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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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Related in: MedlinePlus

Chromatin-immunoprecipitation experiment. (A) Western blot analysis of Myb expression in HD11-E cells grown in the presence or absence of doxycyclin. The black arrow marks the v-MybREV protein. (B) Chromatin immunoprecipitation experiment using HD11-E cells grown in the presence (left part) or absence (right part) of doxycyclin. Immunoprecipitation was carried out using two different Myb-specific antisera (lanes 1 and 2), non-immune serum (lane 3) or no antiserum (lane 4). Lane 5 shows no-template controls and lane 6 shows input controls. PCR primers were specific for the lysozyme gene promoter and the −2.7 kb enhancer. PCR products were analyzed by agarose gel electrophoresis.
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Figure 2: Chromatin-immunoprecipitation experiment. (A) Western blot analysis of Myb expression in HD11-E cells grown in the presence or absence of doxycyclin. The black arrow marks the v-MybREV protein. (B) Chromatin immunoprecipitation experiment using HD11-E cells grown in the presence (left part) or absence (right part) of doxycyclin. Immunoprecipitation was carried out using two different Myb-specific antisera (lanes 1 and 2), non-immune serum (lane 3) or no antiserum (lane 4). Lane 5 shows no-template controls and lane 6 shows input controls. PCR primers were specific for the lysozyme gene promoter and the −2.7 kb enhancer. PCR products were analyzed by agarose gel electrophoresis.

Mentions: To confirm that Myb targets two different cis-acting regions of the lysozyme gene we performed chromatin immunoprecipitation experiments using a stable transfectant of the myelomonocytic HD11 cell line in which the expression of v-MybREV is induced by growing the cells in the presence of doxycyclin. Western blotting showed that the Myb protein was expressed in the presence but not in the absence of doxycyclin (Figure 2A). The chromatin immunoprecipitation experiment illustrated in Figure 2B confirmed the binding of Myb to the lysozyme promoter and the −2.7 kb enhancer following induction by doxycyclin. This experiment, therefore, supported the notion that Myb activates the lysozyme gene by targeting two different regions of the gene.Figure 2.


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)

Chromatin-immunoprecipitation experiment. (A) Western blot analysis of Myb expression in HD11-E cells grown in the presence or absence of doxycyclin. The black arrow marks the v-MybREV protein. (B) Chromatin immunoprecipitation experiment using HD11-E cells grown in the presence (left part) or absence (right part) of doxycyclin. Immunoprecipitation was carried out using two different Myb-specific antisera (lanes 1 and 2), non-immune serum (lane 3) or no antiserum (lane 4). Lane 5 shows no-template controls and lane 6 shows input controls. PCR primers were specific for the lysozyme gene promoter and the −2.7 kb enhancer. PCR products were analyzed by agarose gel electrophoresis.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Chromatin-immunoprecipitation experiment. (A) Western blot analysis of Myb expression in HD11-E cells grown in the presence or absence of doxycyclin. The black arrow marks the v-MybREV protein. (B) Chromatin immunoprecipitation experiment using HD11-E cells grown in the presence (left part) or absence (right part) of doxycyclin. Immunoprecipitation was carried out using two different Myb-specific antisera (lanes 1 and 2), non-immune serum (lane 3) or no antiserum (lane 4). Lane 5 shows no-template controls and lane 6 shows input controls. PCR primers were specific for the lysozyme gene promoter and the −2.7 kb enhancer. PCR products were analyzed by agarose gel electrophoresis.
Mentions: To confirm that Myb targets two different cis-acting regions of the lysozyme gene we performed chromatin immunoprecipitation experiments using a stable transfectant of the myelomonocytic HD11 cell line in which the expression of v-MybREV is induced by growing the cells in the presence of doxycyclin. Western blotting showed that the Myb protein was expressed in the presence but not in the absence of doxycyclin (Figure 2A). The chromatin immunoprecipitation experiment illustrated in Figure 2B confirmed the binding of Myb to the lysozyme promoter and the −2.7 kb enhancer following induction by doxycyclin. This experiment, therefore, supported the notion that Myb activates the lysozyme gene by targeting two different regions of the gene.Figure 2.

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