Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Mar 15;267(1-2):106-15.
doi: 10.1016/j.mce.2007.01.001. Epub 2007 Jan 16.

Estrogen receptor alpha regulates expression of the breast cancer 1 associated ring domain 1 (BARD1) gene through intronic DNA sequence

Amy L Creekmore  1 , Yvonne S Ziegler, Jamie L Bonéy, Ann M Nardulli
Affiliations

Estrogen receptor alpha regulates expression of the breast cancer 1 associated ring domain 1 (BARD1) gene through intronic DNA sequence

Amy L Creekmore et al. Mol Cell Endocrinol. .

Abstract

We have used a chromatin immunoprecipitation (ChIP)-based cloning strategy to isolate and identify genes associated with estrogen receptor alpha (ERalpha) in MCF-7 human breast cancer cells. One of the gene regions isolated was a 288bp fragment from the ninth intron of the breast cancer 1 associated ring domain (BARD1) gene. We demonstrated that ERalpha associated with this region of the endogenous BARD 1 gene in MCF-7 cells, that ERalpha bound to three of five ERE half sites located in the 288bp BARD1 region, and that this 288bp BARD1 region conferred estrogen responsiveness to a heterologous promoter. Importantly, treatment of MCF-7 cells with estrogen increased BARD1 mRNA and protein levels. These findings demonstrate that ChIP cloning strategies can be utilized to successfully isolate regulatory regions that are far removed from the transcription start site and assist in identifying cis elements involved in conferring estrogen responsiveness.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
ERα associates with a 288 bp region from the BARD1 gene within intron 9. Schematic drawing (A) and DNA sequence (B) of the 288 bp region of the BARD1 ninth intron isolated from ERα-immunoprecipitated chromatin. The numbering (+73,683 to +73,971) indicates the distance of the BARD1 gene region from its transcription start site. Black boxes indicate consensus ERE half sites (HS2 and HS4) and gray boxes indicate imperfect ERE half sites (HS1, HS3, and HS5). Arrows show the positions of primers used to produce DNA fragments for ChIP analysis (P1 and P2) and DNAse I footprinting (P3–P5) Bases differing from the consensus ERE half site are in lower case.
Fig. 2
Fig. 2
BARD1 mRNA and protein levels increase when MCF-7 cells are exposed to E2. MCF-7 cells were treated with ethanol vehicle, 10 nM E2 or 100 nM raloxifene (Ral), tamoxifen (Tam), or ICI 182,780 (ICI) as indicated. (A) RNA was isolated and utilized in quantitative real time PCR experiments to determine the levels of BARD1 and 36B4 mRNA. Values were calculated using the ΔΔCt method, which was normalized to 36B4 mRNA levels. Data from three independent experiments were combined and are expressed as the mean-fold increase in BARD1 mRNA levels ± S.E.M. Student's t-tests revealed that samples from MCF-7 cells that had been treated with E2 were statistically different (indicated by an asterisk) from cells that had been treated with ethanol (p ≤ 0.05). (B and C) Whole cell extracts from MCF-7cells that had been treated as indicated were subjected to Western analysis with BARD1 antibody or control β-actin- or Sp1-specific antibody. Each blot shown is representative of three independent experiments. Combined, normalized data is shown graphically as the relative BARD1 protein level (mean ± S.E.M.). Asterisks indicate significant increases in BARD1 protein levels compared to ethanol control (p ≤ 0.05). Error bars are sometimes too small to be visualized.
Fig. 3
Fig. 3
ERα limits DNase I cleavage at three ERE half sites in the ninth intron of the BARD1 gene. Noncoding strand primers were 32P-labeled and used to PCR amplify a 204 bp (A) or 304 bp (B) region of DNA. Labeled DNA was incubated with 0, 25, 100, 250, or 500 fmol of purified, baculovirus-expressed ERα (lanes 2–6) and exposed to DNase I. DNA that had been cleaved at each G residue was included for reference (lanes 1). Hypersensitive sites are indicated by an asterisk.
Fig. 4
Fig. 4
ERα binds specifically to ERE half sites in the ninth intron of the BARD1 gene. Gel mobility shift assays were carried out with 32P-labeled oligos containing 50 bp regions of intron 9 of the BARD1 gene. BARD1 regions containing one or two ERE half sites are represented schematically above the gel. 32P-labeled oligos were incubated with 50 fmol of purified, baculovirus-expressed ERα (lanes 2, 7, and 12). Antibody to ERα (lanes 3, 8, and 13), unlabeled oligos containing the A2 ERE (ERE, lanes 4, 9, and 14), or unlabeled oligos containing nonspecific (NS) DNA sequence (lanes 5, 10, and 15) were included as indicated. Reactions were fractionated on a nondenaturing gel and visualized by audioradiography.
Fig. 5
Fig. 5
Intron nine of the BARD1 gene confers estrogen responsiveness. MDAMB231 breast cancer cells were transfected with an ERα expression vector (ERα), a renilla control vector, and a reporter vector with a TATA sequence alone (TATA) or a TATA sequence combined with the 288 bp region of the BARD1 gene containing wild type DNA sequence (BARD) or one (mHS1, mHS2, mHS4) or three (mHS1,2,4) mutant ERE half sites as indicated in Panels A and B. Cells were treated with ethanol vehicle (−E2) or 10nM E2 (+E2) for 24 h. Data from four independent experiments were combined and are expressed as the mean in relative luciferase units (RLUs)±S.E.M. for all samples. Note that some error bars are too small to visualize. Statistical differences were determined using the Student's t-test as indicated by an asterisk in Panel A. Significant differences were determined by ANOVA in Panel B.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bourdeau V, Deschenes J, Metivier R, Nagai Y, Nguyen D, Bretschneider N, Gannon F, White JH, Mader S. Genome-wide identification of high-affinity estrogen response elements in human and mouse. Mol. Endocrinol. 2004;18:1411–1427. - PubMed
    1. Carroll J, Meyer C, Song J, Li W, Geistlinger T, Eeckhoute J, Brodshy A, Keeton E, Fertuck K, Hall G, Wang Q, Bekiranov S, Sementchenko V, Fox E, Silver P, Gingeras T, Liu X, Brown M. Genome-wide analysis of estrogen receptor binding sites. Nat. Genet. 2006;38:1289–1297. - PubMed
    1. Carroll JS, Liu XS, Brodsky AS, Li W, Meyer CA, Szary AJ, Eeckhoute J, Shao W, Hestermann EV, Geistlinger TR, Fox EA, Silver PA, Brown M. Chromosome-wide mapping of estrogen receptor binding reveals long-range regulation requiring the forkhead protein FoxA1. Cell. 2005;122:33–43. - PubMed
    1. Colditz GA. Relationship between estrogen levels, use of hormone replacement therapy, and breast cancer. J. Natl. Cancer Inst. 1998;90:814–823. - PubMed
    1. Couse JF, Korach KS. Estrogen receptor null mice: what have we learned and where will they lead us? Endocr. Rev. 1999;20:358–417. - PubMed

Publication types

MeSH terms

LinkOut - more resources