title: A Large-Scale Functional Screen to Identify Epigenetic Repressors of Retrotransposon Expression.
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.
authors.affiliation: School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), 1015, Lausanne, Switzerland.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Ecco
authors.affiliation: School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), 1015, Lausanne, Switzerland.|Division of Infection and Immunity, Centre for Medical Molecular Virology, University College London, 90 Gower Street, London, WC1E 6BT, UK.
authors.forename: Helen M
authors.initials: HM
authors.lastname: Rowe
authors.affiliation: School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), 1015, Lausanne, Switzerland. didier.trono@epfl.ch.
authors.forename: Didier
authors.initials: D
authors.lastname: Trono
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D044127:Epigenesis, Genetic; D057890:Epigenomics / Q000379:methods*; D005786:Gene Expression Regulation; D015723:Gene Library; D006801:Humans; D051379:Mice; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D018626:Retroelements; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D012097:Repressor Proteins; D018626:Retroelements; D014157:Transcription Factors
keywords: Epigenetic regulation; Functional screen; GFP repression assay; KRAB-ZFP; Mammalian one-hybrid assay; Protein-DNA interaction; Retroelement control