title: Depletion of Kcnq1ot1 non-coding RNA does not affect imprinting maintenance in stem cells.
abstract: To 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.
authors.affiliation: Department of Obstetrics, University of Western Ontario, Schulich School of Medicine and Dentistry, London, ON N6A 5W9, Canada.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Golding
authors.forename: Lauren S
authors.initials: LS
authors.lastname: Magri
authors.forename: Liyue
authors.initials: L
authors.lastname: Zhang
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Lalone
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Higgins
authors.forename: Mellissa R W
authors.initials: MR
authors.lastname: Mann
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005260:Female; D018392:Genomic Imprinting / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction; D024642:Potassium Channels, Voltage-Gated / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D022661:RNA, Untranslated / Q000235:genetics*; D013234:Stem Cells / Q000378:metabolism*; D024363:Transcription Initiation Site / Q000502:physiology
chemical_list: D024642:Potassium Channels, Voltage-Gated; D022661:RNA, Untranslated