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Techniques for Evaluation of AR Transcriptional Output and Recruitment to DNA

  • Manqi Zhang
  • William C. Krause
  • Irina U. AgoulnikEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1786)

Abstract

Steroid receptors are ligand activated transcription factors whose promoter specificity is regulated by a broad set of coregulators and pioneer factors. Corepressors and coactivators determine receptors’ recruitment to specific regulatory elements and ultimately their transcriptional output. Using androgen receptor (AR) and NCOR1 corepressor as examples, this chapter describes experimental approaches to evaluate recruitment of steroid receptors and their coregulators to DNA and to determine coregulator contribution to the transcriptional output of the receptor. The chromatin immunoprecipitation assay, or ChIP, quantifies protein–DNA interaction in the cellular chromatin environment. Here, we describe a protocol to measure NCOR1 recruitment to AR binding sites of interest using ChIP. Gene Set Enrichment Analysis, GSEA, is a computational technique to determine whether a defined gene set is significantly represented among changes in gene expression between two biological groups. As an example, we examine whether AR repressed genes are significantly represented among genes altered by the NCOR1 knockout.

Key words

NCOR1 Androgen receptor ChIP assay GSEA 

References

  1. 1.
    Narayanan R, Mohler ML, Bohl CE, Miller DD, Dalton JT (2008) Selective androgen receptor modulators in preclinical and clinical development. Nucl Recept Signal 6:e010.  https://doi.org/10.1621/nrs.06010 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Hodgson MC, Shen HC, Hollenberg AN, Balk SP (2008) Structural basis for nuclear receptor corepressor recruitment by antagonist-liganded androgen receptor. Mol Cancer Ther 7(10):3187–3194.  https://doi.org/10.1158/1535-7163.MCT-08-0461 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Yang YC, Banuelos CA, Mawji NR, Wang J, Kato M, Haile S, McEwan IJ, Plymate S, Sadar MD (2016) Targeting androgen receptor activation function-1 with EPI to overcome resistance mechanisms in castration-resistant prostate cancer. Clin Cancer Res 22(17):4466–4477.  https://doi.org/10.1158/1078-0432.CCR-15-2901 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Joseph JD, Wittmann BM, Dwyer MA, Cui H, Dye DA, McDonnell DP, Norris JD (2009) Inhibition of prostate cancer cell growth by second-site androgen receptor antagonists. Proc Natl Acad Sci U S A 106(29):12178–12183.  https://doi.org/10.1073/pnas.0900185106 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Jones A, Hwang DJ, Narayanan R, Miller DD, Dalton JT (2010) Effects of a novel selective androgen receptor modulator on dexamethasone-induced and hypogonadism-induced muscle atrophy. Endocrinology 151(8):3706–3719.  https://doi.org/10.1210/en.2010-0150 CrossRefPubMedGoogle Scholar
  6. 6.
    Kearbey JD, Gao W, Narayanan R, Fisher SJ, Wu D, Miller DD, Dalton JT (2007) Selective Androgen Receptor Modulator (SARM) treatment prevents bone loss and reduces body fat in ovariectomized rats. Pharm Res 24(2):328–335.  https://doi.org/10.1007/s11095-006-9152-9 CrossRefPubMedGoogle Scholar
  7. 7.
    Agoulnik IU, Weigel NL (2008) Androgen receptor coactivators and prostate cancer. Adv Exp Med Biol 617:245–255.  https://doi.org/10.1007/978-0-387-69080-3_23 CrossRefPubMedGoogle Scholar
  8. 8.
    Wang Q, Li W, Liu XS, Carroll JS, Janne OA, Keeton EK, Chinnaiyan AM, Pienta KJ, Brown M (2007) A hierarchical network of transcription factors governs androgen receptor-dependent prostate cancer growth. Mol Cell 27(3):380–392.  https://doi.org/10.1016/j.molcel.2007.05.041 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Kazmin D, Prytkova T, Cook CE, Wolfinger R, Chu TM, Beratan D, Norris JD, Chang CY, McDonnell DP (2006) Linking ligand-induced alterations in androgen receptor structure to differential gene expression: a first step in the rational design of selective androgen receptor modulators. Mol Endocrinol 20(6):1201–1217.  https://doi.org/10.1210/me.2005-0309 CrossRefPubMedGoogle Scholar
  10. 10.
    Wang Q, Li W, Zhang Y et al (2009) Androgen receptor regulates a distinct transcription program in androgen-independent prostate cancer. Cell 138(2):245–256.  https://doi.org/10.1016/j.cell.2009.04.056 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Mahajan NP, Liu Y, Majumder S, Warren MR, Parker CE, Mohler JL, Earp HS, Whang YE (2007) Activated Cdc42-associated kinase Ack1 promotes prostate cancer progression via androgen receptor tyrosine phosphorylation. Proc Natl Acad Sci U S A 104(20):8438–8443.  https://doi.org/10.1073/pnas.0700420104 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Lopez SM, Agoulnik AI, Zhang M et al (2016) Nuclear receptor corepressor 1 expression and output declines with prostate cancer progression. Clin Cancer Res 22(15):3937–3949.  https://doi.org/10.1158/1078-0432.CCR-15-1983 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    He B, Lanz RB, Fiskus W et al (2014) GATA2 facilitates steroid receptor coactivator recruitment to the androgen receptor complex. Proc Natl Acad Sci U S A 111(51):18261–18266.  https://doi.org/10.1073/pnas.1421415111 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Guerrero J, Alfaro IE, Gomez F, Protter AA, Bernales S (2013) Enzalutamide, an androgen receptor signaling inhibitor, induces tumor regression in a mouse model of castration-resistant prostate cancer. Prostate 73(12):1291–1305.  https://doi.org/10.1002/pros.22674 CrossRefPubMedGoogle Scholar
  15. 15.
    Agoulnik IU, Vaid A, Bingman WE 3rd, Erdeme H, Frolov A, Smith CL, Ayala G, Ittmann MM, Weigel NL (2005) Role of SRC-1 in the promotion of prostate cancer cell growth and tumor progression. Cancer Res 65(17):7959–7967.  https://doi.org/10.1158/0008-5472.CAN-04-3541 CrossRefPubMedGoogle Scholar
  16. 16.
    Agoulnik IU, Vaid A, Nakka M et al (2006) Androgens modulate expression of transcription intermediary factor 2, an androgen receptor coactivator whose expression level correlates with early biochemical recurrence in prostate cancer. Cancer Res 66(21):10594–10602.  https://doi.org/10.1158/0008-5472.CAN-06-1023 CrossRefPubMedGoogle Scholar
  17. 17.
    Yan J, Yu CT, Ozen M, Ittmann M, Tsai SY, Tsai MJ (2006) Steroid receptor coactivator-3 and activator protein-1 coordinately regulate the transcription of components of the insulin-like growth factor/AKT signaling pathway. Cancer Res 66(22):11039–11046.  https://doi.org/10.1158/0008-5472.CAN-06-2442 CrossRefPubMedGoogle Scholar
  18. 18.
    Zhang C, Wang L, Wu D et al (2011) Definition of a FoxA1 Cistrome that is crucial for G1 to S-phase cell-cycle transit in castration-resistant prostate cancer. Cancer Res 71(21):6738–6748.  https://doi.org/10.1158/0008-5472.CAN-11-1882 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Sahu B, Laakso M, Ovaska K et al (2011) Dual role of FoxA1 in androgen receptor binding to chromatin, androgen signalling and prostate cancer. EMBO J 30(19):3962–3976.  https://doi.org/10.1038/emboj.2011.328 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Wang D, Garcia-Bassets I, Benner C et al (2011) Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature 474(7351):390–394.  https://doi.org/10.1038/nature10006 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Zwart W, Theodorou V, Kok M, Canisius S, Linn S, Carroll JS (2011) Oestrogen receptor-co-factor-chromatin specificity in the transcriptional regulation of breast cancer. EMBO J 30(23):4764–4776.  https://doi.org/10.1038/emboj.2011.368 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Mei S, Qin Q, Wu Q et al (2017) Cistrome Data Browser: a data portal for ChIP-Seq and chromatin accessibility data in human and mouse. Nucleic Acids Res 45(D1):D658–D662.  https://doi.org/10.1093/nar/gkw983 CrossRefPubMedGoogle Scholar
  23. 23.
    Agoulnik IU, Bingman WE 3rd, Nakka M, Li W, Wang Q, Liu XS, Brown M, Weigel NL (2008) Target gene-specific regulation of androgen receptor activity by p42/p44 mitogen-activated protein kinase. Mol Endocrinol 22(11):2420–2432.  https://doi.org/10.1210/me.2007-0481 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Hodgson MC, Astapova I, Hollenberg AN, Balk SP (2007) Activity of androgen receptor antagonist bicalutamide in prostate cancer cells is independent of NCoR and SMRT corepressors. Cancer Res 67(17):8388–8395.  https://doi.org/10.1158/0008-5472.CAN-07-0617 CrossRefPubMedGoogle Scholar
  25. 25.
    Subramanian A, Tamayo P, Mootha VK et al (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102(43):15545–15550.  https://doi.org/10.1073/pnas.0506580102 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Gene set enrichment analysis GSEA user guide. http://software.broadinstitute.org/gsea/doc/GSEAUserGuideFrame.html. Accessed 16 Jan 2007Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Manqi Zhang
    • 1
  • William C. Krause
    • 2
  • Irina U. Agoulnik
    • 3
    • 4
    • 5
    Email author
  1. 1.Department of Chemistry and BiochemistryFlorida International UniversityMiamiUSA
  2. 2.Department of Cellular and Molecular PharmacologyUniversity of California, San FranciscoSan FranciscoUSA
  3. 3.Biomolecular Sciences InstituteFIUMiamiUSA
  4. 4.Baylor College of MedicineHoustonUSA
  5. 5.Department of Human and Molecular GeneticsHerbert Wertheim College of MedicineMiamiUSA

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