DEVELOPMENT 4203 RESEARCH ARTICLE INTRODUCTION Imprinted genes are expressed from only one of their parental alleles and regulated by epigenetic marks such as DNA methylation and histone tail modifications. These epigenetic marks are parent-of- origin specific and distinguish between the two parental alleles of an imprinted gene (Fournier et al., 2002; Lewis et al., 2004; Li et al., 1993; Reik and Walter, 2001; Umlauf et al., 2004). Most imprinted genes occur in clusters in the mammalian genome. Within a cluster, the imprinting of multiple genes is often regulated in a coordinated fashion, involving imprinting centres that acquire allele-specific DNA methylation in the parental germ cells. To date, there are two principal mechanisms described by which an allele-specific DNA methylation mark can lead to imprinting of a cluster of genes (Lewis and Reik, 2006). The first involves inactivating a chromatin insulator by DNA methylation; distal enhancers are prevented from accessing promoters on the unmethylated allele by a repressive higher order chromatin structure but can activate transcription on the methylated allele. This mechanism regulates the Igf2-H19 imprinting cluster (Bell and Felsenfeld, 2000; Hark et al., 2000; Kanduri et al., 2000; Murrell et al., 2004). The second involves a DNA methylation mark that represses a non-coding RNA transcript on one parental allele. On the other allele the non-coding transcript is expressed leading to the repression of flanking genes by targeting polycomb proteins and repressive histone modifications to the region. This mechanism regulates imprinted X inactivation in mouse placenta and is also likely to occur in the Igf2r and Kcnq1 domains (Heard, 2004; Huynh and Lee, 2003; Lewis et al., 2004; Sleutels et al., 2002; Umlauf et al., 2004). The Kcnq1 imprinted domain lies on distal mouse chromosome 7 and contains one paternally expressed gene, the non-coding RNA Kcnq1ot1, several flanking genes which are paternally repressed in all lineages (we term these ubiquitously imprinted genes) and other flanking genes which are paternally repressed in placental lineages but are not imprinted in embryonic lineages (Engemann et al., 2000; Paulsen et al., 2000). It contains two differentially methylated regions (DMRs): one is a germline imprint which acts as the imprinting centre (IC2) and contains the promoter of the non-coding Kcnq1ot1 gene; the other is a secondary imprint upstream of the cell cycle regulator Cdkn1c which is not established until post- implantation stages of development (Bhogal et al., 2004; Engemann et al., 2000; Fitzpatrick et al., 2002). The other genes in the cluster have no associated differential DNA methylation (Lewis et al., 2004). Allele-specific histone modifications are also present at the locus. In the embryo they are restricted to the DMRs. In extra- embryonic lineages, however, repressive histone modifications mark the entire cluster on the paternal chromosome (with the exception of the Kcnq1ot1 region), while the maternal chromosome is marked by histone modifications known to be associated with active chromatin (Umlauf et al., 2004). The repressive histone methylation marks on the paternal chromosome depend on the presence of the Kcnq1ot1 gene (Lewis et al., 2004), and gene silencing in cis of both ubiquitously and placentally imprinted genes indeed requires the Kcnq1ot1 transcript, or transcriptional elongation at the Kcnq1ot1 promoter (Mancini-Dinardo et al., 2006). There are several mechanistic similarities between imprinting in the Kcnq1 domain and imprinted X chromosome inactivation (Huynh and Lee, 2003; Okamoto et al., 2004). The non-coding RNA Xist is paternally expressed in the preimplantation embryo and Epigenetic dynamics of the Kcnq1 imprinted domain in the early embryo Annabelle Lewis 1, *, Kelly Green 1, *, Claire Dawson 1 , Lisa Redrup 1 , Khanh D. Huynh 2 , Jeannie T. Lee 2 , Myriam Hemberger 1 and Wolf Reik 1,† The mouse Kcnq1 imprinted domain is located on distal chromosome 7 and contains several imprinted genes that are paternally repressed. Repression of these genes is regulated by a non-coding antisense transcript, Kcnq1ot1, which is paternally expressed. Maternal repression of Kcnq1ot1 is controlled by DNA methylation originating in the oocyte. Some genes in the region are imprinted only in the placenta, whereas others are imprinted in both extra-embryonic and embryonic lineages. Here, we show that Kcnq1ot1 is paternally expressed in preimplantation embryos from the two-cell stage, and that ubiquitously imprinted genes proximal to Kcnq1ot1 are already repressed in blastocysts, ES cells and TS cells. Repressive histone marks such as H3K27me3 are present on the paternal allele of these genes in both ES and TS cells. Placentally imprinted genes that are distal to Kcnq1ot1, by contrast, are not imprinted in blastocysts, ES or TS cells. In these genes, paternal silencing and differential histone marks arise during differentiation of the trophoblast lineage between E4.5 and E7.5. Our findings show that the dynamics during preimplantation development of gene inactivation and acquisition of repressive histone marks in ubiquitously imprinted genes of the Kcnq1 domain are very similar to those of imprinted X inactivation. By contrast, genes that are only imprinted in the placenta, while regulated by the same non-coding RNA transcript Kcnq1ot1, undergo epigenetic inactivation during differentiation of the trophoblast lineage. Our findings establish a model for how epigenetic gene silencing by non-coding RNA may depend on distance from the non-coding RNA and on lineage and differentiation specific factors. KEY WORDS: Epigenetics, Imprinting, ES and TS cells, Kcnq1 domain, Mouse Development 133, 4203-4210 (2006) doi:10.1242/dev.02612 1 Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge CB2 4AT, UK. 2 Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School Boston, MA 02114, USA. *These authors contributed equally to this work † Author for correspondence (e-mail: wolf.reik@bbsrc.ac.uk) Accepted 5 September 2006