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First published online 14 January 2009
doi: 10.1242/dev.031328

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The long noncoding RNA Kcnq1ot1 organises a lineage-specific nuclear domain for epigenetic gene silencing

Lisa Redrup¹, Miguel R. Branco¹, Elizabeth R. Perdeaux¹, Christel Krueger¹, Annabelle Lewis^1,*, Fátima Santos¹, Takashi Nagano², Bradley S. Cobb^3,, Peter Fraser² and Wolf Reik^1,4,

¹ Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge CB22 3AT, UK.
² Laboratory of Chromatin and Gene Expression, Epigenetics and Chromatin Programme, The Babraham Institute, Cambridge CB22 3AT, UK.
³ MRC Clinical Sciences Centre, Imperial College London, London W12 0NN, UK.
⁴ Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, UK.

View larger version (29K): [in this window] [in a new window]   Fig. 1. The ncRNA Kcnq1ot1 is 80-121 kb long and localised in the nucleus. (A) The paternally expressed Kcnq1ot1 transcript (wavy arrow) is necessary for silencing of adjacent genes on chromosome 7 (red). Genes imprinted solely in the placenta are indicated in red typescript. Four transcription start sites (TSS) were found by 5'-RACE (left box); the predominant TSS is indicated in black. 3'-RACE (right box) identified a polyadenylation site 121 kb downstream of the TSS (polyadenylation signal underlined). (B) qRT-PCR analysis was performed along the length of the Kcnq1ot1 transcript using wild-type and KvDMR1+/- E13.5 embryos. KvDMR1-dependent transcripts can be detected up to at least 80 kb downstream of the TSS (see also Fig. S1 in the supplementary material). *P<0.05, **P<0.001, P<0.0001. Error bars represent s.e.m. (C) qRT-PCR analysis of nuclear and cytoplasmic fractions from primary mouse embryonic fibroblasts shows that Kcnq1ot1 is mainly located in the nucleus. The 45S and 18S rRNAs are included as controls for predominantly nuclear and cytoplasmic RNAs, respectively. Error bars represent s.e.m.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Silencing by Kcnq1ot1 is not dependent on the RNAi pathway. (A) qRT-PCR analysis of miR-16 abundance in control (Dcrflox/; Meox+/+) E10.5 and mutant (Dcrflox/; MeoxCre/+) E10.5 embryos, normalised to SnoRNA-202. The mature form of miR-16 is present in the mutants at a 50-fold lower level, demonstrating that Dicer action has been drastically reduced in the mutants. Error bars represent s.d. (B) RT-PCR and Sequenom MassArray analysis was performed to determine the percentage of maternal expression for the imprinted genes Phlda2, Cdkn1c, Kcnq1ot1 and Kcnq1 in E10.5 embryos. Means are represented as bars and individual data points as blue dots (one control embryo is shown for each cross). Data are shown for both the B6xSD7 and the reciprocal cross to exclude effects due to sequence variations between the B6 and SD7 alleles. Dicer mutants did not show loss of imprinting.

View larger version (43K): [in this window] [in a new window]   Fig. 3. The Kcnq1ot1 transcript establishes a lineage-specific nuclear domain. (A) RNA-FISH was performed on E12.5 embryo or placental sections using probes for Kcnq1, Xist and Kcnq1ot1 (green; arrows). Nuclei were counterstained with DAPI (blue) and imaged by confocal microscopy. Images have been processed using Volocity for volume rendering. Scale bars: 10 µm. (B) After RNA-FISH, the volumes of signals for Kcnq1, Kcnq1ot1 and Xist were measured in the placenta, showing significantly different distributions (P<0.0001, 2 test), with the Kcnq1ot1 signal occupying an intermediate volume between Kcnq1 and Xist. (C) Measurements of Kcnq1ot1 signal volumes between the embryo and placenta reveal that the Kcnq1ot1 nuclear domain is larger in the placenta (P<0.0001, 2 test), in line with the larger silencing effect in this tissue.

View larger version (71K): [in this window] [in a new window]   Fig. 4. Association of genes with the Kcnq1ot1 nuclear domain creates potential for silencing. (A) After labelling of the Kcnq1ot1 nuclear domain by RNA-FISH (green) on E12.5 embryo and placental sections, DNA-FISH (red) was performed to detect an ubiquitously imprinted gene (Cdkn1c), a gene specifically imprinted in the placenta (Ascl2) and a gene not regulated by Kcnq1ot1 located 300 kb away from the gene cluster (Igf2). Nuclei were counterstained with DAPI (blue) and imaged by confocal microscopy. Panels to the right of each image are a magnification of the indicated areas, showing the RNA-FISH signal (top), DNA-FISH signal (middle) and the corresponding merged image (bottom). Scale bars: 1 µm. (B) For each DNA signal found close to the Kcnq1ot1 domain, 3D distances between the centre of the DNA signal and the edge of the domain were measured. Cdkn1c is found mainly inside and Igf2 mainly outside the RNA domain in both tissues, with Ascl2 positioned near the edge. But whereas in the placenta Ascl2 is closer to the domain than the Kcnq1ot1-unrelated Igf2 gene, their relative positions are not significantly different in the embryo. *P<0.05, ***P<0.001; NS, not significant; Kruskall-Wallis test with Dunn's post-hoc test. Bars on the boxplots represent data points within 1.5 times the interquartile range.

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