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Using Chromatin Immunoprecipitation in Toxicology: A Step-by-Step Guide to Increasing Efficiency, Reducing Variability, and Expanding Applications
Citation:
McCullough, S., D. On, AND E. Bowers. Using Chromatin Immunoprecipitation in Toxicology: A Step-by-Step Guide to Increasing Efficiency, Reducing Variability, and Expanding Applications. Current Protocols in Toxicology. Wiley InterScience, Silver Spring, MD, 72(1):3.14.1-3.14.28, (2017).
Impact/Purpose:
The rapidly emerging field of toxicoepigenetics has enormous potential to provide new insight into adverse outcome pathways and new biomarkers for risk assessment. To realize this potential we must gain a more comprehensive understanding of interactions between environmental factors, toxic exposures, and the epigenome. Here we provide a streamlined ChIP protocol with detailed information on the optimization of critical parameters. This protocol can be readily applied to samples from a wide range of in vitro studies (cell lines and primary cells) and clinical samples (peripheral leukocytes).
Description:
Histone modifications work in concert with DNA methylation to regulate cellular structure, function, and the response to environmental stimuli. More than 130 unique histone modifications have been described to date and chromatin immunoprecipitation (ChIP) allows for the exploration of their associations with the regulatory regions of target genes and other DNA/chromatin-associated proteins across the genome. Many variations of ChIP have been developed in the 30 years since its earliest version came into use, which makes it challenging for users to integrate the procedure into their research programs. Further, the differences in ChIP protocols can confound efforts to increase reproducibility across studies. The streamlined ChIP procedure presented here can be readily applied to samples from a wide range of in vitro studies (cell lines and primary cells), and clinical samples (peripheral leukocytes) in toxicology. We also provide detailed guidance on the optimization of critical protocol parameters, such as chromatin fixation, fragmentation, and immunoprecipitation, to increase efficiency and improve reproducibility. Expanding toxicoepigenetic studies to more readily include histone modifications will facilitate a more comprehensive understanding of the role of the epigenome in environmental exposure effects and the integration of epigenetic data in mechanistic toxicology, adverse outcome pathways, and risk assessment.
