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Identification and regulation of c-Myb target genes in MCF-7 cells.

Quintana AM, Liu F, O'Rourke JP, Ness SA - BMC Cancer (2011)

Bottom Line: By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs.Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1.Our results show that c-Myb associates with a surprisingly large number of promoters in human cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131-0001, USA.

ABSTRACT

Background: The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Although oncogenic versions of c-Myb were first associated with leukemias, over expression or rearrangement of the c-myb gene is common in several types of solid tumors, including breast cancers. Expression of the c-myb gene in human breast cancer cells is dependent on estrogen stimulation, but little is known about the activities of the c-Myb protein or what genes it regulates in estrogen-stimulated cells.

Methods: We used chromatin immunoprecipitation coupled with whole genome promoter tiling microarrays to identify endogenous c-Myb target genes in human MCF-7 breast cancer cells and characterized the activity of c-Myb at a panel of target genes during different stages of estrogen deprivation and stimulation.

Results: By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs. Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1. By studying a panel of these targets to validate the results, we found that estradiol stimulation triggered the association of c-Myb with promoters and that association correlated with increased target gene expression. We studied one target gene, CXCR4, in detail, showing that c-Myb associated with the CXCR4 gene promoter and activated a CXCR4 reporter gene in transfection assays.

Conclusions: Our results show that c-Myb associates with a surprisingly large number of promoters in human cells. The results also suggest that estradiol stimulation leads to large-scale, genome-wide changes in c-Myb activity and subsequent changes in gene expression in human breast cancer cells.

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The CXCR4 promoter is regulated by Myb proteins. (A) Structures of c-Myb and v-Myb proteins. The diagrams depict the structures of the c-Myb and v-Myb proteins, which share conserved domains (shaded) involved in DNA binding and regulation. The oncogenic v-Myb protein is truncated at both ends and has a number of point mutations represented by white dots. The locations of the epitopes for antibodies (Abs) 1493 and 1.1 are indicated. (B) Structure of the CXCR4 gene promoter. The region upstream of the human CXCR4 gene is diagrammed, with putative Myb binding sites indicated by gray boxes. The arrow indicates the start site and direction of transcription. (C) Activation of a CXCR4 reporter gene. A reporter construct containing the CXCR4 promoter upstream of the luciferase reporter gene was co-transfected into HEK293 cells along with control plasmid (vector) or plasmids expressing c-Myb or v-Myb, as indicated. The figure shows reporter gene activity. Error bars show standard deviation of triplicate assays. (D) MCF-7 cells were transduced with a lentivirus expressing FLAG-tagged v-Myb. ChIP was performed with anti-FLAG antibodies (gray Bars) or control IgG (black bars). QPCR was performed with primers specific to CXCR4 and GAPDH. Data is normalized relative to percent of input. Error bars represent standard deviation of triplicate PCR reactions.
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Figure 5: The CXCR4 promoter is regulated by Myb proteins. (A) Structures of c-Myb and v-Myb proteins. The diagrams depict the structures of the c-Myb and v-Myb proteins, which share conserved domains (shaded) involved in DNA binding and regulation. The oncogenic v-Myb protein is truncated at both ends and has a number of point mutations represented by white dots. The locations of the epitopes for antibodies (Abs) 1493 and 1.1 are indicated. (B) Structure of the CXCR4 gene promoter. The region upstream of the human CXCR4 gene is diagrammed, with putative Myb binding sites indicated by gray boxes. The arrow indicates the start site and direction of transcription. (C) Activation of a CXCR4 reporter gene. A reporter construct containing the CXCR4 promoter upstream of the luciferase reporter gene was co-transfected into HEK293 cells along with control plasmid (vector) or plasmids expressing c-Myb or v-Myb, as indicated. The figure shows reporter gene activity. Error bars show standard deviation of triplicate assays. (D) MCF-7 cells were transduced with a lentivirus expressing FLAG-tagged v-Myb. ChIP was performed with anti-FLAG antibodies (gray Bars) or control IgG (black bars). QPCR was performed with primers specific to CXCR4 and GAPDH. Data is normalized relative to percent of input. Error bars represent standard deviation of triplicate PCR reactions.

Mentions: Previous work from our laboratory [18] showed that CXCR4 gene expression increased substantially following ectopic expression of either c-Myb or its oncogenic derivative v-Myb (Figure 5A). We isolated and analyzed the human CXCR4 gene promoter and used it to assemble a luciferase-based reporter gene construct (Figure 5B). As shown in Figure 5C, both c-Myb and v-Myb activated the reporter gene when they were co-transfected into HEK293 cells (which have very low endogenous c-Myb levels) but v-Myb had more activity at the CXCR4 promoter. These results suggest that both c-Myb and v-Myb regulate the expression of the CXCR4 gene by directly binding its promoter. Finally, to confirm that v-Myb also interacted with the endogenous CXCR4 promoter in MCF-7 cells, we generated MCF-7 cells expressing a FLAG epitope-tagged version of v-Myb (Additional file 1: Figure S2), then did a ChIP assay using anti-FLAG antibodies as described in the previous sections. As shown in Figure 5D, the FLAG-tagged v-Myb bound to the CXCR4 promoter, but not to an endogenous control promoter, GAPDH. Taken together, these results confirm that the CXCR4 gene is regulated by both normal and oncogenic variants of c-Myb in human cells.


Identification and regulation of c-Myb target genes in MCF-7 cells.

Quintana AM, Liu F, O'Rourke JP, Ness SA - BMC Cancer (2011)

The CXCR4 promoter is regulated by Myb proteins. (A) Structures of c-Myb and v-Myb proteins. The diagrams depict the structures of the c-Myb and v-Myb proteins, which share conserved domains (shaded) involved in DNA binding and regulation. The oncogenic v-Myb protein is truncated at both ends and has a number of point mutations represented by white dots. The locations of the epitopes for antibodies (Abs) 1493 and 1.1 are indicated. (B) Structure of the CXCR4 gene promoter. The region upstream of the human CXCR4 gene is diagrammed, with putative Myb binding sites indicated by gray boxes. The arrow indicates the start site and direction of transcription. (C) Activation of a CXCR4 reporter gene. A reporter construct containing the CXCR4 promoter upstream of the luciferase reporter gene was co-transfected into HEK293 cells along with control plasmid (vector) or plasmids expressing c-Myb or v-Myb, as indicated. The figure shows reporter gene activity. Error bars show standard deviation of triplicate assays. (D) MCF-7 cells were transduced with a lentivirus expressing FLAG-tagged v-Myb. ChIP was performed with anti-FLAG antibodies (gray Bars) or control IgG (black bars). QPCR was performed with primers specific to CXCR4 and GAPDH. Data is normalized relative to percent of input. Error bars represent standard deviation of triplicate PCR reactions.
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Figure 5: The CXCR4 promoter is regulated by Myb proteins. (A) Structures of c-Myb and v-Myb proteins. The diagrams depict the structures of the c-Myb and v-Myb proteins, which share conserved domains (shaded) involved in DNA binding and regulation. The oncogenic v-Myb protein is truncated at both ends and has a number of point mutations represented by white dots. The locations of the epitopes for antibodies (Abs) 1493 and 1.1 are indicated. (B) Structure of the CXCR4 gene promoter. The region upstream of the human CXCR4 gene is diagrammed, with putative Myb binding sites indicated by gray boxes. The arrow indicates the start site and direction of transcription. (C) Activation of a CXCR4 reporter gene. A reporter construct containing the CXCR4 promoter upstream of the luciferase reporter gene was co-transfected into HEK293 cells along with control plasmid (vector) or plasmids expressing c-Myb or v-Myb, as indicated. The figure shows reporter gene activity. Error bars show standard deviation of triplicate assays. (D) MCF-7 cells were transduced with a lentivirus expressing FLAG-tagged v-Myb. ChIP was performed with anti-FLAG antibodies (gray Bars) or control IgG (black bars). QPCR was performed with primers specific to CXCR4 and GAPDH. Data is normalized relative to percent of input. Error bars represent standard deviation of triplicate PCR reactions.
Mentions: Previous work from our laboratory [18] showed that CXCR4 gene expression increased substantially following ectopic expression of either c-Myb or its oncogenic derivative v-Myb (Figure 5A). We isolated and analyzed the human CXCR4 gene promoter and used it to assemble a luciferase-based reporter gene construct (Figure 5B). As shown in Figure 5C, both c-Myb and v-Myb activated the reporter gene when they were co-transfected into HEK293 cells (which have very low endogenous c-Myb levels) but v-Myb had more activity at the CXCR4 promoter. These results suggest that both c-Myb and v-Myb regulate the expression of the CXCR4 gene by directly binding its promoter. Finally, to confirm that v-Myb also interacted with the endogenous CXCR4 promoter in MCF-7 cells, we generated MCF-7 cells expressing a FLAG epitope-tagged version of v-Myb (Additional file 1: Figure S2), then did a ChIP assay using anti-FLAG antibodies as described in the previous sections. As shown in Figure 5D, the FLAG-tagged v-Myb bound to the CXCR4 promoter, but not to an endogenous control promoter, GAPDH. Taken together, these results confirm that the CXCR4 gene is regulated by both normal and oncogenic variants of c-Myb in human cells.

Bottom Line: By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs.Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1.Our results show that c-Myb associates with a surprisingly large number of promoters in human cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131-0001, USA.

ABSTRACT

Background: The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Although oncogenic versions of c-Myb were first associated with leukemias, over expression or rearrangement of the c-myb gene is common in several types of solid tumors, including breast cancers. Expression of the c-myb gene in human breast cancer cells is dependent on estrogen stimulation, but little is known about the activities of the c-Myb protein or what genes it regulates in estrogen-stimulated cells.

Methods: We used chromatin immunoprecipitation coupled with whole genome promoter tiling microarrays to identify endogenous c-Myb target genes in human MCF-7 breast cancer cells and characterized the activity of c-Myb at a panel of target genes during different stages of estrogen deprivation and stimulation.

Results: By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs. Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1. By studying a panel of these targets to validate the results, we found that estradiol stimulation triggered the association of c-Myb with promoters and that association correlated with increased target gene expression. We studied one target gene, CXCR4, in detail, showing that c-Myb associated with the CXCR4 gene promoter and activated a CXCR4 reporter gene in transfection assays.

Conclusions: Our results show that c-Myb associates with a surprisingly large number of promoters in human cells. The results also suggest that estradiol stimulation leads to large-scale, genome-wide changes in c-Myb activity and subsequent changes in gene expression in human breast cancer cells.

Show MeSH
Related in: MedlinePlus