Abstract
BackgroundThe 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.MethodsWe 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.ResultsBy 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.ConclusionsOur 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.
Highlights
The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells
Expression of the c-myb (MYB) gene is associated with expression of estrogen receptors (ERs) in breast tumors [2,3]. (Note: We use c-Myb and c-myb to distinguish between the protein and gene, respectively.) Regulation by ERs has been implicated in the post-transcriptional regulation of c-myb gene expression [4] and the c-myb gene is involved in recurrent translocations in some breast tumors that are positive for expression of ERs [5]
The one example that we identified was the EPB41 gene, which has a c-Myb binding site identified by Chromatin immunoprecipitation (ChIP)-on-chip and validated by conventional ChIP, the gene was not induced by beta-estradiol stimulation and its expression did not decline when c-Myb expression was knocked down with an shRNA (Figure 4)
Summary
The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Microarray studies have proved to be a powerful tool for studying the activities of Myb proteins, and they have identified dozens of genes that are induced when c-Myb is ectopically over-expressed in MCF-7 breast cancer cells and other cell types [18,19]. It is not clear whether those genes are directly or indirectly regulated by c-Myb, whether they are regulated by c-Myb expressed at its normal levels, or how their regulation is affected by stimulation of ERs or other extra-cellular stimuli
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