Depletion of Kcnq1ot1 non-coding RNA does not affect imprinting maintenance in stem cells

Abstract

SUMMARYTo understand the complex regulation of genomic imprinting it is important to determine how early embryos establish imprinted gene expression across large chromosomal domains. Long non-coding RNAs (ncRNAs) have been associated with the regulation of imprinting domains, yet their function remains undefined. Here, we investigated the mouse Kcnq1ot1 ncRNA and its role in imprinted gene regulation during preimplantation development by utilizing mouse embryonic and extra-embryonic stem cell models. Our findings demonstrate that the Kcnq1ot1 ncRNA extends 471 kb from the transcription start site. This is significant as it raises the possibility that transcription through downstream genes might play a role in their silencing, including Th, which we demonstrate possesses maternal-specific expression during early development. To distinguish between a functional role for the transcript and properties inherent to transcription of long ncRNAs, we employed RNA interference-based technology to deplete Kcnq1ot1 transcripts. We hypothesized that post-transcriptional depletion of Kcnq1ot1 ncRNA would lead to activation of normally maternal-specific protein-coding genes on the paternal chromosome. Post-transcriptional short hairpin RNA-mediated depletion in embryonic stem, trophoblast stem and extra-embryonic endoderm stem cells had no observable effect on the imprinted expression of genes within the domain, or on Kcnq1ot1 imprinting center DNA methylation, although a significant decrease in Kcnq1ot1 RNA signal volume in the nucleus was observed. These data support the argument that it is the act of transcription that plays a role in imprint maintenance during early development rather than a post-transcriptional role for the RNA itself.

The placental imprintome and imprinted gene function in the trophoblast glycogen cell lineage

“…Using this approach, Okae et al established reciprocal F1 TS cells between B6 and JF1 mice and demonstrated imprinted expression of the Megs Tspan32 and Tfpi2, after confirming appropriate maintenance of imprinted expression at 10 known imprinted genes. Together with similar recent studies carried out on reciprocal [B6xCAST]F1 TS cells (Golding et al, 2011), the imprinted expression of 20 genes have now been confirmed in the trophoblast lineage using TS cells (Table 1). Those results are important since they establish that epigenetic imprinting marks are stably maintained in TS cells in culture, which therefore represent an important model system to study imprinted gene regulation and function in this lineage.…”

“…The genes are sorted by chromosomal position. The data from three recent studies, including two genome-wide RNA-seq analyses of placental imprinted genes, are summarized (Golding et al, 2011;Okae et al, 2011;Wang et al, 2011). …”

Long noncoding RNA-mediated maintenance of DNA methylation and transcriptional gene silencing

“…As the relaxation of imprinting of genes within the Kcnq1 locus remains similar between the deletions encompassing Kcnq1 ICR (3.0 kb), Kcnq1ot1 promoter (246 bp) or the truncation of the Kcnq1ot1 transcript that retains 3.0 kb ICR (Mancini-Dinardo et al, 2006), the observed loss of transcriptional silencing of UIGs is mainly due to conditional depletion of Kcnq1ot1 RNA rather than other functional elements within the Kcnq1 ICR. In a recent investigation, by posttranscriptionally depleting Kcnq1ot1 RNA using a short hairpin RNA (shRNA) in cultured ES cells it was demonstrated that Kcnq1ot1 ncRNA is not required to maintain imprinted silencing of the UIGs (Golding et al, 2011). As these experiments were performed in cultured cells, and for a snapshot of time, it is not clear how well they represent the in vivo situation.…”

A role for chromatin topology in imprinted domain regulation

“…The Kcnq1ot1 imprinted domain spans a 1 Mb region in mice and is characterized by a maternally methylated ICR and two paternally methylated secondary somatic DMRs (at and upstream of the Cdkn1c gene) (Yatsuki et al 2002;Bhogal et al 2004;Lewis et al 2004;Umlauf et al 2004;Golding et al 2011;Mohammad et al 2012) (Fig. 2).…”