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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.
Bart Deplancke, Guido Van Mierlo, Judith Franziska Kribelbauer
Jonas Caspar De Tribolet-Hardy
Didier Trono, Jacques Fellay, Priscilla Turelli, Christian Axel Wandall Thorball, Evaristo Jose Planet Letschert, Julien Léonard Duc, Romain Forey, Bara Khubieh, Sandra Eloise Kjeldsen, Alexandre Coudray, Michaël Imbeault, Cyril David Son-Tuyên Pulver, Jonas Caspar De Tribolet-Hardy