Abstract
Estrogen receptor-alpha (ERalpha) and its ligand estradiol play critical roles in breast cancer growth and are important therapeutic targets for this disease. Using chromatin immunoprecipitation (ChIP)-on-chip, ligand-bound ERalpha was recently found to function as a master transcriptional regulator via binding to many cis-acting sites genome-wide. Here, we used an alternative technology (ChIP cloning) and identified 94 ERalpha target loci in breast cancer cells. The ERalpha-binding sites contained both classic estrogen response elements and nonclassic binding sequences, showed specific transcriptional activity in reporter gene assay, and interacted with the key transcriptional regulators, including RNA polymerase II and nuclear receptor coactivator-3. The great majority of the binding sites were located in either introns or far distant to coding regions of genes. Forty-three percent of the genes that lie within 50 kb to an ERalpha-binding site were regulated by estradiol. Most of these genes are novel estradiol targets encoding receptors, signaling messengers, and ion binders/transporters. mRNA profiling in estradiol-treated breast cancer cell lines and tissues revealed that these genes are highly ERalpha responsive both in vitro and in vivo. Among estradiol-induced genes, Wnt11 was found to increase cell survival by significantly reducing apoptosis in breast cancer cells. Taken together, we showed novel genomic binding sites of ERalpha that regulate a novel set of genes in response to estradiol in breast cancer. Our findings suggest that at least a subset of these genes, including Wnt11, may play important in vivo and in vitro biological roles in breast cancer.
Highlights
Estrogen receptor-a (ERa; ref. 1) is a ligand-activated nuclear receptor that regulates transcription of estrogen-responsive genes important for cell growth, differentiation, and malignant transformation in various target cells [2]
The chromatin immunoprecipitation (ChIP)-PCR procedure was optimized to achieve amplification using an ERa antibody to detect proteins bound to the promoter of a prototypical ERa target gene, Myc, in the absence of any nonspecific IgG binding after 40 cycles of PCR (Supplementary Fig. S1)
Once these conditions were reproducibly achieved in five consecutive experiments, DNA fragments immunoprecipitated by the ERa antibody were extracted, cloned, and sequenced
Summary
Estrogen receptor-a (ERa; ref. 1) is a ligand-activated nuclear receptor that regulates transcription of estrogen-responsive genes important for cell growth, differentiation, and malignant transformation in various target cells [2]. Significant progress has been made in understanding the role of ERa as a transcription factor that regulates the expression of target genes by directly binding to an estrogen response element Using chromatin immunoprecipitation (ChIP)-on-chip, this group mapped a large number of ERa-binding sites on a chromosome and genome-wide scale, identifying novel cis-regulatory sites and target genes in MCF-7 breast cancer cells [6, 7]. The majority of these binding sites were distant from the transcription start sites of regulated genes [6, 7]
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