In genomic imprinting, one of the two parental alleles of an autosomal gene is silenced epigenetically by a cis-acting mechanism. A bidirectional silencer for a 400-kilobase region that contains three imprinted, maternally expressed protein-coding genes (Igf2r/Slc22a2/Slc22a3) has been shown by targeted deletion to be located in a sequence of 3.7 kilobases, which also contains the promoter for the imprinted, paternally expressed non-coding Air RNA. Expression of Air is correlated with repression of all three genes on the paternal allele; however, Air RNA overlaps just one of these genes in an antisense orientation. Here we show, by inserting a polyadenylation signal that truncates 96% of the RNA transcript, that Air RNA is required for silencing. The truncated Air allele maintains imprinted expression and methylation of the Air promoter, but shows complete loss of silencing of the Igf2r/Slc22a2/Slc22a3 gene cluster on the paternal chromosome. Our results indicate that non-coding RNAs have an active role in genomic imprinting.

The 11-zinc finger protein CCCTC-binding factor (CTCF) is a highly conserved protein, involved in imprinting, longrange chromatin interactions and transcription. To investigate its function in vivo, we generated mice with a conditional Ctcf knockout allele. Consistent with a previous report, we find that ubiquitous ablation of the Ctcf gene results in early embryonic lethality. Tissue-specific inactivation of CTCF in thymocytes specifically hampers the differentiation of ab T cells and causes accumulation of late double-negative and immature single-positive cells in the thymus of mice. These cells are normally large and actively cycling, and contain elevated amounts of CTCF. In Ctcf knockout animals, however, these cells are small and blocked in the cell cycle due to increased expression of the cyclin-CDK inhibitors p21 and p27. Taken together, our results show that CTCF is required in a dose-dependent manner and is involved in cell cycle progression of ab T cells in the thymus. We propose that CTCF positively regulates cell growth in rapidly dividing thymocytes so that appropriate number of cells are generated before positive and negative selection in the thymus.

Imprinting of the maternally-expressed Igf2r gene is controlled by an intronic imprint control element (ICE) known as Region2 that contains the promoter of the noncoding Air RNA, whose transcript overlaps the silenced paternal Igf2r promoter in an antisense orientation. Two novel imprinted genes, Slc22a2 and Slc22a3 are described here that lie 110 and 155 kb 3 to Igf2r and that are not overlapped by the Air transcript but are regulated by the Igf2r-ICE, as previously shown for Igf2r. These results identify a new cluster of imprinted genes whose repression by the bidirectional action of the Region2-ICE is independent of transcript overlap by the Air RNA. Cis-acting epigenetic mechanisms that regulate gene expression are of critical importance in understanding how gene regulation is controlled in development and disease, but are currently understood only at a basic level. Of the many mammalian systems currently used to investigate epigenetic mechanisms, parental-specific gene expression induced by genomic imprinting has the best potential to identify relevant DNA sequences and modifications, since both the active and silent parental alleles are retained in the same nuclear environment and can be directly compared (for reviews, see Reik and Walter 2001;Sleutels and Barlow 2001). At present, 50 imprinted genes have been identified with approximately equal numbers of maternally and paternally expressed examples . This may represent a near complete collection, since the total number of imprinted genes, predicted to be in the range of 200, should now be reduced by 60%, following the reappraisal of the total number of genes in the mammalian genome (Claverie 2001).A large body of evidence indicates that imprinted genes are regulated by cis-linked DNA imprint control elements (ICEs) that are epigenetically modified by an imprint in either the maternal germ line (for maternally imprinted genes) or paternal germ line (for paternally imprinted genes). Experimental data also indicate that DNA methylation is the imprint or at least is involved in maintaining the imprint for most but not all genes (Caspary et al. 1998;Jackson-Grusby et al. 2001). In contrast to early expectations, the data clearly show that imprints can act either as epigenetic activators to activate one allele of an imprinted gene, or as epigenetic repressors to silence one allele (Obata et al. 1998;Kato et al. 1999;Jackson-Grusby et al. 2001). The observation that imprinted genes are clustered within a chromosomal region has led to the suggestion that the ICE may also serve as an epigenetic domain regulator that controls the imprinted expression of more than one imprinted gene. To date, seven clusters have been identified in the mouse genome that are also conserved in the human genome . The clusters show generally similar behavior whereby a majority of the clustered genes are expressed from one parental chromosome and only a minority from the reciprocal parental chromosome. Two well-known examples are the Prader-Willi syndrome (PWS) region (human 15q11-15 syn...