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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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Estrogen changes c-Myb activity. (A) MCF-7 cells were deprived of estrogen for 48 hr then stimulated with 10 nM 17-beta-estradiol for 6, 12 or 24 hours. QPCR was used to measure the levels of CCNB1, CXCR4, JUN, KLF4 and EPB41 RNAs. Error bars show standard deviation in triplicate PCR reactions, and results are relative to the estrogen-deprived cells. (B) MCF-7 cells were transduced with retroviral vectors expressing doxycycline inducible shRNAs (scrambled or c-myb specific). Each cell line was induced for 24 hr with doxycycline and the relative expression of c-myb, CCNB1, CXCR4, JUN, KLF4 and EPB41 RNAs was measured by QPCR. Data are normalized to the scrambled control shRNA.
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Figure 4: Estrogen changes c-Myb activity. (A) MCF-7 cells were deprived of estrogen for 48 hr then stimulated with 10 nM 17-beta-estradiol for 6, 12 or 24 hours. QPCR was used to measure the levels of CCNB1, CXCR4, JUN, KLF4 and EPB41 RNAs. Error bars show standard deviation in triplicate PCR reactions, and results are relative to the estrogen-deprived cells. (B) MCF-7 cells were transduced with retroviral vectors expressing doxycycline inducible shRNAs (scrambled or c-myb specific). Each cell line was induced for 24 hr with doxycycline and the relative expression of c-myb, CCNB1, CXCR4, JUN, KLF4 and EPB41 RNAs was measured by QPCR. Data are normalized to the scrambled control shRNA.

Mentions: To address the impact of c-Myb binding to the target gene promoters, we performed a time course experiment with cells deprived of estrogen or treated with beta-estradiol for up to 24 hr and analyzed the relative expression of each of the genes characterized above. Interestingly, each of the target genes described above displayed a different pattern of expression in the time course experiment. Compared to the estrogen-deprived cells, the CCNB1 gene showed no response at 6 hr but was about 5-fold induced at 12 hr and returned to baseline expression by 24 hr (Figure 4A). CXCR4 was about 3-fold induced at 6 and at 12 hr, but was nearly 7-fold induced by 24 hr. JUN showed no induction at 6 or 12 hr, but was strongly activated at 24 hr. The KLF4 gene showed no response at 6 hr, but was induced 4-fold by 12 hr and more than 8-fold at 24 hr. Only the EPB41 gene failed to be activated following beta-estradiol stimulation. Although c-Myb was bound to each of these target promoters after addition of beta-estradiol, the genes responded quite differently, suggesting that, at least for some genes, changes in the binding of c-Myb to the promoters was not sufficient to cause increased expression.


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

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

Estrogen changes c-Myb activity. (A) MCF-7 cells were deprived of estrogen for 48 hr then stimulated with 10 nM 17-beta-estradiol for 6, 12 or 24 hours. QPCR was used to measure the levels of CCNB1, CXCR4, JUN, KLF4 and EPB41 RNAs. Error bars show standard deviation in triplicate PCR reactions, and results are relative to the estrogen-deprived cells. (B) MCF-7 cells were transduced with retroviral vectors expressing doxycycline inducible shRNAs (scrambled or c-myb specific). Each cell line was induced for 24 hr with doxycycline and the relative expression of c-myb, CCNB1, CXCR4, JUN, KLF4 and EPB41 RNAs was measured by QPCR. Data are normalized to the scrambled control shRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3038977&req=5

Figure 4: Estrogen changes c-Myb activity. (A) MCF-7 cells were deprived of estrogen for 48 hr then stimulated with 10 nM 17-beta-estradiol for 6, 12 or 24 hours. QPCR was used to measure the levels of CCNB1, CXCR4, JUN, KLF4 and EPB41 RNAs. Error bars show standard deviation in triplicate PCR reactions, and results are relative to the estrogen-deprived cells. (B) MCF-7 cells were transduced with retroviral vectors expressing doxycycline inducible shRNAs (scrambled or c-myb specific). Each cell line was induced for 24 hr with doxycycline and the relative expression of c-myb, CCNB1, CXCR4, JUN, KLF4 and EPB41 RNAs was measured by QPCR. Data are normalized to the scrambled control shRNA.
Mentions: To address the impact of c-Myb binding to the target gene promoters, we performed a time course experiment with cells deprived of estrogen or treated with beta-estradiol for up to 24 hr and analyzed the relative expression of each of the genes characterized above. Interestingly, each of the target genes described above displayed a different pattern of expression in the time course experiment. Compared to the estrogen-deprived cells, the CCNB1 gene showed no response at 6 hr but was about 5-fold induced at 12 hr and returned to baseline expression by 24 hr (Figure 4A). CXCR4 was about 3-fold induced at 6 and at 12 hr, but was nearly 7-fold induced by 24 hr. JUN showed no induction at 6 or 12 hr, but was strongly activated at 24 hr. The KLF4 gene showed no response at 6 hr, but was induced 4-fold by 12 hr and more than 8-fold at 24 hr. Only the EPB41 gene failed to be activated following beta-estradiol stimulation. Although c-Myb was bound to each of these target promoters after addition of beta-estradiol, the genes responded quite differently, suggesting that, at least for some genes, changes in the binding of c-Myb to the promoters was not sufficient to cause increased expression.

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