Chapter title |
A Large-Scale Functional Screen to Identify Epigenetic Repressors of Retrotransposon Expression
|
---|---|
Chapter number | 25 |
Book title |
Transposons and Retrotransposons
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3372-3_25 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3370-9, 978-1-4939-3372-3
|
Authors |
Ecco, Gabriela, Rowe, Helen M, Trono, Didier, Gabriela Ecco, Helen M. Rowe, Didier Trono Ph.D., Didier Trono |
Editors |
Jose L. Garcia-Pérez |
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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