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Chromatin Immunoprecipitation of HIF-α in Breast Tumor Cells Using Wild Type and Loss of Function Models

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Hypoxia

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1742))

Abstract

Chromatin immunoprecipitation (ChIP) is a powerful method to determine whether a protein of interest binds to specific regulatory elements of the genome. Herein, we outline protocols optimized to detect binding of Hypoxia-Inducible Factor (HIF)-1α or HIF-2α to putative hypoxia response elements (HREs) within HIF target genes expressed in breast tumor epithelial cells.

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References

  1. Gilmour DS, Lis JT (1985) In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster. Mol Cell Biol 5(8):2009–2018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Gilmour DS, Pflugfelder G, Wang JC, Lis JT (1986) Topoisomerase I interacts with transcribed regions in Drosophila cells. Cell 44(3):401–407

    Article  CAS  PubMed  Google Scholar 

  3. Gilmour DS, Rougvie AE, Lis JT (1991) Protein-DNA cross-linking as a means to determine the distribution of proteins on DNA in vivo. Methods Cell Biol 35:369–381

    Article  CAS  PubMed  Google Scholar 

  4. Semenza GL (2010) Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene 29(5):625–634. https://doi.org/10.1038/onc.2009.441

    Article  CAS  PubMed  Google Scholar 

  5. Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, Kriegsheim A, Hebestreit HF, Mukherji M, Schofield CJ, Maxwell PH, Pugh CW, Ratcliffe PJ (2001) Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292(5516):468–472

    Article  CAS  PubMed  Google Scholar 

  6. Wenger RH, Stiehl DP, Camenisch G (2005) Integration of oxygen signaling at the consensus HRE. Sci STKE 306:re12. https://doi.org/10.1126/stke.3062005re12

    Google Scholar 

  7. Schodel J, Oikonomopoulos S, Ragoussis J, Pugh CW, Ratcliffe PJ, Mole DR (2011) High-resolution genome-wide mapping of HIF-binding sites by ChIP-seq. Blood 117(23):e207–e217. https://doi.org/10.1182/blood-2010-10-314427

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Tsai YP, Wu KJ (2012) Hypoxia-regulated target genes implicated in tumor metastasis. J Biomed Sci 19:102. https://doi.org/10.1186/1423-0127-19-102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hu CJ, Wang LY, Chodosh LA, Keith B, Simon MC (2003) Differential roles of hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha in hypoxic gene regulation. Mol Cell Biol 23(24):9361–9374

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Stiehl DP, Bordoli MR, Abreu-Rodriguez I, Wollenick K, Schraml P, Gradin K, Poellinger L, Kristiansen G, Wenger RH (2011) Non-canonical HIF-2alpha function drives autonomous breast cancer cell growth via an AREG-EGFR/ErbB4 autocrine loop. Oncogene 31(18):2283–2297. https://doi.org/10.1038/onc.2011.417

    Article  PubMed  Google Scholar 

  11. Liao D, Corle C, Seagroves TN, Johnson RS (2007) Hypoxia-inducible factor-1alpha is a key regulator of metastasis in a transgenic model of cancer initiation and progression. Cancer Res 67(2):563–572. https://doi.org/10.1158/0008-5472.CAN-06-2701

    Article  CAS  PubMed  Google Scholar 

  12. Schwab LP, Peacock DL, Majumdar D, Ingels JF, Jensen LC, Smith KD, Cushing RC, Seagroves TN (2012) Hypoxia-inducible factor 1alpha promotes primary tumor growth and tumor-initiating cell activity in breast cancer. Breast Cancer Res 14(1):R6. https://doi.org/10.1186/bcr3087

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Raval RR, Lau KW, Tran MG, Sowter HM, Mandriota SJ, Li JL, Pugh CW, Maxwell PH, Harris AL, Ratcliffe PJ (2005) Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma. Mol Cell Biol 25(13):5675–5686. https://doi.org/10.1128/MCB.25.13.5675-5686.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Chi JT, Wang Z, Nuyten DS, Rodriguez EH, Schaner ME, Salim A, Wang Y, Kristensen GB, Helland A, Borresen-Dale AL, Giaccia A, Longaker MT, Hastie T, Yang GP, van de Vijver MJ, Brown PO (2006) Gene expression programs in response to hypoxia: cell type specificity and prognostic significance in human cancers. PLoS Med 3(3):e47

    Article  PubMed  PubMed Central  Google Scholar 

  15. Semenza GL (2002) HIF-1 and tumor progression: pathophysiology and therapeutics. Trends Mol Med 8(4 Suppl):S62–S67

    Article  CAS  PubMed  Google Scholar 

  16. Lin EY, Jones JG, Li P, Zhu L, Whitney KD, Muller WJ, Pollard JW (2003) Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases. Am J Pathol 163(5):2113–2126. https://doi.org/10.1016/S0002-9440(10)63568-7

    Article  PubMed  PubMed Central  Google Scholar 

  17. Brooks DL, Schwab LP, Krutilina R, Parke DN, Sethuraman A, Hoogewijs D, Schorg A, Gotwald L, Fan M, Wenger RH, Seagroves TN (2016) ITGA6 is directly regulated by hypoxia-inducible factors and enriches for cancer stem cell activity and invasion in metastatic breast cancer models. Mol Cancer 15:26. https://doi.org/10.1186/s12943-016-0510-x

    Article  PubMed  PubMed Central  Google Scholar 

  18. Wang GL, Semenza GL (1995) Purification and characterization of hypoxia-inducible factor 1. J Biol Chem 270(2):1230–1237

    Article  CAS  PubMed  Google Scholar 

  19. Oosthuyse B, Moons L, Storkebaum E, Beck H, Nuyens D, Brusselmans K, Van Dorpe J, Hellings P, Gorselink M, Heymans S, Theilmeier G, Dewerchin M, Laudenbach V, Vermylen P, Raat H, Acker T, Vleminckx V, Van Den Bosch L, Cashman N, Fujisawa H, Drost MR, Sciot R, Bruyninckx F, Hicklin DJ, Ince C, Gressens P, Lupu F, Plate KH, Robberecht W, Herbert JM, Collen D, Carmeliet P (2001) Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat Genet 28(2):131–138. https://doi.org/10.1038/88842

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by NIH grant CA138488 and the Department of Defense Breast Cancer Research Program award BC083846 (to TNS). The HIF-1 WT and KO PyMT cells were originally created by Dr. Luciana P. Schwab, and the HIF shRNA knockdown MDA-MB-231 cell line models were provided by Dr. Roland Wenger of the University of Zurich. We also thank Dr. Meiyun Fan at the University of Tennessee Health Science Center for providing us with technical advice and for assisting us with troubleshooting these protocols during their optimization.

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Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Center for Cancer Research, University of Tennessee Health Science Center, Memphis, TN, USA

    Danielle L. Brooks & Tiffany N. Seagroves

  2. National Cancer Institute, Women’s Malignancies Branch, Bethesda, MD, USA

    Danielle L. Brooks

Authors
  1. Danielle L. Brooks
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  2. Tiffany N. Seagroves
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Corresponding author

Correspondence to Tiffany N. Seagroves .

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Editors and Affiliations

  1. Department of Neurosurgery, Clinical Neurosciences Center, University of Utah, Salt Lake City, Utah, USA

    L. Eric Huang

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Brooks, D.L., Seagroves, T.N. (2018). Chromatin Immunoprecipitation of HIF-α in Breast Tumor Cells Using Wild Type and Loss of Function Models. In: Huang, L. (eds) Hypoxia. Methods in Molecular Biology, vol 1742. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7665-2_7

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  • DOI: https://doi.org/10.1007/978-1-4939-7665-2_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7664-5

  • Online ISBN: 978-1-4939-7665-2

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