain of AMV-v-Myb affect the ability to recognize binding site 3 of the −2.7 kb enhancer, we performed electrophoretic mobility shift assays using bacterially expressed v-Myb proteins containing or lacking AMV-specific amino acid substitutions within the DNA-binding domain. The experiment shown in Figure 6A demonstrated that bacterially expressed v-Myb, lacking AMV-specific amino acid substitutions within the DNA-binding domain, was indeed able to recognize Myb-binding site 3 in vitro. Binding to this site was weaker than to the Myb binding site A of the mim-1 promoter, which was expected because binding site 3 of the lysozyme enhancer differs significantly from the Myb consensus binding motif. Mutation of binding site 3 abolished binding of Myb, consistent with the observation that mutation of this site abolished the activation of the enhancer by Myb. In the experiment illustrated in Figure 6B we compared the ability of both versions of Myb to bind to the mim-1 A site and to Myb-binding site 3. Both proteins recognized the mim-1 A site equally well; however, the v-Myb protein containing the AMV-specific amino acid substitutions was virtually unable to bind to Myb-binding site 3. This observation clearly demonstrated that the amino acid substitutions in the DNA-binding domain of AMV v-Myb have affected the sequence specificity of the protein in such a way that it no longer recognizes Myb-binding site 3 of the lysozyme enhancer. This lack of binding provides a straightforward explanation for the inability of v-MybAMV to activate the lysozyme enhancer.Figure 6.

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