The Airn lncRNA does not require any DNA elements within its locus to silence distant imprinted genes

Abstract

Long non-coding (lnc) RNAs are numerous and found throughout the mammalian genome, and many are thought to be involved in the regulation of gene expression. However, the majority remain relatively uncharacterised and of uncertain function making the use of model systems to uncover their mode of action valuable. Imprinted lncRNAs target and recruit epigenetic silencing factors to a cluster of imprinted genes on the same chromosome, making them one of the best characterized lncRNAs for silencing distant genes in cis . In this study we examined silencing of the distant imprinted gene Slc22a3 by the lncRNA Airn in the Igf2r imprinted cluster in mouse. Previously we proposed that imprinted lncRNAs may silence distant imprinted genes by disrupting promoter-enhancer interactions by being transcribed through the enhancer, which we called the enhancer interference hypothesis. Here we tested this hypothesis by first using allele-specific chromosome conformation capture (3C) to detect interactions between the Slc22a3 promoter and the locus of the Airn lncRNA that silences it on the paternal chromosome. In agreement with the model, we found interactions enriched on the maternal allele across the entire Airn gene consistent with multiple enhancer-promoter interactions. Therefore, to test the enhancer interference hypothesis we devised an approach to delete the entire Airn gene. However, the deletion showed that there are no essential enhancers for Slc22a2 , Pde10a and Slc22a3 within the Airn gene, strongly indicating that the Airn RNA rather than its transcription is responsible for silencing distant imprinted genes. Furthermore, we found that silent imprinted genes were covered with large blocks of H3K27me3 on the repressed paternal allele. Therefore we propose an alternative hypothesis whereby the chromosome interactions may initially guide the lncRNA to target imprinted promoters and recruit repressive chromatin, and that these interactions are lost once silencing is established.

Placental imprinting: Emerging mechanisms and functions

“…To understand how lncRNAs could target such large genomic regions, the roles of chromosome folding, lncRNA abundance, and CpG islands in the targeting of Airn and Kcnq1ot1 lncRNAs was assessed in trophoblast stem cells [51]. The authors found that chromosome folding brought distal regions into close proximity of the transcribed lncRNA on the paternal allele [51], consistent with allelic differences in folding of the Airn/Igf2r locus seen in vivo [45]. CpG islands, in particular, enabled PRC targeting and spreading along the silenced paternal alleles within these imprinted domains [51].…”

“…In somatic tissues, the extent of imprinting is restricted to a 'core' subset of genes within relatively close proximity to the gDMRs. However, in the placenta, numerous genes, as far away as 7.7 megabases (Mb), are silenced on the paternal allele ( Fig 2B) [36,37,39,43,45]. To date, placental-specific imprinted gene expression has been observed for 15 genes distal of Kcnq1ot1/Kcnq1 and 12 genes distal of Airn/Igf2r (Fig 1).…”

“…A targeted deletion of the entire Airn gene indicated that the Airn lncRNA, rather than its genomic features, was essential for allelic silencing of nonoverlapped genes on the paternal allele [45]. To understand how lncRNAs could target such large genomic regions, the roles of chromosome folding, lncRNA abundance, and CpG islands in the targeting of Airn and Kcnq1ot1 lncRNAs was assessed in trophoblast stem cells [51].…”

Smchd1is a maternal effect gene required for autosomal imprinting

“…For some non-canonical imprinted genes (Smoc1, Jade1, Sfmbt2), it has recently been shown that the H3K27me3 imprint is lost during preimplantation development and through an unknown mechanism the imprint is transferred into a secondary DNA methylation imprint (39). By contrast, the Kcnq1 cluster, the Igf2r-Airn clus-ter and the Magel2 region of the Snrpn cluster gain H3K27me3 in post-implantation tissues, dependent on the germline DMR imprint (39), and for the Kcnq1 and Igf2r-Airn clusters through the action of imprinted long noncoding RNAs (40)(41)(42)(43). Interestingly, Dnmt1 knockout studies have shown that while imprinting of genes located centrally in the Kcnq1 cluster is maintained via germline DMR methylation, genes affected by maternal deletion of Smchd1, Tssc4 and Ascl2, are maintained via histone modifications (44)(45)(46).…”

Screening and evaluation of key genes in contributing to pathogenesis of hepatic fibrosis based on microarray data

“…More and more evidence has indicated that, rather than being transcriptional noise, lots of non-coding RNAs (ncRNAs) can affect the expression levels of target genes [7,8]. Long non-coding RNAs (lncRNAs), one of a ncRNAs, which play critical roles in transcription, splicing and translation [9]. However, up to now, compared with mRNAs, the function of lncRNAs has not been well annotated [10,11].…”