Unknown,Transcriptomics,Genomics,Proteomics

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Chromatin immunoprecipitation profiling of human breast cancer cell lines and tissues to identify novel estrogen receptor-{alpha} binding sites and estradiol target genes

ABSTRACT: Estrogen receptor-{alpha} (ER{alpha}) 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 ER{alpha} 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 ER{alpha} target loci in breast cancer cells. The ER{alpha}-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 ER{alpha}-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 ER{alpha} 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 ER{alpha} 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. Experiment Overall Design: This Series currently contains the gene expression data accompanying Zhihong Lin et al. Cancer Research 67,5017-5024(2007). MCF7 cells were treated with vehicle or E2 at a concentration of 10E-9 mol/L for 3 and 6 h. All experiments were performed in triplicate.

ORGANISM(S): Homo sapiens

SUBMITTER: Zhihong Lin

PROVIDER: E-GEOD-11506 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Novel estrogen receptor-alpha binding sites and estradiol target genes identified by chromatin immunoprecipitation cloning in breast cancer.

Lin Zhihong Z Reierstad Scott S Huang Chiang-Ching CC Bulun Serdar E SE

Cancer research 20070501 10

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 contai ...[more]

PMID: 17510434

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Project description:Estrogen receptors play critical roles in both the normal physiological, and disease states of numerous tissues, including breast and uterus. Estrogen receptor alpha (ER) can activate or repress the expression of target genes upon estrogen stimulation. In order to better understand the transcriptional network of ER in breast and uterus, we generated genome wide maps ofM-BM- ER binding sites (ERBS) and gene expression profiles in breast cancer cells (MCF7 and T47D) and uterine cancer cells (ECC1 and Ishikawa) through ChIP-Seq and microarray techniques. Surprisingly, we identified large scale differences in the numbers of ERBS between these cell lines when treated with E2 (17-M-NM-2 estradiol). Besides identification of common and unique ERBS between breast and uterine cancer cell types., our data also suggest that both cell types could recruit a large set of common co-operating transcription factors (Co-TFs) and a few unique Co-TFs as well. Besides the genes that are commonly regulatedM-BM- between the different cell lines, there are a number of genes that are differentially regulated in different cell types. Gene pathway analyses of E2 target genes suggest that ER regulates many biological pathways and processes in both tissue-type dependent and independent manners. Our results showed that cell lines derived from same tissue display a greater similarity for both profiles of ERBS and gene expression, and that the differential profiles of ER and preferential recruitment of some Co-TFs are the main determinants for the differential regulation of E2 signaling in breast and uterine cancer cells. In order to explore common and distinctive features of ERM-NM-1 (estrogen receptor alpha) binding profiles between breast and uterus, we generated eight ChIP-Seq libraries for the four cell lines (MCF7, T47D, ECC1 and Ishikawa) under two different treatments (E2, ethanol). In addition, we generated four control libraries for the four cell lines. For all treatment libraries, we generated about 7-12 million unique tags each. ER antibody catalog number is (Santa Cruz,sc-543).

Project description:The interplay between mitogenic and proinflammatory signaling pathways play key roles in determining the phenotypes and clinical outcomes of breast cancers. We have used global nuclear run-on coupled with deep sequencing to characterize the immediate transcriptional responses of MCF-7 breast cancer cells treated with estradiol, TNF?, or both. In addition, we have integrated these data with chromatin immunoprecipitation coupled with deep sequencing for estrogen receptor alpha (ER?), the pioneer factor FoxA1 and the p65 subunit of the NF-?B transcription factor. Our results indicate extensive transcriptional interplay between these two signaling pathways, which is observed for a number of classical mitogenic and proinflammatory protein-coding genes. In addition, GRO-seq has allowed us to capture the transcriptional crosstalk at the genomic locations encoding for long non-coding RNAs, a poorly characterized class of RNAs which have been shown to play important roles in cancer outcomes. The synergistic and antagonistic interplay between estrogen and TNF? signaling at the gene level is also evident in the patterns of ER? and NF-?B binding, which relocalize to new binding sites that are not occupied by either treatment alone. Interestingly, the chromatin accessibility of classical ER? binding sites is predetermined prior to estrogen treatment, whereas ER? binding sites gained upon co-treatment with TNF? require NF-?B and FoxA1 to promote chromatin accessibility de novo. Our data suggest that TNF? signaling recruits FoxA1 and NF-?B to latent ER? enhancer locations and directly impact ER? enhancer accessibility. Binding of ER? to latent enhancers upon co-treatment, results in increased enhancer transcription, target gene expression and altered cellular response. This provides a mechanistic framework for understanding the molecular basis for integration of mitogenic and proinflammatory signaling in breast cancer. Using GRO-seq and ChIP-seq (ER, FoxA1 and p65) to assay the molecular crosstalk of MCF-7 cells treated with E2, TNFa or both E2+TNFa.

Dataset Information

Chromatin immunoprecipitation profiling of human breast cancer cell lines and tissues to identify novel estrogen receptor-{alpha} binding sites and estradiol target genes

Publications

Novel estrogen receptor-alpha binding sites and estradiol target genes identified by chromatin immunoprecipitation cloning in breast cancer.

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