A Downstream CpG Island Controls Transcript Initiation and Elongation and the Methylation State of the Imprinted Airn Macro ncRNA Promoter

A CpG island (CGI) lies at the 5′ end of the Airn macro non-protein-coding (nc) RNA that represses the flanking Igf2r promoter in cis on paternally inherited chromosomes. In addition to being modified on maternally inherited chromosomes by a DNA methylation imprint, the Airn CGI shows two unusual organization features: its position immediately downstream of the Airn promoter and transcription start site and a series of tandem direct repeats (TDRs) occupying its second half. The physical separation of the Airn promoter from the CGI provides a model to investigate if the CGI plays distinct transcriptional and epigenetic roles. We used homologous recombination to generate embryonic stem cells carrying deletions at the endogenous locus of the entire CGI or just the TDRs. The deleted Airn alleles were analyzed by using an ES cell imprinting model that recapitulates the onset of Igf2r imprinted expression in embryonic development or by using knock-out mice. The results show that the CGI is required for efficient Airn initiation and to maintain the unmethylated state of the Airn promoter, which are both necessary for Igf2r repression on the paternal chromosome. The TDRs occupying the second half of the CGI play a minor role in Airn transcriptional elongation or processivity, but are essential for methylation on the maternal Airn promoter that is necessary for Igf2r to be expressed from this chromosome. Together the data indicate the existence of a class of regulatory CGIs in the mammalian genome that act downstream of the promoter and transcription start

Long-range DNase I hypersensitivity mapping reveals the imprinted Igf2r and Air promoters share cis-regulatory elements

Epigenetic mechanisms restrict the expression of imprinted genes to one parental allele in diploid cells. At the Igf2r/Air imprinted cluster on mouse chromosome 17, paternal-specific expression of the Air noncoding RNA has been shown to silence three genes in cis: Igf2r, Slc22a2, and Slc22a3. By an unbiased mapping of DNase I hypersensitive sites (DHS) in a 192-kb region flanking Igf2r and Air, we identified 21 DHS, of which nine mapped to evolutionarily conserved sequences. Based on the hypothesis that silencing effects of Air would be directed towards cis regulatory elements used to activate genes, DHS are potential key players in the control of imprinted expression. However, in this 192-kb region only the two DHS mapping to the Igf2r and Air promoters show parental specificity. The remaining 19 DHS were present on both parental alleles and, thus, have the potential to activate Igf2r on the maternal allele and Air on the paternal allele. The possibility that the Igf2r and Air promoters share the same cis-acting regulatory elements, albeit on opposite parental chromosomes, was supported by the similar expression profiles of Igf2r and Air in vivo. These results refine our understanding of the onset of imprinted silencing at this cluster and indicate the Air noncoding RNA may specifically target silencing to the Igf2r promoter

Imprinted <i>Igf2r</i> silencing depends on continuous <i>Airn</i> lncRNA expression and is not restricted to a developmental window

The imprinted Airn macro long non-coding (lnc) RNA is an established example of a cis-silencing lncRNA. Airn expression is necessary to initiate paternal-specific silencing of the Igf2r gene, which is followed by gain of a somatic DNA methylation imprint on the silent Igf2r promoter. However, the developmental requirements for Airn initiation of Igf2r silencing and the role of Airn or DNA methylation in maintaining stable Igf2r repression have not been investigated. Here, we use inducible systems to control Airn expression during mouse embryonic stem cell (ESC) differentiation. By turning Airn expression off during ESC differentiation, we show that continuous Airn expression is needed to maintain Igf2r silencing, but only until the paternal Igf2r promoter is methylated. By conditionally turning Airn expression on, we show that Airn initiation of Igf2r silencing is not limited to one developmental ‘window of opportunity’ and can be maintained in the absence of DNA methylation. Together, this study shows that Airn expression is both necessary and sufficient to silence Igf2r throughout ESC differentiation and that the somatic methylation imprint, although not required to initiate or maintain silencing, adds a secondary layer of repressive epigenetic information.</jats:p

Extra-embryonic-specific imprinted expression is restricted to defined lineages in the post-implantation embryo

AbstractA subset of imprinted genes in the mouse have been reported to show imprinted expression that is restricted to the placenta, a short-lived extra-embryonic organ. Notably, these so-called “placental-specific” imprinted genes are expressed from both parental alleles in embryo and adult tissues. The placenta is an embryonic-derived organ that is closely associated with maternal tissue, and as a consequence, maternal contamination can be mistaken for maternal-specific imprinted expression. The complexity of the placenta, which arises from multiple embryonic lineages, poses additional problems in accurately assessing allele-specific repressive epigenetic modifications in genes that also show lineage-specific silencing in this organ. These problems require that extra evidence be obtained to support the imprinted status of genes whose imprinted expression is restricted to the placenta. We show here that the extra-embryonic visceral yolk sac (VYS), a nutritive membrane surrounding the developing embryo, shows a similar “extra-embryonic–lineage-specific” pattern of imprinted expression. We present an improved enzymatic technique for separating the bilaminar VYS and show that this pattern of imprinted expression is restricted to the endoderm layer. Finally, we show that VYS “extra-embryonic–lineage-specific” imprinted expression is regulated by DNA methylation in a similar manner as shown for genes showing multi-lineage imprinted expression in extra-embryonic, embryonic, and adult tissues. These results show that the VYS is an improved model for studying the epigenetic mechanisms regulating extra-embryonic–lineage-specific imprinted expression

Identification of the human homolog of the imprinted mouse Air non-coding RNA

AbstractGenomic imprinting is widely conserved amongst placental mammals. Imprinted expression of IGF2R, however, differs between mice and humans. In mice, Igf2r imprinted expression is seen in all fetal and adult tissues. In humans, adult tissues lack IGF2R imprinted expression, but it is found in fetal tissues and Wilms' tumors where it is polymorphic and only seen in a small proportion of tested samples. Mouse Igf2r imprinted expression is controlled by the Air (Airn) ncRNA whose promoter lies in an intronic maternally-methylated CpG island. The human IGF2R gene carries a homologous intronic maternally-methylated CpG island of unknown function. Here, we use transfection and transgenic studies to show that the human IGF2R intronic CpG island is a ncRNA promoter. We also identify the same ncRNA at the endogenous human locus in 16–40% of Wilms' tumors. Thus, the human IGF2R gene shows evolutionary conservation of key features that control imprinted expression in the mouse

<i>Airn</i> Transcriptional Overlap, But Not Its lncRNA Products, Induces Imprinted <i>Igf2r</i> Silencing

Long Noncoding RNAs The past 5 years have uncovered thousands of long (&gt;100 nucleotides) noncoding RNAs (lncRNAs) outpacing our understanding of their functions and mechanisms in regulating the genome. Lee (p. 1435 ) reviews the known and suspected means by which these intriguing molecules control gene expression locally and at great distances, considers potential universal roles for lncRNAs, and warns that classifying these intriguing molecules may be quite challenging given their diversity. The very long noncoding RNA, Airn , silences the Igf2r (insulin-like growth factor 2 receptor) imprinted gene cluster in mammals. Latos et al. (p. 1469 ) show in mouse cells that, rather than recruiting enzymes that modify histones to repress the locus, it is the act of transcription of Airn (and not the Airn gene product) that results in the silencing. </jats:p

Active and repressive chromatin are interspersed without spreading in an imprinted gene cluster in the mammalian genome

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The methylation-free state of the paternal ICE depends on the CGI.

<p>(A) DNA blot assaying methylation of the <i>Airn</i> promoter MluI site as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5A</a>, in undifferentiated ES cells carrying a paternal <i>CGIΔ</i> or wildtype (<i>+</i>) allele. The 5.0 kb band identified by probe MEi (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5A</a> map) indicates a gain of methylation on the <i>CGIΔ</i> paternal allele. This band is weaker in cells with lower passage numbers that still retain the selection cassette (<i>S12/CGIΔ+cas</i>-1,-2) compared to cells that have been in culture for 8 more passages (<i>S12/CGIΔ</i>-1A,-1B,-2A,-2B) with a deleted selection cassette. The lower panel confirms this by showing a matching loss of the unmethylated 1.1 kb fragment specific to the paternal allele in cells with a higher passage number. Both panels were from the same blot and the intervening area lacking any hybridisation signal removed. (B) DNA blot as in (A) assaying <i>Airn</i> promoter MluI methylation during ES cell differentiation showing that the level of paternal methylation on the <i>CGIΔ</i> allele in undifferentiated ES (d0) cells (5 kb band) does not change in differentiated d5 and d14 cells. Probe MEi is a 1 kb EcoRI-MluI fragment shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5A</a> map. (C) Bisulfite sequencing of two undifferentiated <i>S12/CGIΔ</i> ES cell clones using primers spanning the deletion that specifically amplify the paternal <i>CGIΔ</i> allele, confirms the strong gain of DNA methylation, but also shows that some alleles are more methylated than others (details as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5B</a>).</p