ARTICLE Imprinted Chromatin around DIRAS3 Regulates Alternative Splicing of GNG12-AS1, a Long Noncoding RNA Malwina Niemczyk, 1 Yoko Ito, 1 Joanna Huddleston, 1 Anna Git, 1 Sayeda Abu-Amero, 2 Carlos Caldas, 1 Gudrun E. Moore, 2 Lovorka Stojic, 1 and Adele Murrell 1, * Imprinted gene clusters are regulated by long noncoding RNAs (lncRNAs), CCCTC binding factor (CTCF)-mediated boundaries, and DNA methylation. DIRAS3 (also known as ARH1 or NOEY1) is an imprinted gene encoding a protein belonging to the RAS superfamily of GTPases and is located within an intron of a lncRNA called GNG12-AS1. In this study, we investigated whether GNG12-AS1 is imprinted and coregulated with DIRAS3. We report that GNG12-AS1 is coexpressed with DIRAS3 in several tissues and coordinately downregulated with DIRAS3 in breast cancers. GNG12-AS1 has several splice variants, all of which initiate from a single transcription start site. In placenta tissue and normal cell lines, GNG12-AS1 is biallelically expressed but some isoforms are allele-specifically spliced. Cohesin plays a role in allele-specific splicing of GNG12-AS1. In breast cancer cell lines with loss of DIRAS3 imprinting, DIRAS3 and GNG12-AS1 are silenced in cis and the remaining GNG12-AS1 transcripts are predominantly monoallelic. The GNG12-AS1 locus, which includes DIRAS3, provides an example of imprinted cotranscriptional splicing and a potential model system for studying the long-range effects of CTCF-cohesin binding on splicing and transcriptional interference. Introduction DIRAS family, GTP-binding RAS-like protein 3 (DIRAS3 [MIM 605193]) is imprinted, widely expressed in many adult tissues, and downregulated in several cancers, including breast and ovarian cancers. 1–3 As a tumor sup- pressor, DIRAS3 has been reported to have a role in cell migration, 4–7 proliferation, 8,9 apoptosis, 10,11 and auto- phagy. 10,12 At the DIRAS3 locus, a long noncoding RNA (lncRNA) named LOC100289178, or GNG12-AS1, covers a 370 kb genomic region that starts near the GNG12 promoter, runs through DIRAS3 in an antisense orientation, and ter- minates within G-protein-coupled receptor 177 (GPR177, also known as WLS [MIM 611514]) (Figure 1). DIRAS3 is located within an intron of GNG12-AS1. The arrangement of the two genes is similar to that found in imprinted retro- gene pairs, where a gene is retrotransposed from the X chromosome into a host gene. The retrotransposed gene is imprinted and, in some cases, so is the host gene. 13,14 Although DIRAS3 is not derived from the X chromosome and is therefore not a retrogene, it is nonetheless possible that GNG12-AS1 might also be imprinted similarly to a ret- rogene host. Imprinted genes are associated with differentially meth- ylated regions (DMRs), which comprise promoters and insulator/ boundary elements. 15 It has been shown that CCCTC binding factor (CTCF) and cohesin play a role in regulating imprinting by binding to the unmethylated allele at insulator/boundary elements and a subset of pro- moter DMRs. 15 At several imprinted gene clusters, a pro- moter DMR regulates the expression of an lncRNA that silences adjacent genes in cis. 16 LncRNAs (transcripts of 200 bp and more) are widespread throughout the genome and include ‘‘long intergenic noncoding RNAs’’ (lincRNAs) and macro noncoding RNAs, which are inefficiently pro- cessed and unspliced. 17 The imprinted GNAS, KCNQ1, and mouse Igf2r loci are examples of loci where allele-spe- cific silencing of adjacent genes is mediated by lncRNAs. 17 Imprinted lncRNAs such as Airn and KCNQ1OT1 silence their target genes by recruiting chromatin modifiers: Airn recruits H3K9me3 methyltransferase G9a to the mouse Igf2r locus, 18 and KCNQ1OT1 at the KCNQ1 locus binds both G9a and polycomb repressive complex 2 (PRC2). 19 It has been proposed that the regulation of gene expres- sion in cis by lncRNA at Igf2r, Gnas, and Copg2 imprinted gene clusters might be through the act of transcrip- tional interference without the involvement of a mature RNA. 16 ‘‘Transcriptional interference’’ implies interference between transcriptional machineries along adjacent sequences. 20,21 At the Igf2r locus, it has recently been confirmed that imprinted silencing of Igf2r only requires a transcriptional overlap of Airn at the Igf2r promoter to interfere with recruitment of RNA polymerase. 22 At nonimprinted loci, lncRNAs are increasingly being shown to have a role in epigenetic gene regulation. Exam- ples include HOTTIP and Mistral, which activate the nearby genes at HOXA clusters by recruiting the trimethy- lation of lysine 4 on histone H3 (H3K4me3) methyltrans- ferase MLL1. 23,24 LncRNAs might also regulate genes in trans, as demonstrated by the HOTAIR transcript, which is expressed from the HOXC cluster and recruits PRC2 to silence the HOXD cluster on a separate chromosome. 25 In this study, we characterize GNG12-AS1 and its imprinting state at the DIRAS3 locus in normal and cancer cell lines. We report that it is transcribed from a single 1 Cancer Research UK, Robinson Way, Cambridge CB2 0RE, UK; 2 Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK *Correspondence: adele.murrell@cruk.cam.ac.uk http://dx.doi.org/10.1016/j.ajhg.2013.06.010. Ó2013 by The American Society of Human Genetics. 224 The American Journal of Human Genetics 93, 224–235, August 8, 2013 Open access under CC BY-NC-ND license.