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Depletion of Kcnq1ot1 Non-Coding RNA Does Not Affect Imprinting Maintenance in Stem Cells Michael C

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Depletion of Kcnq1ot1 Non-Coding RNA Does Not Affect Imprinting Maintenance in Stem Cells Michael C DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE 3667 Development 138, 3667-3678 (2011) doi:10.1242/dev.057778 © 2011. Published by The Company of Biologists Ltd Depletion of Kcnq1ot1 non-coding RNA does not affect imprinting maintenance in stem cells Michael C. Golding1,2,3,*,†, Lauren S. Magri1,2,3,†, Liyue Zhang2,3, Sarah A. Lalone1,2,3, Michael J. Higgins4 and Mellissa R. W. Mann1,2,3,‡ SUMMARY 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. KEY WORDS: Genomic imprinting, Noncoding RNA, RNA interference, Kcnq1ot1, Mouse INTRODUCTION paternally expressed ncRNA, Kcnq1ot1, and eight maternally Genomic imprinting is a specialized transcriptional regulatory expressed protein-coding genes, including Slc22a18 (solute carrier mechanism that restricts expression to the maternally or paternally family 22a, member 18), Cdkn1c (cyclin-dependent kinase inherited allele (Verona et al., 2003; Barlow and Bartolomei, 2007). inhibitor 1c), Kcnq1 (potassium voltage-gated channel, KQT-like Imprinted genes often cluster together in large domains that are subfamily, member 1) and Ascl2 (achaete-scute homolog 2) (Fig. coordinately regulated by cis-acting regions known as imprinting 1). The Kcnq1ot1 transcription start site (TSS) is located within the centers (ICs) or imprinting control regions (ICRs). ICRs harbor ICR (Lee et al., 1999; Mitsuya et al., 1999; Smilinich et al., 1999; gamete-derived parental allelic marks and are responsible for Mancini-DiNardo et al., 2003). When methylated on the maternal imprinted gene regulation over hundreds of kilobases in a allele, Kcnq1ot1 is silent (Mancini-DiNardo et al., 2003). On the bidirectional manner. Interestingly, imprinting domains are paternal allele, the ICR is unmethylated and Kcnq1ot1 is associated with a non-coding RNA (ncRNA) that may regulate transcribed. Paternal deletion of the Kcnq1ot1 ICR results in imprinted gene expression for the entire cluster (Barlow and domain-wide loss of imprinting, demonstrating broad control by Bartolomei, 2007). Generally, imprinted ncRNAs are transcribed the ICR of imprinted gene regulation (Fitzpatrick et al., 2002). from the unmethylated ICR and can range from 2.2 to possibly Activation of the normally silent paternal alleles of the protein- more than 1000 kb in length and thus are duly referred to as long coding genes is also seen when Kcnq1ot1 is truncated, possibly or macro ncRNAs. indicating a functional role for the macro ncRNA in imprinted The Kcnq1ot1/KCNQ1OT1 (potassium voltage-gated channel, domain regulation (Mancini-DiNardo et al., 2006; Shin et al., member 1, overlapping transcript 1) imprinting domain is located 2008). on mouse chromosome 7 and human 11p15.5 (Verona et al., 2003; The mechanism by which Kcnq1ot1 represses the expression of Barlow and Bartolomei, 2007). This domain contains one genes located more than 300 kb away is not completely understood, although various models have been proposed. These include a role for the ncRNA in nucleating silent chromatin, similar 1 Departments of Obstetrics and Gynecology and Biochemistry, University of Western to Xist in X inactivation; a role for Kcnq1ot1 transcription in Ontario, Schulich School of Medicine and Dentistry, London, ON N6A 5W9, Canada. 2Children’s Health Research Institute, London, ON N6C 2V5, Canada. 3Lawson initiating chromatin silencing through transcriptional interference, Health Research Institute, London, ON N6C 2V5, Canada. 4Department of Molecular repressive chromatin compartmentalization or chromatin looping; and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA. or a combination of these mechanisms (Mancini-DiNardo et al., *Present address: Department of Veterinary Physiology, College of Veterinary 2003; Lewis et al., 2004; Lewis et al., 2006; Pauler et al., 2007; Medicine, Texas A&M University, College Station, TX 77843, USA Pandey et al., 2008; Shin et al., 2008; Terranova et al., 2008; † These authors contributed equally to this work Koerner et al., 2009; Nagano and Fraser, 2009). Important ‡Author for correspondence ([email protected]) regulatory elements have been identified by mutational analysis but Accepted 17 June 2011 such studies do not differentiate between these various possibilities. DEVELOPMENT 3668 RESEARCH ARTICLE Development 138 (17) Deletion studies of the Kcnq1ot1 ICR domain indicate that the Kcnq1ot1 at 82 kb (K82), or with an shRNA targeting the luciferase gene ICR, a functional promoter and/or transcription are required for (LUC) (Golding et al., 2010). K43 shRNA, which starts at Kcnq1ot1 domain imprinting (Fitzpatrick et al., 2002; Mancini-DiNardo et position 43,804, 5Ј-TGCTGTTGACAGTGAGCGACCAGAGTTTGT - al., 2006). Truncation studies suggest that it is the transcript or CTTTCATAAATAGTGAAGCCACAGATGTATTTATGAAAGACAAA- CTCTGGGTGCCTACTGCCTCGGA-3Ј, was processed into the 22mer transcription that has a role in silencing, as premature termination Ј Ј of Kcnq1ot1 results in the derepression of imprinted protein-coding 5 -CCCAGAGTTTGTCTTTCATAAA-3 ; and K82 shRNA, which begins at Kcnq1ot1 position 82,541, 5Ј-TGCTGTTGACAGTGAGCGATGGCT- genes within their domains (Mancini-DiNardo et al., 2006; Shin et TAAGCTGATCAATTAATAGTGAAGCCACAGATGTATTAATTGAT- al., 2008). However, these studies are unable to differentiate the CAGCTTAAGCCACTGCCTACTGCCTCGGA-3Ј was processed into function of the transcript from that of transcription. Additional the 22mer 5Ј-GTGGCTTAAGCTGATCAATTAA-3Ј. Production of complexity arises from tissue-specific differential imprinted gene recombinant virus, infection, puromycin selection and passage of cells regulation (Caspary et al., 1998; Lewis et al., 2004; Umlauf et al., were performed as described (Golding et al., 2010). Cells were collected 2004; Shin et al., 2008; Weaver et al., 2010). Osbpl5, Tssc4, Cd81 between passages 7 and 27. For the shRNA depletion studies, multiple and Ascl2 are imprinted only in the placenta, whereas Phlda2, independent cell lines were generated and independent biological Slc22a18, Cdkn1c and Kcnq1 are imprinted in embryonic and replicates of wild-type (WT), LUC, K82 and K43 samples were used; six placental tissues (Fig. 1). Thus, functional studies should include to ten each for XEN cell samples, four each for ES cell samples, and four these different lineages in the analyses. to six each for TS cell samples. Robust depletion was observed in K43- transduced stem cells (82-93% depleted) compared with control cells (see Because long ncRNAs may be central regulators of imprinted Fig. S1 in the supplementary material). Importantly, there was considerable domains, it is important to elucidate their modes of action. In this sample overlap for Kcnq1ot1 length analysis, allelic expression analysis study, we investigated the mouse Kcnq1ot1 ncRNA and its role in and fluorescence in situ hybridization. imprinted gene regulation, specifically during preimplantation NIH 3T3 cells were obtained from the American Type Culture development by utilizing embryonic and extra-embryonic stem Collection (Manassas, VA, USA). Custom siRNAs targeting the putative cells. Our findings demonstrate that the Kcnq1ot1 ncRNA Kcnq1ot1 ncRNA were designed using RNAi Codex terminates 471 kb from the TSS, exists primarily as a full-length (http://hannonlab.cshl.edu/GH_siRNA.html) and synthesized by transcript and originates from the Kcnq1ot1 ICR. The length of Dharmacon (Thermo Scientific, Lafayette, CO, USA). si7, 5Ј- GGCAUACUGUCCAUACGUAUU-3Ј, starts at Kcnq1ot1 position 6933, Kcnq1ot1 is conserved in various tissues and at different Ј Ј developmental time points, suggesting that Kcnq1ot1 length does and si463, 5 -CCGAGCAGAUGAUACAGUAUU-3 , starts at Kcnq1ot1 position 463,032. Control siRNAs were an siGLO Green Transfection not contribute to differential tissue-specific silencing. However, the Indicator to monitor transfection success and a scrambled sequence non- length
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