Imprinted Chromatin Around DIRAS3 Regulates Alternative Splicing of GNG12-AS1, a Long Noncoding RNA

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 Murrell1,*

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 200 bp and more) are widespread throughout the genome and include ‘‘long intergenic noncoding RNAs’’ (lincRNAs) DIRAS family, GTP-binding RAS-like protein 3 (DIRAS3 and macro noncoding RNAs, which are inefficiently pro- [MIM 605193]) is imprinted, widely expressed in many cessed and unspliced.17 The imprinted GNAS, KCNQ1, adult tissues, and downregulated in several cancers, and mouse Igf2r loci are examples of loci where allele-spe- including breast and ovarian cancers.1–3 As a tumor sup- cific silencing of adjacent genes is mediated by lncRNAs.17 pressor, DIRAS3 has been reported to have a role in Imprinted lncRNAs such as Airn and KCNQ1OT1 silence cell migration,4–7 proliferation,8,9 apoptosis,10,11 and auto- their target genes by recruiting chromatin modifiers: Airn phagy.10,12 recruits H3K9me3 methyltransferase G9a to the mouse At the DIRAS3 locus, a long noncoding RNA (lncRNA) Igf2r locus,18 and KCNQ1OT1 at the KCNQ1 locus binds named LOC100289178, or GNG12-AS1, covers a 370 kb both G9a and polycomb repressive complex 2 (PRC2).19 genomic region that starts near the GNG12 promoter, It has been proposed that the regulation of gene expres- runs through DIRAS3 in an antisense orientation, and ter- sion in cis by lncRNA at Igf2r, Gnas, and Copg2 imprinted minates within G-protein-coupled receptor 177 (GPR177, gene clusters might be through the act of transcrip- also known as WLS [MIM 611514]) (Figure 1). DIRAS3 is tional interference without the involvement of a mature located within an intron of GNG12-AS1. The arrangement RNA.16 ‘‘Transcriptional interference’’ implies interference of the two genes is similar to that found in imprinted retro- between transcriptional machineries along adjacent gene pairs, where a gene is retrotransposed from the X sequences.20,21 At the Igf2r locus, it has recently been chromosome into a host gene. The retrotransposed gene confirmed that imprinted silencing of Igf2r only requires is imprinted and, in some cases, so is the host gene.13,14 a transcriptional overlap of Airn at the Igf2r promoter to Although DIRAS3 is not derived from the X chromosome interfere with recruitment of RNA polymerase.22 and is therefore not a retrogene, it is nonetheless possible At nonimprinted loci, lncRNAs are increasingly being that GNG12-AS1 might also be imprinted similarly to a ret- shown to have a role in epigenetic gene regulation. Exam- rogene host. ples include HOTTIP and Mistral, which activate the Imprinted genes are associated with differentially meth- nearby genes at HOXA clusters by recruiting the trimethy- ylated regions (DMRs), which comprise promoters and lation of lysine 4 on histone H3 (H3K4me3) methyltrans- insulator/ boundary elements.15 It has been shown that ferase MLL1.23,24 LncRNAs might also regulate genes in CCCTC binding factor (CTCF) and cohesin play a role in trans, as demonstrated by the HOTAIR transcript, which regulating imprinting by binding to the unmethylated is expressed from the HOXC cluster and recruits PRC2 to allele at insulator/boundary elements and a subset of pro- silence the HOXD cluster on a separate chromosome.25 moter DMRs.15 At several imprinted gene clusters, a pro- In this study, we characterize GNG12-AS1 and its moter DMR regulates the expression of an lncRNA that imprinting state at the DIRAS3 locus in normal and cancer silences adjacent genes in cis.16 LncRNAs (transcripts of cell lines. We report that it is transcribed from a single

1Cancer Research UK, Robinson Way, Cambridge CB2 0RE, UK; 2Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ajhg.2013.06.010. Ó2013 by The American Society of Human Genetics. All rights reserved.

224 The American Journal of Human Genetics 93, 224–235, August 8, 2013 Figure 1. Organization of DIRAS3 and the GNG12-AS1 Locus (A) The organization of the entire GNG12- AS1 locus (chr1: 68,297,971–68,668,670 [UCSC genome browser hg19) shows CTCF binding sites (designated a–k), CpG islands, and the positions of SNPs for DIRAS3 and GNG12-AS1. The known exons of GNG12-AS1 are enlarged for clarity and marked 1–9. (B) Enlarged view of DIRAS3 and its sur- rounding region. Exon 2 of GNG12-AS1 is 11 kb downstream of DIRAS3, and exon 3 is 21 kb upstream. Differentially methylated DIRAS3-associated regions DMR1, DMR2, and DMR3 are shown. DMR1 corresponds to the DIRAS3 promoter, DMR2 corresponds to the transcription start site (TSS), and DMR3 corresponds to exon 2 of DIRAS3. Binding site CTCF-f corresponds to DIRAS3 DMR1, and binding site CTCF-e corresponds to DIRAS3 DMR3. (C) Schematic representation of imprinted DIRAS3. DIRAS3-associated DMRs are shown as lollipops (black represents methylated, and white represents unmethylated). CTCF binds only to the unmethylated DMR1. The arrow on the paternal allele indicates expression and direction of DIRAS3. Abbreviations are as follows: M, maternal allele; and P, paternal allele.

Cell Lines and Reagents Human mammary epithelial cells (HB2 cell line) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) supplemented with 10% fetal promoter on both alleles and that specific isoforms are bovine serum (FBS), 10 ml/l penicillin-streptomycin solution spliced on one allele only. Cohesin plays a role in this (P/S), 5 mg/ml insulin (Sigma), and 1 mg/ml hydrocortisone allele-specific splicing. We further show that transcription (HC, Sigma). SUM159 cells were cultured in Ham’s F-12 m of DIRAS3 and GNG12-AS1 is coordinately downregulated medium (GIBCO) supplemented with 5% FBS, 5 g/ml insulin, m in breast cancers. Where DIRAS3 is epigenetically silenced and 1 g/ml HC; MCF7, K562, and Hs27 cell lines were cultured in DMEM with 10% FBS and 10 ml/l P/S. CAL51 in cancer cell lines, transcription of GNG12-AS1 is downre- cells were cultured in RPMI 1640 with 10% FBS, and MCF10A gulated allele specifically and becomes predominantly cells were cultured in MEGM SingleQuots (Lonza). Cell lines monoallelic. were cultured at 37C. For 5-azacytidine treatment, 5-aza- 20deoxycytidine (5-aza) (A3656, Sigma) was added to the m 27 Material and Methods medium at 1 M for 48 hr, as previously described. Alpha- amanitin (Sigma) was used at 5 mg/ml for 24 hr. Antibodies, siRNAs, and primers used in this study are in Table S1–S4 (avail- Human Tissue DNA and RNA Samples able online). Placenta DNA and RNA samples were obtained from G.E.M. at the Institute of Child Health at University College London. Samples were collected with informed consent under the RNA Interference guidelines of the Hammersmith and Queen Charlotte’s and Cell lines were transfected with 50 nM of siRNAs targeting CTCF Chelsea Hospitals Trust Research Ethics Committee (registration (catalog no. M-020165-01), Rad21 (catalog no. M-006832-01), no. 2001/6029). Total RNA from human tissues used in experi- and DIRAS3 (catalog no. M-008660-01) with the use of ments was commercially obtained from Ambion. DIRAS3 and DharmaFECT 3 and/or DharmaFECT 4 (Dharmacon) as per the GNG12AS1 expression analyses utilized 79 breast tumor samples manufacturer’s instructions. Control siRNA was siGENOME Non- and 36 normal breast samples obtained under ethical approval Targeting siRNA Pool #2 (Dharmacon, catalog no. D-001206-14- from the Molecular Taxonomy of Breast Cancer International 20). The cells were collected for RNA and protein analysis 48 hr Consortium.26 after transfection.

The American Journal of Human Genetics 93, 224–235, August 8, 2013 225 Nuclear Fractionation 50 and 30 Rapid Amplification of cDNA Ends For nuclear fractionation, HB2 cells were harvested and resus- 50 and 30 rapid amplification of cDNA ends (RACE) was performed pended in 1 ml PBS, 1 ml buffer C1 (cell-lysis buffer, QIAGEN), with the RLM RACE Kit (Ambion). Primers for 30 and 50 RACE are and 3 ml water and incubated for 15 min on ice. Then cells were listed in Table S7. centrifuged at 2,500 rpm for 15 min, the supernatant was dis- carded, and the nuclear pellet was kept for RNA extraction. Primers ChIP Assays used for GNG12-AS1 were against exon 7 (Table S3). ChIP assays for RNAPII, CTCF, cohesin, and trimethylation of lysine 36 on histone H3 (H3K36me3) were performed as described RNA Isolation and Quantitative Real-Time PCR previously32 with the use of antibodies as listed in Table S1. The RNA was extracted and reverse transcribed with the RNeasy Kit input and the immunoprecipitated materials were quantified by and the QuantiTect Reverse Transcription Kit (QIAGEN). Quantita- QubitFluorometer (Life Technologies) with the dsDNA BR Assay tive real-time PCR was performed on a 7900HT Fast Real-Time PCR Kit (Invitrogen). Per ChIP experiment, 20–50 mg of chromatin System (Applied Biosystems) with Fast SYBR Green Master Mix and 5 mg of antibody were used. The qPCR data were corrected for (Invitrogen). We used PCR Miner software28 to calculate both DNA amount, and enrichment was normalized against the input the cycle threshold of each PCR and the amplification efficiency according to the formula 2 [Ct(Ab) log2(DF)] [Ct(input) log2(DF)]. of each primer pair. To determine the expression levels of The data were further normalized against the negative-control re- GNG12-AS1 transcripts, we used the assay for exons 7 and 8. To gion FOXA1 for CTCF and RAD21 ChIP. RNAPII and H3K36me3 determine the relative abundance of specific splice variants of ChIP experiments were normalized against positive-control re- GNG12-AS1 transcripts, we used expression primers to exon- gions GAPDH transcription start site (TSS) and GAPDH exon 6, exon junctions. Expression levels were normalized to GAPDH respectively. Each ChIP experiment contained at least three biolog- expression (Table S3). ical replicates, primers for which are listed in Table S4.

Cell Lysis and Immunoblot Statistics Cells were harvested and washed twice with PBS, resuspended in Unless otherwise indicated, an independent two-sample t test was lysis buffer (50 mM Tris-HCl, pH 8, 125 mM NaCl, 1% NP-40, carried out for the statistical analysis in all experiments. Signifi- 2 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail [Roche]) cance of the paired siRNA experiments and paired 5-aza experi- and incubated on ice for 25 min. The proteins were denatured, ments was calculated with a paired two-sample t test. reduced, and separated by SDS-PAGE, and immunoblotting Values from all experiments were used for calculation of the was performed as described previously.29 Secondary antibodies means and their respective SEMs. p values < 0.05 were considered were conjugated with peroxidase, and immunobands were de- statistically significant. Correlation coefficients (R) (Figure S1) tected with a Supersignal West Dura HRP Detection Kit (Thermo- were calculated with Pearson tests. Scientific). Results Analysis of Allelic Expression Cell lines, chromatin immunoprecipitation (ChIP) DNA, and Confirmation of Genomic Imprinting of DIRAS3 in placenta samples were genotyped for exonic SNPs identified in Selected Normal and Breast Cancer Cell Lines the UCSC Genome Browser (hg19). Informative (i.e., heterozy- DIRAS3 is not present in the rodent lineage, so we selected gous) samples were used in the analysis of allelic expression by pyrosequencing (PSQ) and Sanger sequencing. For PSQ allelic five human cell lines to study its effects on GNG12-AS1 analysis of GNG12-AS1, long-range PCRs were performed with expression (Table S8). First, we identified four human primers located at exons 1 and 9. For analysis of splice variants cell lines with heterozygous SNPs in exon 2 of DIRAS3 containing exon 2, long-range PCRs with primers located in exons (Table S8)—two normal cell lines (HB2 [breast epithelial] 2 and 9 were performed. Each biological sample was assayed with and Hs27 [foreskin fibroblast]) and two breast cancer cell the same SNP for both major and exon 2 isoforms. Terms ‘‘allele 1’’ lines (SUM159 and CAL51)—and examined allelic expres- and ‘‘allele 2’’ were used for unifying results between different cell sion of DIRAS3. We confirmed that the normal cell lines lines. For allelic expression of transcripts terminating at exon 7, have imprinting of DIRAS3 transcription (monoallelic) rs11209218 was utilized. Primers for PSQ assays are listed in Table and that the cancer cell lines have loss of imprinting S5. For allele-specific enrichment of RNA polymerase II (RNAPII) (LOI), manifested as basal low levels of biallelic expression and CTCF binding, Sanger sequencing was carried out with the (Figure S2A). The expression levels of DIRAS3 in normal quantitative real-time PCR products obtained after ChIP (primers are listed in Table S4). cell lines (Hs27, MCF10A, and HB2) were higher than those in cancer cell lines (SUM159, CAL51, and MCF7) (Figure 2A). DIRAS3 is normally expressed only from the Analysis of DNA Methylation paternal allele. The maternal allele of DIRAS3 is silenced Analysis of DNA methylation was done by bisulfite conversion and has a differentially methylated promoter (DMR2) with the EZ DNA Methylation-Gold Kit (Zymo Research) and sub- sequent PSQ as previously described.30 PSQ assays for DIRAS3 re- and an upstream CpG island containing a CTCF binding 33 gions DMR1, DMR2, and DMR3 are listed in Table S6. Methylation site (DMR1). We therefore investigated the methylation analysis by DNA immunoprecipitation was done as previously state of these DIRAS3 DMRs in our cell lines. None of the described31 with an antibody (mAb-006) from Diagenode. Primers cell lines had SNPs at the DMRs, so we used a PSQ assay to are listed in Table S4. determine the overall percentage of methylation at the

226 The American Journal of Human Genetics 93, 224–235, August 8, 2013 Figure 2. DIRAS3 and GNG12-AS1 Are Coexpressed in Several Tissues and Reduced in Breast Cancer (A) DIRAS3 expression in a panel of tissues and in cell lines. The dotted line marks the background DIRAS3 expression in cell lines with loss of imprinting. (B) GNG12-AS1 expression in tissues and cell lines. Expression levels were normalized to GAPDH. Error bars represent the SDs from at least three biological experiments. Because of the wide expression range, the y axis is split. (C) Nuclear localization of GNG12-AS1 in HB2 cells. U1 RNA is a control nuclear RNA, and RPS18 is a control cytoplasmic RNA. Values represent the median of three replicate experiments 5 SD. (D) Coordinate reduction of DIRAS3 and GNG12-AS1 in normal breast (n ¼ 72) and tumor breast (n ¼ 36). ****p < 0.0001.

CAL51, and MCF7 are good models for loss of DIRAS3 imprinting.

GNG12-AS1 Has Several Splice Variants Using RT-PCR with primers located in exons 1 and 9, we confirmed that GNG12-AS1 is expressed in a human cDNA tissue panel and human cell lines. RT-PCR products from human kidney and the HB2 cell line were cloned and sequenced. We found that GNG12-AS1 has multiple splice variants (Figure S3A). 50 and 30 RACE with primers located in exons 7 and 9(Figures S4A–S4E) showed a single TSS that lies within exon 1 (and starts at chr1: 68,298,000 [hg19]) and three polyadenylation sites, as indicated in Figure S3A. Exons 7 and 8 seemed to be present in most of the transcripts. Analysis of exon inclusion by mea- DMRs. Two of the normal cell lines (HB2 and Hs27) surement of the ratios of splice junctions relative to exons showed the expected 50% methylation at DMR1 and 7 and 8 indicated that transcripts with splice junctions DMR2, which is in accordance with normal imprinting from exons 1 to 3 and from exons 1 to 2 are less frequent at the locus. The cancer cell lines (SUM159, MCF7, and (Figure S3B). CAL51) were hypermethylated at DMR1. SUM159 and CAL51 had 50% methylation at DMR2, and MCF7 had GNG12-AS1 Is Coexpressed with DIRAS3 in a Wide hypermethylation at DMR2 (Figure S2B and Table S8). Range of Tissues and Cell Lines The CpG island located within the DIRAS3 gene body To determine whether GNG12-AS1 and DIRAS3 are coex- has been described as a DMR (DMR3).33 However, we pressed, we performed RT-PCR by using intron-spanning have previously found it to be hypermethylated in primers in a panel of tissues and cell lines (Figures 2A normal tissues, suggesting that if it is a DMR, it is likely and 2B). DIRAS3 is highly expressed in the ovaries, to be tissue specific.15 Similarly, DMR3 was found to be pancreas, testes, cervix, and breasts, whereas GNG12-AS1 hypermethylated in all of our cell lines (Figure S2B). These has the highest expression in the pancreas, small intestine, data confirm that the HB2 and Hs27 cell lines are suitable testes, liver, breasts, cervix, and adipose tissue. We models for normal DIRAS3 imprinting and that SUM159, found that the expression of GNG12-AS1 is significantly

The American Journal of Human Genetics 93, 224–235, August 8, 2013 227 Figure 3. GNG12-AS1 Is a RNAPII Tran- script with an H3K36me3 Signature Char- acteristic of a Transcribed Locus (A) A schematic representation of the GNG12-AS1 locus shows the relative loca- tion of GNG12-AS1 exons (above the line) and the locations of primers used for ChIP analysis (bars below the line). (B) RNAPII enrichment occurred throughout the GNG12-AS1 locus in HB2 and SUM159 cells. Note the signiﬁcant reduction of RNAPII enrichment at DIRAS3 for SUM159 cells. In the left panel, representative chromatograms for SNP analysis indicate biallelic enrichment in HB2 cells and monoallelic enrichment in SUM159 cells. (C) Enrichment of H3K36me3 in HB2 and SUM159 cells and representative chromatograms indicating allelic enrichment for SNP analysis. Error bars represent the SEM from three independent biological experiments for HB2 cells and three independent experiments for SUM159 cells.

the 1p31 region, where DIRAS3 is located, is often deleted in breast cancer, we checked our samples for loss of heterozygosity (LOH). We did not ﬁnd LOH at this locus in our samples (data not shown), suggesting that the whole region is subject to epigenetic silencing in breast cancer.

GNG12-AS1 Is an RNAPII Transcript with H3K36me3 Signatures of Transcribed Gene LncRNAs exhibit chromatin signatures similar to those of transcribed coding genes, such as enrichment of RNAPII, H3K4me3, and H3K36me3.34,35 correlated with DIRAS3 expression in tissues (R ¼ 0.607, To determine whether GNG12-AS1 is an RNAPII tran- Pearson correlation) and cell lines (R ¼ 0.960, Pearson cor- script, we treated HB2 cells with alpha-amanitin, which relation) (Figures S1A and S1B). We also found a correlation is known to inhibit RNAPII at low concentrations (5 mg/ between GNG12-AS1 and GNG12 expression in tissues (R ¼ ml).36 After 24 hr, MYC, a positive RNAPII transcript con- 0.553, Pearson correlation) and cell lines (R ¼ 0.912, Pear- trol, was fully downregulated, whereas the RNAPI tran- son correlation), which might be explained by the overlap script, RPS18, was unaffected. GNG12-AS1 transcription of their promoters (Figures S1C and S1D). GNG12-AS1 was also inhibited (Figure S5), suggesting that it is indeed overlaps the 30 end of WLS. However, we observed no cor- an RNAPII transcript. relation between GNG12-AS1 and WLS expression (Figures We next compared RNAPII enrichment at GNG12-AS1 S1E and S1F). We also investigated the cellular localization between normal cells with imprinted DIRAS3 (HB2) and of GNG12-AS1 and found that it is predominantly present cancer cells with loss of imprinted DIRAS3 expression in the nucleus (Figure 2C). (SUM159). The antibody used for ChIP recognizes both DIRAS3 expression is known to be reduced in breast can- serine 2 and serine 5 phosphorylated forms of RNAPII cer; therefore, we examined expression levels of DIRAS3 and has a preference for serine 5. We found that RNAPII and GNG12-AS1 in a panel of tumor cDNAs with matched was enriched at the TSSs of GNG12 and GNG12AS1,as normal tissue from individuals with breast cancer. We well as 1.6 and 2.2 kb downstream of the TSS in the first found that there was significantly less expression of both intron of GNG12-AS1, confirming that it is transcribed by DIRAS3 and the GNG12-AS1 in cancer tissues than in RNAPII in both cell lines (Figures 3A and 3B). At the normal breast tissues (p < 0.0001) (Figure 2D). Because DIRAS3 region, enrichment of RNAPII was only found in

228 The American Journal of Human Genetics 93, 224–235, August 8, 2013 Figure 4. GNG12-AS1 Transcripts Con- taining Exon 2 Are Monoallelic (A) GNG12-AS1 splice variants containing exon 2 in HB2 cells. The majority of these transcripts are expressed from allele A, as determined by cloning of PCR products and PSQ. These transcripts also contain exon 3. Rare transcripts with exon 2 but without exon 3 are produced only from allele G. (B and C) Allele-speciﬁc expression, analyzed by PSQ, of GNG12-AS1 major isoforms in cell lines and placenta (B) and allele-speciﬁc expression, analyzed by PSQ, of GNG12-AS1 isoforms containing exon 2 in the same cell lines and placenta (C). Error bars indicate the means 5 SDs of three or more replicates. In (B) and (C), two different SNPs were assessed, and the results are therefore presented as alleles 1 and 2. The same SNPs were used in the assay of transcripts 1–9 (major biallelic transcripts) and transcripts 2–9 (monoallelic transcripts containing exon 2). (D) Maternal expression of GNG12-AS1 transcripts with exon 2 in two representative placenta triads (parents and placenta) indicates expression from the maternal allele. (E) Proportion of transcript variants in normal and cancer cell lines, as determined by qPCR. Relative abundance of the transcripts was normalized to GAPDH and related to expression of exons 7 and 8. n R 3 independent replicate experiments.

H3K36me3 as observed with RNAPII. Thus, in HB2 cells, both GNG12-AS1 alleles have H3K36me3, whereas in SUM159 cells, the enrichment is skewed toward one allele. Together, these data indicate that GNG12-AS1 has a chromatin signature similar to that of other lncRNAs and point the normal HB2 cells and not in the SUM159 cancer cells, toward allelic differences in transcription between normal which accords with the differences in DIRAS3 imprinting and cancer cells (see below). and low levels of DIRAS3 expression in SUM159. Very low RNAPII enrichment was seen at GNG12-AS1 exons 2 Some Splice Variants of GNG12-AS1 Are Allele Specific and 3, which fits with their low frequency of inclusion To determine whether GNG12-AS1 is imprinted, we utilized into GNG12-AS1 transcripts. Allele-specific analyses using SNPs located in exons 7 and 9 and examined the cloned se- a SNP located in exon 9 showed RNAPII occupation at quences from the HB2 cell line. We found that most splice GNG12-AS1 on both alleles in the normal cell line HB2 variants are biallelic in this cell line. Additionally, splice var- (Figures 3A and 3B). However, in the cancer cell line iants with alternative termination of transcription at exons SUM159, we unexpectedly observed that occupation of 7 and 8d were found to be biallelic (data not shown). In RNAPII was biased toward one allele, suggesting that contrast, we found that splice variants containing exon 2 GNG12-AS1 might be expressed in a monoallelic manner are predominantly monoallelic (allele A in HB2 cells) (Fig- in SUM159 cells (Figure 3B). ure 4A). However, different allele specificities were found H3K36me3 ChIP experiments in the same cell lines depending on whether exon 3 was also included in the confirmed that GNG12-AS1 is enriched with H3K36me3 isoforms containing exon 2. Exon 3 is absent in a small pro- within the gene body (Figure 3C). SNP analysis of precipi- portion (10%) of isoforms containing exon 2, and these tated material indicated similar allele-specific binding for transcripts originate from allele G in the HB2 cell line.

The American Journal of Human Genetics 93, 224–235, August 8, 2013 229 DMR1, Figure S2A and S2B), we expected to find that the exon 2 isoforms are biallelic. Instead, we consistently found that all the GNG12-AS1 transcripts are predominantly monoallelic and on the same allele as the exon 2 isoforms (change in biallelic 50% to 50% ratio to at least 70% to 30% ratio in cancer cell lines [Figures 4B and 4C]). Given that we found these exon 2 isoforms to be of maternal origin in the placenta, we extrapolate that in cancer cell lines the monoallelic expression is biased toward the maternal allele upon silencing of the paternally expressed DIRAS3 allele. The monoallelic expression in the cancer cell lines is consistent with the allele-specific enrichment observed with RNAPII and H3K36me3 ChIP experiments (Figure 3). To exclude the possibility that the observed allele bias for GNG12-AS1 was due to an increase in exon 2 isoforms, we analyzed the proportions of the variant isoforms in normal (HB2) and cancer Figure 5. Demethylation of DMRs Restores Imprinted DIRAS3 (SUM159) cell lines. We found no increase in the exon 2 Expression in Breast Cancer Cell Line SUM159 and Reduces isoforms in the SUM159 cell line, confirming that splicing GNG12-AS1 Expression choice is not responsible for monoallelic expression of (A) Methylation of DIRAS3 DMRs in control (nontreated) and 5-aza-treated SUM159 cells. GNG12-AS1 in cancer cell lines (Figure 4E). These results (B) Relative expression of DIRAS3, GNG12, WLS, and GNG12-AS1 indicate that neoplastic silencing of the normally active in control and 5-aza-treated cells. Expression levels were normal- DIRAS3 allele also reduces transcription of the GNG12- ized to GAPDH. Because of the large expression differences, the AS1 lncRNA in cis but does not affect its transcription on y axis is split. (C) Allelic expression of DIRAS3 in SUM159 cells in control and the homologous chromosome with the constitutively 5-aza-treated cells. silent imprinted DIRAS3 allele. This interpretation could (D) Allelic expression of GNG12-AS1 major isoforms in SUM159 explain the apparent gain of imprinting of GNG12-AS1 cells upon 5-aza treatment. in cancer cell lines. (E) Allelic expression of GNG12-AS1 transcripts containing exon 2 in SUM159 cells. Error bars represent the SDs from at least four independent biological replicates. The statistical significance Demethylation of a Cancer Cell Line with Loss of of the individual bars is compared to their respective control DIRAS3 Imprinting Leads to Upregulation of DIRAS3 < < < bars; *p 0.05, **p 0.01, ***p 0.001. Expression and a Decrease in the Levels of GNG12-AS1 Transcripts We then developed assays to quantitatively detect allele In the breast cancer cell lines, despite predominantly ratios for the major population of GNG12-AS1 isoforms monoallelic GNG12-AS1 expression, the GNG12-AS1 pro- (captured with long PCR assays with primers in exons 1 moter was unmethylated (Figure S6). To examine whether and 9) (Figure 4B) and for isoforms containing exon 2 we could change allele-specific transcription of GNG12- (captured with primers in exons 2 and 9 Figure 4C). Using AS1 by reducing DNA methylation at the DIRAS3 DMRs, these assays, we were able to show that the normal breast we treated the SUM159 cell line with 5-aza. SUM159 was cell lines HB2 and MCF10a, as well as placenta tissue, are hypermethylated at all DMRs, and after 5-aza treatment, biallelic for the major transcripts and monoallelic for the the methylation at DMR1, DMR2, and DMR3 was exon 2 isoforms (Figures 4B and 4C). Genetic analyses in decreased to 50%, 40%, and 40%, respectively (Figure 5A). three placenta trios indicated that the monoallelic isoforms DIRAS3 expression was increased by 10-fold predomi- of GNG12-AS1 are expressed from the maternal allele, nantly on one allele, so it appeared to be similar to where DIRAS3 is silenced (Figure 4D). Maternal contamina- imprinted expression in normal cells (Figures 5B and 5C). tion in placenta samples was excluded by SNP analysis of The skewed upregulation of DIRAS3 expression after maternal markers (results not shown). Because there was 5-aza treatment suggests that the constitutively silent no evidence of methylation at the CpG island upstream imprinted allele is resistant to 5-aza treatment. of the GNG12-AS1 promoter region in our cell lines (Figures Demethylation of the DIRAS3 locus and concomitant S6A–S6D), it seems likely that transcription initiates on upregulation of DIRAS3 expression resulted in a significant both alleles and that allele-specific splicing occurs at the reduction of GNG12-AS1 expression. However, there was intron that contains the imprinted silenced DIRAS3. no effect on GNG12 or WLS (Figure 5B), suggesting that increased transcription of DIRAS3 might attenuate tran- GNG12-AS1 Is Predominantly Monoallelic in Breast scription of the lncRNA. Indeed, GNG12-AS1 allelic expres- Cancer Cell Lines with Loss of DIRAS3 Imprinting sion changed from predominantly monoallelic (70% to In the cancer cell lines (SUM159, Cal51, and MCF7), which 30% ratio of C to T) to completely monoallelic (reaching had loss of DIRAS3 imprinting (i.e., hypermethylation at a 94% to 6% ratio of C to T), indicating that the reduction

230 The American Journal of Human Genetics 93, 224–235, August 8, 2013 of transcription occurs at the allele that already has We then analyzed CTCF-cohesin binding profiles at reduced transcription in the cancer cell line. Because GNG12-AS1 in HB2 and SUM159 cells. We used the GNG12-AS1 repression occurs on the same chromosome CTCF-b site as a positive reference for normalization and as the increased DIRAS3 expression, this could suggest found that CTCF and the cohesin subunit RAD21 bind transcriptional interference from the upregulated DIRAS3 strongly to the CTCF-b, CTCF-c, and CTCF-i sites in both allele (Figures 5D and 5E). cell lines (Figures S10A and S10B). The CTCF-k site was Treating the normal HB2 cell line with 5-aza further less strongly enriched with CTCF in these cells and had shows that expression levels of GNG12-AS1 and DIRAS3 no enrichment of RAD21 in the SUM159 cells. This weaker are interrelated. In this cell line, 5-aza had a smaller effect CTCF enrichment might have been due to DNA methyl- on DNA methylation levels, confirming that the con- ation, as mentioned above. At sites DMR1 (CTCF-f) and stitutively silent DIRAS3 is resistant to 5-aza treatment CTCF-g, CTCF and RAD21 were bound in the normal (Figure S7A). The treatment with 5-aza had an inhibitory HB2 cell line and not in the cancer cell line (Figure S10). effect on DIRAS3 expression, presumably as a result of a Although it was expected that in the cancer cell line hyper- slight decrease in methylation at the somatic DMR3 within methylation and loss of DIRAS3 imprinting would exclude the gene body (Figure S7A and S7B). As expected, DIRAS3 CTCF binding at the DMRs, the additional lack of CTCF expression remained monoallelic (Figure S7C). Interest- binding to the CTCF-g site was unexpected. CTCF-g is ingly, reduced expression of DIRAS3 was accompanied located 7 kb upstream of DMR1, and the differential bind- by a slight increase in expression of GNG12-AS1. How- ing between the cell lines at this site suggests that it might ever, GNG12-AS1 expression remained biallelic, and tran- either be tissue specific or absent in cancer cell lines. scripts with exon 2 remained monoallelic (Figures S7D Inspection of CTCF binding profiles for several cell lines and S7E). on the UCSC Genome Browser indicated that CTCF-g is DIRAS3 has been shown to physically interact with generally a strong binding site for CTCF. Some cancer cell STAT3, and changes in DIRAS3 expression might therefore lines, such as MCF7, HeLaS3, or CACO2 (in which CTCF affect transcription of STAT3 target genes.37 In order to binding to both DMR1 and DMR3 is low), similarly exhibit examine whether the above observed changes in GNG12- no CTCF binding at CTCF-g. Other cell lines (e.g., HCT16, AS1 transcription are directly in response to increased WERI-Rb-1, and human embryonic kidney 293) that have DIRAS3 transcription or indirectly due to DIRAS3 signaling CTCF binding at DMR3 also have CTCF peaks at CTCF-g pathways, we used siRNA to deplete DIRAS3 in normal cells (Figure S11). These data indicate that CTCF binding within (HB2 and HS27) and examined the effect on expression of DIRAS3 and the CTCF-g site might be functionally linked. GNG12-AS1. We found no change in GNG12-AS1 expres- The presence of CTCF at DIRAS3 DMR1 might positively sion after depletion of DIRAS3 (Figure S8). Because regulate DIRAS3 expression, as evidenced by the recruit- siRNA-mediated silencing targets genes posttranscription- ment of CTCF to DMR1 upon 5-aza-mediated demethyla- ally, these results indicate that the GNG12-AS1 transcript tion in SUM159 cells (Figure S12). is not regulated by DIRAS3. The results further support a To determine the effect of CTCF binding on expression hypothesis that the act of increased DIRAS3 transcription of DIRAS3 and GNG12-AS1, we knocked down CTCF and in cells treated with 5-aza affects GNG12-AS1 expression. cohesin with siRNAs in three different cell lines (HB2, HS27, and SUM159), as shown in Figure 6A (left panel). CTCF and Cohesin Binding at the DIRAS3 DMRs Is In all three cell lines, DIRAS3 expression was reduced (by Required for DIRAS3 Expression, but Not for that of 65% in HB2, by 73% in Hs27, and by 40% in SUM159 GNG12-AS1 [6Figure B]). The decrease in DIRAS3 expression in The UCSC Genome Browser includes several annotated SUM159 cells (albeit less than in normal cell lines) was un- CTCF binding sites within the transcription unit of expected, given that the CTCF binding site at DMR1 is GNG12-AS1 (Figure 1A). At DIRAS3, both DMR1 and hypermethylated. It’s likely that the low basal levels of DMR3 have CTCF binding sites (CTCF-f and CTCF-e, DIRAS3 in SUM159 cells are associated with the small pro- respectively, in Figure 1A). We confirmed that at DMR1, portion of unmethylated DMR1 sites. In the normal cell CTCF binds to the unmethylated allele in two normal lines, the decrease in DIRAS3 expression after CTCF or cell lines (Figure S9A). We examined whether any of the cohesin knockdown indicates that CTCF and cohesin CTCF sites are within CpG islands and found that, in addi- binding at DMR1 positively regulates DIRAS3 expression tion to DMR1 and DMR3, CTCF-k is also a CpG island on the paternal allele. Despite the reduction of DIRAS3 (within GNG12-AS1 in intron i8b/9). This region has fully expression after CTCF knockdown, GNG12-AS1 transcript methylated DNA in HB2 and SUM159 cells (data not levels and allelic expression were unchanged (Figure 6C shown), indicating that it is not an allele-specific DMR. and 6D). GNG12-AS1 levels appeared to not be affected CTCF ChIP analysis and sequencing of informative SNPs by CTCF knockdown, which is surprising because the at CTCF sites b, c, g, and i indicate that these CTCF sites lncRNA is transcribed through several CTCF sites. are bound on both alleles in the HB2 cell line (Figures Although CTCF has previously been reported to play a S9B and S9C). Thus, DMR1 is the only region likely to role in cotranscriptional splicing,38 we did not consistently have allele-specific CTCF binding within the locus. find an increase in exon 2 isoforms upon CTCF depletion.

The American Journal of Human Genetics 93, 224–235, August 8, 2013 231 Figure 6. CTCF and Cohesin Binding at the DIRAS3 DMRs Regulates DIRAS3 Expression Cohesin is required for exon 2 skipping near the active DIRAS3 allele. (A) Immunoblots showing the efficiency of CTCF and RAD21 knockdown by siRNA in HB2, HS27, and SUM159 cell lines. (B) DIRAS3 expression levels decreased upon CTCF and cohesin (Rad21) knockdown in all cell lines. (C) Overall GNG12-AS1 expression levels did not change upon CTCF and cohesin (Rad21) knockdown. (D) Allelic expression of the GNG12-AS1 major isoforms did not change after siRNA-mediated CTCF and cohesin depletion in HB2 cells. (E) Expression of GNG12-AS1 isoforms with exon 2 increased in normal cell lines upon cohesin depletion, but not with CTCF depletion. Cancer cells were unaffected. (F) Allelic expression of GNG12-AS1 exon 2 isoforms in CTCF and cohesin-siRNA- treated HB2 cells. A significant change in allele ratio was found after cohesin depletion. At least three technical replicates were performed for each biological experiment. Error bars represent the SDs from at least three independent biological experiments. The statistical significance of the individual bars is compared to their respective control bars; *p < 0.05, **p < 0.01, ***p < 0.001.

propose that the inclusion of exon 2 requires (1) that transcription of GNG12-AS1 be above a threshold level and (2) that cohesin must be absent at DMR1 and DMR3. SUM159 cells lack cohesin at DMR1 and DMR3 on both alleles but express GNG12-AS1 on one allele only and hence include exon 2 in a monoallelic fashion. However, we found that cohesin knockdown resulted in a Normal cells have biallelic expression of GNG12-AS1 but 2-fold increase of GNG12-AS1 exon 2 isoforms (Figure 6E) have no cohesin binding at the DMRs on one allele, which in the normal cell lines, HB2 and Hs27, but not in cancer enables the inclusion of exon 2 during splicing. cell line SUM159. SNP analysis in the HB2 cell line revealed a shift in allele ratios toward biallelic expression of the exon 2 isoform (Figure 6F). These results might suggest Discussion that cohesin-mediated stabilized looping at the locus has a role in determining whether an exon is included during Figure 7 summarizes our findings and demonstrates the cotranscriptional splicing. We do not have an immediate relationship between the imprinted DIRAS3 and the explanation for the changes in allele-specific splicing lncRNA GNG12-AS1. In our studies, GNG12-AS1 was not with cohesin depletion and not with CTCF. It might be imprinted in non-cancer cells. However, one caveat is that despite a strong reduction of CTCF protein levels, that we only studied cell lines and placenta tissues, and CTCF binding to DNA was only marginally reduced by therefore our results do not preclude GNG12-AS1’s being RNAi, as previously observed.39 imprinted in a tissue-specific manner. In cancer cell lines, From the above data and under the assumption that monoallelic GNG12-AS1 transcription is associated with exon 2 isoforms of GNG12-AS1 are from the maternal loss of DIRAS3 imprinting. This intriguing observation re- allele, where DIRAS3 is silenced through imprinting, we quires genetic and expression analyses in primary tumor

232 The American Journal of Human Genetics 93, 224–235, August 8, 2013 specific transcriptional termination has been described at the imprinted retrogene H13-Mcts244 and Herc3-Nap1l545 loci. The GNG12-AS1 locus is an unusual and intriguing example of parent-of-origin-specific splicing. Splicing has been linked to RNAPII pausing at CTCF binding sites.38 However, exon 2 of GNG12-AS1, where transcription is likely to pause, does not have a CTCF binding site. Thus, if CTCF has a role in alternative splicing of GNG12-AS1, it would have to act from a more distant CTCF site, such as at DMR1 or another site. Knockdown of cohesin resulted in a decrease in DIRAS3 expression and additionally in biallelic splicing of exon 2 on both alleles, indicating that cohesin has a role in exon 2 skipping. CTCF and cohesin occur at the same sites at this locus, and because CTCF binding is required for the recruitment of cohesin,46 it is surprising that depletion of CTCF does not have the same effect on exon 2 splicing as cohesin does. Cohesin and CTCF have recently been shown to have distinct roles despite their co-occupancy.47 One explanation of our observation of the cohesin effect on splicing might be that it is more difficult to effectively re- move CTCF from chromatin and that although reduced Figure 7. GNG12-AS1 Expression and Splicing Model in Normal CTCF binding is sufficient to inhibit transcription of and Cancer Cells DIRAS3, there is still enough cohesin recruited to enable (A) In normal cells, GNG12-AS1 was expressed from both maternal exon 2 skipping. Serendipitously, the reduction in DIRAS3 (‘‘M’’) and paternal (‘‘P’’) alleles, and inclusion of exon 2 occurred on the maternal allele. transcription with CTCF knockdown not only excludes a (B) In cancer cell lines, GNG12-AS1 expression was inhibited on direct role for CTCF in exon 2 splicing but also potentially the paternal allele, and the lncRNA was predominantly expressed excludes effects of DIRAS3 transcription. from the maternal allele. The expression of transcripts with exon 2 Determining the function of GNG12-AS1 will require remained monoallelic from the maternal allele. further studies. Because it is a nuclear transcript, it might have both trans and cis functions in gene regulation. In tissues for determining whether neoplastic silencing of addition, its variety of isoforms could potentially provide DIRAS3 is accompanied by monoallelic expression of additional diversity in its regulatory repertoire. This locus GNG12-AS1 in vivo. is also a good model system for future studies of long-range Our preliminary screen in breast cancer tissues strongly effects of CTCF-cohesin binding on splicing and allele indicated that expression of GNG12-AS1 and DIRAS3 is exclusion. In this work, we have shown that an imprinted coordinately reduced in breast cancer. We have excluded gene can influence the splicing of a nonimprinted gene. loss of heterozygosity at this locus in our samples, so the GNG12-AS1, which includes DIRAS3, is a model locus for silencing is likely to occur via an epigenetic mechanism the study of a novel role for cohesin in the regulation of that potentially spreads across the locus. This acquired alternative splicing. silencing mechanism during neoplasia is unlikely to be the same as the innate imprinted silencing that occurs in Supplemental Data the maternal germline. Indeed, the constitutively silenced maternal DIRAS3 allele was found to be more resistant to Supplemental Data include 12 figures and 8 tables and can be 5-aza-mediated DNA demethylation in the cell lines stud- found with this article online at http://www.cell.com/AJHG/. ied here, suggesting that differential epigenetic silencing marks are present on the imprinted and the neoplastic Acknowledgments silenced alleles. Moreover, it seems that a gain of DNA methylation alone is not sufficient to silence GNG12-AS1 The authors wish to thank G.E.M. and S.A.-A. at the Institute of in cis, given that 5-aza treatment upregulated DIRAS3 Child Health at University College London for the placenta tissue and DNA. We would like to acknowledge the support of The expression levels without restoring biallelic GNG12-AS1 University of Cambridge, Cancer Research UK, and Hutchison expression. Interestingly, the DIRAS3 region has been iden- Whampoa Limited. tified as a contiguous region of genes suppressed by long- 40 range epigenetic silencing in prostate cancer. Received: February 21, 2013 RNA processing is increasingly being shown to occur co- Revised: April 19, 2013 transcriptionally, and chromatin structure has been shown Accepted: June 7, 2013 to play a role in alternative splicing.41–43 Parent-of-origin- Published: July 18, 2013

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