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Chromatin immunoprecipitation assay detects er recruitment to gene specific promoters in uterus

Abstract

Chromatin immunoprecipitation (ChIP) technique allows detection of proteins that bind to chromatin. While this technique has been applied extensively in cell-based studies, its tissue-based application remains poorly explored. We are specifically interested in examining estrogen-dependent transcriptional mechanism in respect of recruitment of estrogen receptor-alpha (ERα), a ligand-activated transcription factor, to uterine gene promoters in mice. Recent genearray studies, utilizing ERα knock-out vs. wild-type mice, have revealed that estrogen regulates numerous uterine genes temporally and most importantly via ERα during the phase-II response, including three well characterized genes viz., lactoferrin (Ltf), progesterone receptor (Pgr) and cyclinD1 (Ccnd1). Here, utilizing systematic ChIP studies, we demonstrate endogenous recruitment of ERα to above uterine gene promoters following estradiol-17β (E2) injection in mice.

Abbreviations

ChIP:

chromatin immunoprecipitation

ERα:

estrogen receptor-alpha

Ltf :

lactoferrin

Pgr :

progesterone receptor

Ccnd1:

cyclin D1

IP:

immunoprecipitation

References

  1. Hearnes JM, Mays DJ, Schavolt KL, Tang L, Jiang X, Pietenpol JA. Chromatin immunoprecipitation-based screen to identify functional genomic binding sites for sequence-specific transactivators. Mol Cell Biol 2005; 25:10148–10158.

    Article  PubMed  CAS  Google Scholar 

  2. Couse JF, Korach KS. Estrogen receptor null mice: what have we learned and where will they lead us? Endo Rev 1999; 20(3):358–417.

    Article  CAS  Google Scholar 

  3. Tsai MJ, O’Malley BW. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Ann Rev Biochem 1994; 63:451–486.

    Article  PubMed  CAS  Google Scholar 

  4. Ray S, Hou X, Zhou H-E, Wang H, Das SK. Bip is a molecular link between the phase-I and phase-II estrogenic responses in uterus. Mol Endocrinol 2006; 20(8):1825–1837.

    Article  PubMed  CAS  Google Scholar 

  5. Hewitt SC, Deroo BJ, Hansen K, Collins J, Grissom S, Afshari CA, Korach KS. Estrogen receptordependent genomic responses in the uterus mirror the biphasic physiological response to estrogen. Mol Endocrinol 2003; 17:2070–2083.

    Article  PubMed  CAS  Google Scholar 

  6. Das SK, Taylor JA, Korach KS, Paria BC, Dey SK, Lubahn DB. Estrogenic responses in estrogen receptor-α deficient mice reveal a novel estrogen signaling pathway. Proc Natl Acad Sci USA 1997; 94:12786–12791.

    Article  PubMed  CAS  Google Scholar 

  7. Das SK, Tan J, Raja S, Halder J, Paria BC, Dey SK. Estrogen targets genes involved in protein processing, calcium homeostasis and Wnt signaling in the mouse uterus independent of estrogen receptor-α and - β. J Biol Chem 2000; 275:28834–28842.

    Article  PubMed  CAS  Google Scholar 

  8. Hou X, Tan Y, Li M, Dey SK, Das SK. Canonical Wnt Signaling Is Critical to Estrogen Mediated Uterine Growth. Mol Endocrinol 2004; 18:3035–3049.

    Article  PubMed  CAS  Google Scholar 

  9. Tan J, Paria BC, Dey SK, Das SK. Differential uterine expression of estrogen and progesterone receptors correlates with uterine preparation for implantation and decidualization in the mouse. Endocrinology 1999; 140:5310–5321.

    Article  PubMed  CAS  Google Scholar 

  10. Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS, Gustafsson JA, Smithies O. Generation and reproductive phenotypes of mice lacking estrogen receptor β. Proc Natl Acad Sci USA 1998; 95:15677–15682.

    Article  PubMed  CAS  Google Scholar 

  11. Klein-Hitpass L, Ryffel GU, Heitlinger E, Cato AC. A 13 bp palindrome is a functional estrogen responsive element and interacts specifically with estrogen receptor. Nucleic Acids Res 1988; 16:647–663.

    Article  PubMed  CAS  Google Scholar 

  12. Sukovich DA, Mukherjee R, Benfield PA. A novel, celltype-specific mechanism for estrogen receptormediated gene activation in the absence of an estrogen-responsive element. Mol Cell Biol 1994; 14:7134–7143.

    PubMed  CAS  Google Scholar 

  13. O’Lone R, Frith MC, Karlsson EK, Hansen U. Genomic targets of nuclear estrogen receptors. Mol Endocrinol 2004; 18:1859–1875.

    Article  PubMed  CAS  Google Scholar 

  14. Webb P, Nguyen P, Valentine C, Lopez GN, Kwok GR, McInerney E, Katzenellenbogen BS, Enmark E, Gustafsson JA, Nilsson S, Kushner PJ. The estrogen receptor enhances AP-1 activity by two distinct mechanisms with different requirements for receptor transactivation functions. Mol Endocrinol 1999; 13:1672–1685.

    Article  PubMed  CAS  Google Scholar 

  15. Das SK, Tan J, Johnson DC, Dey SK. Differential spatiotemporal regulation of lactoferrin and progesterone receptor genes in the mouse uterus by primary estrogen, catechol estrogen, and xenoestrogen. Endocrinology 1998; 139: 2905–2915.

    Article  PubMed  CAS  Google Scholar 

  16. Tong W, Pollard JW. Progesterone inhibits estrogeninduced cyclin D1 and cdk4 nuclear translocation, cyclin E- and cyclin A-cdk2 kinase activation, and cell proliferation in uterine epithelial cells in mice. Mol Cell Biol 1999; 19:2251–2264

    PubMed  CAS  Google Scholar 

  17. Liu Y, Teng CT. Estrogen response module of the mouse lactoferrin gene contains overlapping chicken ovalbumin upstream promoter transcription factor and estrogen receptor-binding elements. Mol Endocrinol 1992; 6:355–364.

    Article  PubMed  CAS  Google Scholar 

  18. Planas-Silva MD, Shang Y, Donaher JL, Brown M, Weinberg RA. AIB1 enhances estrogen dependent induction of cyclin D1 expression. Cancer Res 2001; 61:3858–3862.

    PubMed  CAS  Google Scholar 

  19. Petz LN, Ziegler YS, Schultz JR, Kim H, Kemper JK, Nardulli AM. Differential regulation of the human progesterone receptor gene through an estrogen response element half site and Sp1 sites. J Steroid Biochem Mol Biol 2004; 88:113–122.

    Article  PubMed  CAS  Google Scholar 

  20. Kazi AA, Jones JM, Koos RD. Chromatin immunoprecipitation analysis of gene expression in the rat uterus in vivo: estrogen-induced recruitment of both estrogen receptor alpha and hypoxiainducible factor 1 to the vascular endothelial growth factor promoter. Mol Endocrinol 2005; 19:2006–2019.

    Article  PubMed  CAS  Google Scholar 

  21. Rahman MA, Li M, Li P, Wang H, Dey SK, Das SK. Hoxa-10 deficiency alters region-specific gene expression and perturbs differentiation of natural killer cells during decidualization. Dev Biol 2006; 290:105–117.

    Article  PubMed  CAS  Google Scholar 

  22. Orlando V. Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation. Trends Biochem Sci 2000; 25:99–104.

    Article  PubMed  CAS  Google Scholar 

  23. Yahata T, Shao W, Endoh H, Hur J, Coser KR, Sun H, Ueda Y, Kato S, Isselbacher KJ, Brown M, Shioda T. Selective coactivation of estrogen-dependent transcription by CITED1 CBP/p300-binding protein. Genes Dev 2001; 15:2598–2612.

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Sanjoy K. Das.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Ray, S., Das, S.K. Chromatin immunoprecipitation assay detects er recruitment to gene specific promoters in uterus. Biol. Proced. Online 8, 69–76 (2006). https://doi.org/10.1251/bpo120

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  • DOI: https://doi.org/10.1251/bpo120

Indexing terms

  • Chromatin
  • Immunoprecipitation
  • Estrogen Receptor Alpha