Analysis of the c-KIT ligand promoter using chromatin immunoprecipitation
Por:
Zhang P., Rojas A., Blechacz B.
Publicada:
1 ene 2017
Resumen:
Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation of the DNA-protein interaction, followed by immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-ß (TGF-ß)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF). © 2017 Journal of Visualized Experiments.
Filiaciones:
Zhang P.:
Department of Gastroenterology, Hepatology and Nutrition, The University of Texas MD Anderson Cancer Center, United States
Rojas A.:
Department of Gastroenterology, Hepatology and Nutrition, The University of Texas MD Anderson Cancer Center, United States
Blechacz B.:
Department of Gastroenterology, Hepatology and Nutrition, The University of Texas MD Anderson Cancer Center, United States
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