Silencing and transcriptional properties of the imprinted Airn ncRNA are independent of the endogenous promoter

Abstract

The Airn macro ncRNA is the master regulator of imprinted expression in the Igf2r imprinted gene cluster where it silences three flanking genes in cis. Airn transcription shows unusual features normally viewed as promoter specific, such as impaired post-transcriptional processing and a macro size. The Airn transcript is 108 kb long, predominantly unspliced and nuclear localized, with only a minority being variably spliced and exported. Here, we show by deletion of the Airn ncRNA promoter and replacement with a constitutive strong or weak promoter that splicing suppression and termination, as well as silencing activity, are maintained by strong Airn expression from an exogenous promoter. This indicates that all functional regions are located within the Airn transcript. DNA methylation of the maternal imprint control element (ICE) restricts Airn expression to the paternal allele and we also show that a strong active promoter is required to maintain the unmethylated state of the paternal ICE. Thus, Airn expression not only induces silencing of flanking mRNA genes but also protects the paternal copy of the ICE from de novo methylation.

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

“…We previously reported a tetracycline (Tet)-inducible Airn allele (Stricker et al, 2008). However, owing to gain of Tet-Airn DNA methylation, these cells were not suitable for further experiments (supplementary material Fig.…”

“…We initially attempted to control endogenous Airn expression using a TetOn system (Stricker et al, 2008). However, the Tetdriven Airn promoter was modified by DNA methylation and the effects of inducing Airn expression could be assayed only in a subset of cells.…”

“…S4). PstI digestion of amplified cDNA from undifferentiated ESCs, which express Igf2r biallelically, yields an undigested maternal band and two paternal PstI-cut fragments ( (Stricker et al, 2008;Wutz et al, 2001). To determine whether Airn is required to maintain Igf2r silencing, we 1187 RESEARCH ARTICLE Igf2r silencing in development turned Airn expression off at day 5, day 9 or day 13 of differentiation, after Igf2r silencing has occurred (seen in the untreated 'no TAM' day 5-17 controls).…”

GnRHa Treatment of Cryptorchid Boys Affects Genes Involved in Hormonal Control of the HPG Axis and Fertility

“…The Airn macro ncRNA is the master regulator of imprinted expression in the Igf2 receptor ( Igfr ) imprinted gene cluster, where it silences 3 flanking genes in cis [Stricker et al, 2008]. Airn expression not only induces silencing of flanking mRNA genes but also protects the paternal copy of ICE from de novo methylation.…”

Macro lncRNAs

“…74 However, replacement of the Airn promoter with the mouse Pgk1 promoter showed that the endogenous promoter does not control the low splicing capacity of the Airn lncRNA. 75 Both the Airn and Kcnq1ot1 macro lncRNAs are unusual in that their CpG island lies immediately downstream of the promoter and although they lack sequence similarities, they each contain a series of tandem direct repeats that have been suggested to guide epigenetic modifications such as the acquisition of the maternal-specific methylation imprint. 76 In support of this, the Airn tandem direct repeats have recently been shown to be necessary for the oocyte-specific DNA methylation imprint.…”