Abstract
Deposition of epigenetic marks is an important layer of the transcriptional control of retrotransposons, especially during early embryogenesis. Krüppel-associated box domain zinc finger proteins (KRAB-ZFPs) are one of the largest families of transcription factors, and collectively partake in this process by tethering to thousands of retroelement-containing genomic loci their cofactor KAP1, which acts as a scaffold for a heterochromatin-inducing machinery. However, while the sequence-specific DNA binding potential of the poly-zinc finger-containing KRAB-ZFPs is recognized, very few members of the family have been assigned specific targets. In this chapter, we describe a large-scale functional screen to identify the retroelements bound by individual murine KRAB-ZFPs. Our method is based on the automated transfection of a library of mouse KRAB-ZFP-containing vectors into 293T cells modified to express GFP from a PGK promoter harboring in its immediate vicinity a KAP1-recruiting retroelement-derived sequence. Analysis is then performed by plate reader and flow cytometry fluorescence readout. Such large-scale DNA-centered functional approach can not only help to identify the trans-acting factors responsible for silencing retrotransposons, but also serve as a model for dissecting the transcriptional networks influenced by retroelement-derived cis-acting sequences.
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Acknowledgements
We thank P. Turelli and C. Delattre-Gubelman for advice, C. Raclot and S. E. Offner for technical support, M. Chambon and J. B. Chapalay from the EPFL Biomolecular Screening Facility for advice and help with the robotics, and the EPFL Flow Cytometry Core Facility for FACS sorting. This work was supported by funds from the Swiss National Science Foundation and from the European Research Council.
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Ecco, G., Rowe, H.M., Trono, D. (2016). A Large-Scale Functional Screen to Identify Epigenetic Repressors of Retrotransposon Expression. In: Garcia-Pérez, J. (eds) Transposons and Retrotransposons. Methods in Molecular Biology, vol 1400. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3372-3_25
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DOI: https://doi.org/10.1007/978-1-4939-3372-3_25
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