Abstract
Assays of luciferase gene activity are a sensitive and quantitative reporter system suited to high-throughput screening. We adapted a luciferase assay to a screening strategy for identifying factors that reactivate epigenetically silenced genes. This epigenetic luciferase reporter is subject to endogenous gene silencing mechanisms on the inactive X chromosome (Xi) in primary mouse cells and thus captures the multilayered nature of chromatin silencing in development. Here, we describe the optimization of an Xi-linked luciferase reactivation assay in 384-well format and adaptation of the assay for high-throughput siRNA and chemical screening. Xi-luciferase reactivation screening has applications in stem cell biology and cancer therapy. We have used the approach described here to identify chromatin-modifying proteins and to identify drug combinations that enhance the gene reactivation activity of the DNA demethylating drug 5-aza-2′-deoxycytidine.
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Acknowledgments
This work was supported National Research Service Award AG039179 to A.K. and by funds from the Iris Cantor-UCLA Women’s Health Center Executive Advisory Board to K.P./A.K. K.P. is supported by the NIH (DP2OD001686 and P01 GM099134), CIRM (RN1-00564, RB3-05080, and RB4-06133), and the Jonsson Comprehensive Cancer Center and the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA. We are grateful to Winnie Hwong for technical assistance, and to Stephen T. Smale for experimental advice.
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Keegan, A., Plath, K., Damoiseaux, R. (2018). High-Throughput Screening of a Luciferase Reporter of Gene Silencing on the Inactive X Chromosome. In: Damoiseaux, R., Hasson, S. (eds) Reporter Gene Assays. Methods in Molecular Biology, vol 1755. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7724-6_6
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DOI: https://doi.org/10.1007/978-1-4939-7724-6_6
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