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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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Identification of a functional Myb-binding site in the −2.7 kb enhancer. (A) QT6 cells were transfected with the reporter gene pTATA-2.7 and expression vector for v-MybREV (wt-Myb), the N186A mutation of v-MybREV (mut-Myb) or empty expression vector (no Myb), as indicated below the columns. To control the transfection efficiencies cells were additionally transfected with the β-galactosidase plasmid pCMVβ. Luciferase and β-galactosidase activities were determined 24 h after transfection. The luciferase activity was normalized to the β-galactosidase activity and is expressed in arbitrary units. The activity of the reporter gene in the absence of v-Myb was designated as 1. Thin lines show standard deviations. The insert at the top shows a western blot analysis of v-Myb expression in cells transfected with the same expression vectors. (B) The figure at the top illustrates the position of Ets (E), C/EBP (C) and potential binding sites for Myb (M) in the −2.7 kb enhancer. The sequences of the four potential Myb-binding sites are shown below. Results of reporter gene assays, performed in QT6 cells, are shown at the bottom. pTATA (no enh.), pTATA-2.7 (wt) or derivatives of this plasmid carrying point mutations in one of the four Myb-binding sites (mut1-4) were transfected with expression vector for v-MybREV. Transfections were analyzed as in (A). (C) Reporter gene assays were performed in QT6 cells using the full-length or a truncated −2.7 kb enhancer construct. Cells were additionally transfected with expression vector for v-MybREV (black bars) or empty expression vector (white bars). Transfections were analyzed as in (A).
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Figure 4: Identification of a functional Myb-binding site in the −2.7 kb enhancer. (A) QT6 cells were transfected with the reporter gene pTATA-2.7 and expression vector for v-MybREV (wt-Myb), the N186A mutation of v-MybREV (mut-Myb) or empty expression vector (no Myb), as indicated below the columns. To control the transfection efficiencies cells were additionally transfected with the β-galactosidase plasmid pCMVβ. Luciferase and β-galactosidase activities were determined 24 h after transfection. The luciferase activity was normalized to the β-galactosidase activity and is expressed in arbitrary units. The activity of the reporter gene in the absence of v-Myb was designated as 1. Thin lines show standard deviations. The insert at the top shows a western blot analysis of v-Myb expression in cells transfected with the same expression vectors. (B) The figure at the top illustrates the position of Ets (E), C/EBP (C) and potential binding sites for Myb (M) in the −2.7 kb enhancer. The sequences of the four potential Myb-binding sites are shown below. Results of reporter gene assays, performed in QT6 cells, are shown at the bottom. pTATA (no enh.), pTATA-2.7 (wt) or derivatives of this plasmid carrying point mutations in one of the four Myb-binding sites (mut1-4) were transfected with expression vector for v-MybREV. Transfections were analyzed as in (A). (C) Reporter gene assays were performed in QT6 cells using the full-length or a truncated −2.7 kb enhancer construct. Cells were additionally transfected with expression vector for v-MybREV (black bars) or empty expression vector (white bars). Transfections were analyzed as in (A).

Mentions: To further understand the mechanism by which Myb activates the −2.7 kb lysozyme enhancer we examined the enhancer for Myb-binding sites which mediate the stimulation by Myb. As a first step we performed a transactivation experiment using a mutant of v-MybREV which lacks DNA-binding activity. In this mutant a single amino acid residue (Asp186), which is directly involved in contacts with specific bases of the Myb recognition motif and has been shown to be crucial for the specific DNA-binding activity of Myb (36,37), was mutated to alanine. As shown in Figure 4A, the mutant Myb protein failed to stimulate the enhancer, consistent with the notion that one or several Myb-binding sites mediate the effect of Myb. We then examined the sequence of the enhancer for potential Myb-binding sites. There are four GTT- (or AAC- in the reverse orientation) motifs within the enhancer sequence, which conform to the central core of the Myb-binding site. One of these motifs (designated as site 1 in Figure 4B) showed a good match to the Myb consensus site (PyAACT/GG) whereas the other sites were more distantly related. We mutated each of these motifs and examined the ability of v-MybREV to activate the mutated enhancers. Figure 4B shows that only mutation of site 3 significantly reduced the stimulation of the enhancer by Myb. This suggested that binding site 3 plays a key role in the Myb-dependent activation of the enhancer. We also constructed a truncated version of the enhancer and found that its stimulation by v-MybREV was essentially identical to that of the full-length enhancer (Figure 4C).Figure 4.


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)

Identification of a functional Myb-binding site in the −2.7 kb enhancer. (A) QT6 cells were transfected with the reporter gene pTATA-2.7 and expression vector for v-MybREV (wt-Myb), the N186A mutation of v-MybREV (mut-Myb) or empty expression vector (no Myb), as indicated below the columns. To control the transfection efficiencies cells were additionally transfected with the β-galactosidase plasmid pCMVβ. Luciferase and β-galactosidase activities were determined 24 h after transfection. The luciferase activity was normalized to the β-galactosidase activity and is expressed in arbitrary units. The activity of the reporter gene in the absence of v-Myb was designated as 1. Thin lines show standard deviations. The insert at the top shows a western blot analysis of v-Myb expression in cells transfected with the same expression vectors. (B) The figure at the top illustrates the position of Ets (E), C/EBP (C) and potential binding sites for Myb (M) in the −2.7 kb enhancer. The sequences of the four potential Myb-binding sites are shown below. Results of reporter gene assays, performed in QT6 cells, are shown at the bottom. pTATA (no enh.), pTATA-2.7 (wt) or derivatives of this plasmid carrying point mutations in one of the four Myb-binding sites (mut1-4) were transfected with expression vector for v-MybREV. Transfections were analyzed as in (A). (C) Reporter gene assays were performed in QT6 cells using the full-length or a truncated −2.7 kb enhancer construct. Cells were additionally transfected with expression vector for v-MybREV (black bars) or empty expression vector (white bars). Transfections were analyzed as in (A).
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Figure 4: Identification of a functional Myb-binding site in the −2.7 kb enhancer. (A) QT6 cells were transfected with the reporter gene pTATA-2.7 and expression vector for v-MybREV (wt-Myb), the N186A mutation of v-MybREV (mut-Myb) or empty expression vector (no Myb), as indicated below the columns. To control the transfection efficiencies cells were additionally transfected with the β-galactosidase plasmid pCMVβ. Luciferase and β-galactosidase activities were determined 24 h after transfection. The luciferase activity was normalized to the β-galactosidase activity and is expressed in arbitrary units. The activity of the reporter gene in the absence of v-Myb was designated as 1. Thin lines show standard deviations. The insert at the top shows a western blot analysis of v-Myb expression in cells transfected with the same expression vectors. (B) The figure at the top illustrates the position of Ets (E), C/EBP (C) and potential binding sites for Myb (M) in the −2.7 kb enhancer. The sequences of the four potential Myb-binding sites are shown below. Results of reporter gene assays, performed in QT6 cells, are shown at the bottom. pTATA (no enh.), pTATA-2.7 (wt) or derivatives of this plasmid carrying point mutations in one of the four Myb-binding sites (mut1-4) were transfected with expression vector for v-MybREV. Transfections were analyzed as in (A). (C) Reporter gene assays were performed in QT6 cells using the full-length or a truncated −2.7 kb enhancer construct. Cells were additionally transfected with expression vector for v-MybREV (black bars) or empty expression vector (white bars). Transfections were analyzed as in (A).
Mentions: To further understand the mechanism by which Myb activates the −2.7 kb lysozyme enhancer we examined the enhancer for Myb-binding sites which mediate the stimulation by Myb. As a first step we performed a transactivation experiment using a mutant of v-MybREV which lacks DNA-binding activity. In this mutant a single amino acid residue (Asp186), which is directly involved in contacts with specific bases of the Myb recognition motif and has been shown to be crucial for the specific DNA-binding activity of Myb (36,37), was mutated to alanine. As shown in Figure 4A, the mutant Myb protein failed to stimulate the enhancer, consistent with the notion that one or several Myb-binding sites mediate the effect of Myb. We then examined the sequence of the enhancer for potential Myb-binding sites. There are four GTT- (or AAC- in the reverse orientation) motifs within the enhancer sequence, which conform to the central core of the Myb-binding site. One of these motifs (designated as site 1 in Figure 4B) showed a good match to the Myb consensus site (PyAACT/GG) whereas the other sites were more distantly related. We mutated each of these motifs and examined the ability of v-MybREV to activate the mutated enhancers. Figure 4B shows that only mutation of site 3 significantly reduced the stimulation of the enhancer by Myb. This suggested that binding site 3 plays a key role in the Myb-dependent activation of the enhancer. We also constructed a truncated version of the enhancer and found that its stimulation by v-MybREV was essentially identical to that of the full-length enhancer (Figure 4C).Figure 4.

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