EMBO Fellows Meeting 2012

Joana Marques
EMBO Fellows Meeting 2012
A stable RNAi knockdown system to study epigenetic regulators of pluripotency in
mouse ES cells
Abstract
Embryonic stem (ES) cells are in a pluripotent state that is epigenetically regulated by DNA methylation and
other chromatin modifications. We are using a stable and inducible RNAi knockdown system in mouse ES cells
to interrogate the function of potential epigenetic regulators of pluripotency. In this ICE (Inducible Cassette
Exchange) system (Iacovino et al., 2011), we use a genetically engineered mES cell line (A2lox.cre) that allows
the stable integration, by Cre-mediated site-specific recombination, of an shRNA-mir cassette near the
constitutively active HPRT locus under a tetracycline-regulatable promoter. The system has been successfully
used to generate a stable Tet1 shRNA cell line that allowed us to investigate the function of Tet1 and DNA
hydroxymethylation in regulating the balance between pluripotent/lineage commitment states (Ficz et al.,
2011). Several pluripotency genes were downregulated together with Tet1, namely Ecat1, Esrrb, Klf2, Fbxo15,
Tcl1 and Zfp42, showing increased levels of DNA methylation. Recovery of Tet1 expression resulted in
restoration of expression and methylation levels of the above pluripotency genes. These results suggest that
Tet1 actively regulates the pluripotent state, possibly by maintaining appropriate levels of DNA methylation at
pluripotency genes. We are now establishing stable shRNA lines for several potential epigenetic regulators of
pluripotency, identified by a genome-wide promoter methylation analysis and showing high levels of
expression in key developmental stages for epigenetic reprogramming.
References:
Iacovino et al., Stem Cells 2011; 29:1580-1587; Ficz et al., Nature 2011; 473:398-402.
Lab of Developmental Genetics and Imprinting, Babraham Institute, UK; Neurosciences Research Domain, ICVS, University
of Minho, Portugal
14-17 June 2012, Heidelberg, Germany