title: Chromatin Immunoprecipitation Assay to Identify Genomic Binding Sites of Regulatory Factors.
abstract: DNA-protein interactions are vital to fundamental cellular events including transcription, replication, DNA repair, and recombination. Thus, their study holds the key to our understanding of mechanisms underlying normal development and homeostasis as well as disease. Transcriptional regulation is a highly complex process that involves recruitment of numerous factors resulting in formation of multi-protein complexes at gene promoters to regulate gene expression. The studied proteins can be, for example, transcription factors, epigenetic regulators, co-activators, co-repressors, or ligand-activated nuclear receptors as estrogen receptor-α (ERα) bound either directly to the DNA or indirectly by interaction with other DNA-bound factors. Chromatin immunoprecipitation (ChIP) assay is a powerful method to study interactions of proteins and a specific genomic DNA region. Recruitment of ERα to promoters of estrogen-dependent genes is a common mechanism to activate or enhance gene transcription in breast cancer thus promoting tumor progression. In this chapter, we demonstrate a stepwise protocol for ChIP assay using binding of ERα to its genomic targets after stimulation with 17β-estradiol (E2) in breast cancer cells as an example.
authors.affiliation: Division of Experimental and Translational Oncology, Institute of Immunology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany.|TRON gGmbH-Translational Oncology, University Medical Center of the Johannes Gutenberg University Mainz, Freiligrathstrabe, 55131, Mainz, Germany.
authors.forename: Meike
authors.initials: M
authors.lastname: Wagner
authors.affiliation: Institute of Molecular Biology (IMB), Mainz, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Jung
authors.affiliation: TRON gGmbH-Translational Oncology, University Medical Center of the Johannes Gutenberg University Mainz, Freiligrathstrabe, 55131, Mainz, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Koslowski
authors.affiliation: Ganymed Pharmaceuticals AG, Mainz, Germany.
authors.forename: Özlem
authors.initials: Ö
authors.lastname: Türeci
authors.affiliation: Institute of Molecular Biology (IMB), Mainz, Germany. v.tiwari@imb-mainz.de.
authors.forename: Vijay K
authors.initials: VK
authors.lastname: Tiwari
authors.affiliation: Division of Experimental and Translational Oncology, Institute of Immunology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany. ugur.sahin@tron-mainz.de.|TRON gGmbH-Translational Oncology, University Medical Center of the Johannes Gutenberg University Mainz, Freiligrathstrabe, 55131, Mainz, Germany. ugur.sahin@tron-mainz.de.|BioNTech AG, Mainz, Germany. ugur.sahin@tron-mainz.de.
authors.forename: Ugur
authors.initials: U
authors.lastname: Sahin
mesh_terms: D001665:Binding Sites; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D061986:MCF-7 Cells; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D057188:Workflow
chemical_list: D002843:Chromatin; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D004958:Estradiol
keywords: Cancer; ChIP assay; Chromatin; DNA–protein interactions; Estrogen receptor α; Estrogen signal ing; Gene regulation