Limits...
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.

Show MeSH

Related in: MedlinePlus

Myb-dependent activity of chicken lysozyme reporter genes. The lysozyme gene upstream region is shown schematically at the top. Black boxes indicate the promoter (P) or enhancer sequences (numbered boxes) that are covered by reporter genes. (A, B) HD11 cells were transfected with the indicated reporter genes and expression vector for v-MybREV (black columns) or empty vector (white columns). To control the transfection efficiency cells were additionally transfected with the β-galactosidase plasmid pCMVβ. Cells were analyzed for luciferase and β-galactosidase activities 24 h after transfection. The columns show the average luciferase activity (arbitrary units) normalized to the β-galactosidase activity. Thin lines show standard deviations. Panel B shows an identical experiment except that expression vector for v-MybAMV was used. (C) QT6 cells with the indicated reporter genes and expression vectors for v-MybAMV (hatched columns), v-MybREV (black columns) or empty expression vector (white columns). (D) Western blot of cells transfected with expression vectors for v-MybAMV (lane 1) and v-MybREV (lane 2) using Myb-specific antiserum (28). The arrow marks the v-Myb protein. (E) Schematic illustration of the structure of v-MybAMV and v-MybREV. Arrows mark point mutations of v-MybAMV relative to c-Myb. The three point mutation in the DNA-binding domain (DBD) that affects the ability of v-Myb to activate the lysozyme gene are highlighted with the numbering referring to c-Myb.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2175353&req=5

Figure 1: Myb-dependent activity of chicken lysozyme reporter genes. The lysozyme gene upstream region is shown schematically at the top. Black boxes indicate the promoter (P) or enhancer sequences (numbered boxes) that are covered by reporter genes. (A, B) HD11 cells were transfected with the indicated reporter genes and expression vector for v-MybREV (black columns) or empty vector (white columns). To control the transfection efficiency cells were additionally transfected with the β-galactosidase plasmid pCMVβ. Cells were analyzed for luciferase and β-galactosidase activities 24 h after transfection. The columns show the average luciferase activity (arbitrary units) normalized to the β-galactosidase activity. Thin lines show standard deviations. Panel B shows an identical experiment except that expression vector for v-MybAMV was used. (C) QT6 cells with the indicated reporter genes and expression vectors for v-MybAMV (hatched columns), v-MybREV (black columns) or empty expression vector (white columns). (D) Western blot of cells transfected with expression vectors for v-MybAMV (lane 1) and v-MybREV (lane 2) using Myb-specific antiserum (28). The arrow marks the v-Myb protein. (E) Schematic illustration of the structure of v-MybAMV and v-MybREV. Arrows mark point mutations of v-MybAMV relative to c-Myb. The three point mutation in the DNA-binding domain (DBD) that affects the ability of v-Myb to activate the lysozyme gene are highlighted with the numbering referring to c-Myb.

Mentions: To understand how Myb activates the expression of the chicken lysozyme gene we analyzed the influence of Myb on luciferase reporter genes containing the promoter or known cis-regulatory sequences of the chicken lysozyme gene. We used an expression vector encoding a modified version of v-Myb in which most of the AMV-specific amino acid substitutions had been reverted by replacing part of the coding region with the corresponding sequence of the c-myb gene. The structure of this modified v-Myb (referred to as v-MybREV) is shown schematically in Figure 1E. We have shown before that this modified version of v-Myb activates the endogenous chicken lysozyme gene, in contrast to v-MybAMV, which does not activate the gene (12). Reporter gene experiments were performed in the myelomonocytic cell line HD11 which lacks endogenous v-Myb or c-Myb. As shown in Figure 1A, v- MybREV strongly activated the lysozyme promoter and the reporter gene containing the −2.7 kb lysozyme enhancer. The −6.1 kb lysozyme enhancer was weakly activated and the −3.9 kb enhancer was not activated at all. We also performed experiments in parallel with the expression vector for v-MybAMV, reasoning that the direct comparison of the effects of both versions of v-Myb might provide clues about why the AMV version of v-Myb does not activate the lysozyme gene. As shown in Figure 1B, both Myb proteins displayed similar activities with the notable exception of the −2.7 kb enhancer which was not affected significantly by v-MybAMV. This difference was not due to different expression levels of both proteins (Figure 1D). Taken together, these experiments identified the promoter and the −2.7 kb enhancer as major Myb-responsive elements of the lysozyme gene. In addition, they demonstrated that the two versions of v-Myb display significantly different activities at the −2.7 kb lysozyme enhancer. These differences were also observed when co-transfections were performed in a fibroblast cell line, confirming that the presence of the amino acid substitutions in v-MybAMV has abolished the ability of the protein to stimulate the −2.7 kb enhancer (Figure 1C).Figure 1.


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)

Myb-dependent activity of chicken lysozyme reporter genes. The lysozyme gene upstream region is shown schematically at the top. Black boxes indicate the promoter (P) or enhancer sequences (numbered boxes) that are covered by reporter genes. (A, B) HD11 cells were transfected with the indicated reporter genes and expression vector for v-MybREV (black columns) or empty vector (white columns). To control the transfection efficiency cells were additionally transfected with the β-galactosidase plasmid pCMVβ. Cells were analyzed for luciferase and β-galactosidase activities 24 h after transfection. The columns show the average luciferase activity (arbitrary units) normalized to the β-galactosidase activity. Thin lines show standard deviations. Panel B shows an identical experiment except that expression vector for v-MybAMV was used. (C) QT6 cells with the indicated reporter genes and expression vectors for v-MybAMV (hatched columns), v-MybREV (black columns) or empty expression vector (white columns). (D) Western blot of cells transfected with expression vectors for v-MybAMV (lane 1) and v-MybREV (lane 2) using Myb-specific antiserum (28). The arrow marks the v-Myb protein. (E) Schematic illustration of the structure of v-MybAMV and v-MybREV. Arrows mark point mutations of v-MybAMV relative to c-Myb. The three point mutation in the DNA-binding domain (DBD) that affects the ability of v-Myb to activate the lysozyme gene are highlighted with the numbering referring to c-Myb.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175353&req=5

Figure 1: Myb-dependent activity of chicken lysozyme reporter genes. The lysozyme gene upstream region is shown schematically at the top. Black boxes indicate the promoter (P) or enhancer sequences (numbered boxes) that are covered by reporter genes. (A, B) HD11 cells were transfected with the indicated reporter genes and expression vector for v-MybREV (black columns) or empty vector (white columns). To control the transfection efficiency cells were additionally transfected with the β-galactosidase plasmid pCMVβ. Cells were analyzed for luciferase and β-galactosidase activities 24 h after transfection. The columns show the average luciferase activity (arbitrary units) normalized to the β-galactosidase activity. Thin lines show standard deviations. Panel B shows an identical experiment except that expression vector for v-MybAMV was used. (C) QT6 cells with the indicated reporter genes and expression vectors for v-MybAMV (hatched columns), v-MybREV (black columns) or empty expression vector (white columns). (D) Western blot of cells transfected with expression vectors for v-MybAMV (lane 1) and v-MybREV (lane 2) using Myb-specific antiserum (28). The arrow marks the v-Myb protein. (E) Schematic illustration of the structure of v-MybAMV and v-MybREV. Arrows mark point mutations of v-MybAMV relative to c-Myb. The three point mutation in the DNA-binding domain (DBD) that affects the ability of v-Myb to activate the lysozyme gene are highlighted with the numbering referring to c-Myb.
Mentions: To understand how Myb activates the expression of the chicken lysozyme gene we analyzed the influence of Myb on luciferase reporter genes containing the promoter or known cis-regulatory sequences of the chicken lysozyme gene. We used an expression vector encoding a modified version of v-Myb in which most of the AMV-specific amino acid substitutions had been reverted by replacing part of the coding region with the corresponding sequence of the c-myb gene. The structure of this modified v-Myb (referred to as v-MybREV) is shown schematically in Figure 1E. We have shown before that this modified version of v-Myb activates the endogenous chicken lysozyme gene, in contrast to v-MybAMV, which does not activate the gene (12). Reporter gene experiments were performed in the myelomonocytic cell line HD11 which lacks endogenous v-Myb or c-Myb. As shown in Figure 1A, v- MybREV strongly activated the lysozyme promoter and the reporter gene containing the −2.7 kb lysozyme enhancer. The −6.1 kb lysozyme enhancer was weakly activated and the −3.9 kb enhancer was not activated at all. We also performed experiments in parallel with the expression vector for v-MybAMV, reasoning that the direct comparison of the effects of both versions of v-Myb might provide clues about why the AMV version of v-Myb does not activate the lysozyme gene. As shown in Figure 1B, both Myb proteins displayed similar activities with the notable exception of the −2.7 kb enhancer which was not affected significantly by v-MybAMV. This difference was not due to different expression levels of both proteins (Figure 1D). Taken together, these experiments identified the promoter and the −2.7 kb enhancer as major Myb-responsive elements of the lysozyme gene. In addition, they demonstrated that the two versions of v-Myb display significantly different activities at the −2.7 kb lysozyme enhancer. These differences were also observed when co-transfections were performed in a fibroblast cell line, confirming that the presence of the amino acid substitutions in v-MybAMV has abolished the ability of the protein to stimulate the −2.7 kb enhancer (Figure 1C).Figure 1.

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