Silencing and transcriptional properties of the imprinted Airn ncRNA are independent of the endogenous promoter

Abstract

The Airn macro ncRNA is the master regulator of imprinted expression in the Igf2r imprinted gene cluster where it silences three flanking genes in cis. Airn transcription shows unusual features normally viewed as promoter specific, such as impaired post-transcriptional processing and a macro size. The Airn transcript is 108 kb long, predominantly unspliced and nuclear localized, with only a minority being variably spliced and exported. Here, we show by deletion of the Airn ncRNA promoter and replacement with a constitutive strong or weak promoter that splicing suppression and termination, as well as silencing activity, are maintained by strong Airn expression from an exogenous promoter. This indicates that all functional regions are located within the Airn transcript. DNA methylation of the maternal imprint control element (ICE) restricts Airn expression to the paternal allele and we also show that a strong active promoter is required to maintain the unmethylated state of the paternal ICE. Thus, Airn expression not only induces silencing of flanking mRNA genes but also protects the paternal copy of the ICE from de novo methylation.

Figure 1

Deletion and replacement of Airn promoter constructs. (A) The Airn promoter lies in an antisense orientation in Igf2r intron 2. A 959-bp fragment is deleted in the APD, APD-PGK and APD-TET alleles (thick line). The main transcription start site (T1) and downstream CpG island are unchanged. SD: Airn splice donor. A 3.65-bp PacI–BamHI fragment that has been genetically defined as the imprint control element (ICE) for the Igf2r imprinted cluster is indicated by the dotted line (Wutz et al, 2001). (B) Three constructs were used for targeting the Airn promoter deletion (APD) and the two promoter replacement alleles containing a PGK promoter (APD-PGK) or a TET promoter (APD-TET). Msi, Psi, OT2.4: Southern blot probes; hygro: hygromycin cassette. (C) Southern blot showing homologous targeting indicated by a 7.4-kb band. (D) Southern blot using the SfuI methyl-sensitive enzyme shows paternal targeting indicated by a 1.2 kb (APD) or 3.5 kb (APD-PGK, APD-TET) band. (E) Southern blot shows successful Cre recombination by loss of the unrecombined allele (APD 2.3 kb, APD-PGK 5.5 kb and APD-TET 5.4 kb) and gain of the recombined allele (APD 3.1 kb, APD-PGK 3.7 kb, APD-TET 3.6 kb). (F) Undifferentiated ES cells show biallelic expression of Igf2r and no expression of Airn. The Airn CpG island is methylated (Me) on the maternal allele (mat). Differentiation with retinoic acid (RA) leads to Airn upregulation, repression of Igf2r and methylation of the Igf2r CpG island on the paternal allele (pat) (PAL, manuscript submitted).

Figure 2

Airn expression changes in promoter deletion and replacement cells. (A) Map of the wild-type Airn promoter (WT), the Airn promoter deletion (APD) and replacement alleles (APD-PGK and APD-TET). Airn TQM F/R: QPCR assay used in (B, C). Numbered black boxes: Igf2r exons. Black oval: Airn CpG island, grey oval: Igf2r CpG island. The positions of the Airn and Igf2r transcription start sites with respect to the CpG island were determined by 5′RACE (Seidl et al, 2006). (B) QPCR showing Airn expression in targeted ES cells differentiated for 0, 5 or 14 days. Control undifferentiated ES cells (S12/+) lack Airn expression (day 0). Differentiation for 5 or 14 days increases Airn expression strongly. S12/APD cells lack Airn expression at all time points. S12/APD-PGK cells show moderate Airn expression (50% of 5 day control) in undifferentiated cells (d0) that increases to WT levels by day 5. S12/APD-TET cells show weak Airn expression under non-induced conditions but only in differentiated cells (3.8% at 5 days, 6.5% at 14 days relative to 14 day control). *Set to 100. Error bars represent standard deviation of three technical replicates (one biological replica). (C) QPCR comparing Airn expression in three biological replicates of S12/+ and S12/APD-PGK ES cells, differentiated for 0, 5 or 14 days, shows equal Airn expression at 5 days in S12/+ and S12/APD-PGK that is reduced at 14 days to 15% of control levels. Details as in (B). (D) 5′RACE products to visualize Airn transcription starts in 14-day differentiated S12/+ and S12/PGK cells. (E) Sequence showing locations of published Airn (T1) and PGK (T1(PGK)) transcription start sites and two transcription start sites of PGK-driven Airn (T1(AirPGK) and T2(AirPGK)). Rev 1, Rev 2: RACE primers; underlined: PGK promoter sequence; black font: PGK-Airn transcript; black arrow: transcript direction.

Figure 3

The PGK promoter expresses full-length Airn. (A) Map showing unspliced Airn (black arrow) and the known Airn splice variants and primers used in the splice variant-specific QPCR. The unspliced and spliced transcripts use the same transcription start sites and all four spliced variants use the same 53 bp exon 1. SV3 extends beyond the mapped end of the unspliced Airn transcript. A common forward primer (FP1) and Taqman probe (Probe) are combined with different reverse primers (RP21 (SV1), RP6 (SV1a), RP5 (SV2) and RP4 (SV3)). (B) Splice variant-specific QPCR. S12/+ control cells mostly lack expression of all splice variants at day 0 and show maximum expression at day 5. Spliced variants represent <5% of total Airn transcripts (Seidl et al, 2006); however, for purposes of comparison with the targeted alleles, the expression levels of unspliced and splice variants transcripts in S12/+ cells at day 5 is set to 100 (^*). S12/APD cells and S12/APD-TET lack Airn splice variants at day 0, whereas S12/APD-PGK shows low expression levels at day 0. Expression of splice variants increases in S12/APD-PGK and S12/APD-TET ES cells at day 5. Mean values and standard deviations of two biological replicates are shown. (C) cDNA hybridization experiment on a custom PCR genomic tiling array (Regha et al, 2007). Top: map of the Igf2r imprinted cluster contained on the tiling array (note that only the promoter regions of Slc22a2 and Slc22a3 were included on the array). The three lower tracks on the y axis show RNA signal ratios of S12/+, S12/APD-PGK, S12/APD-TET relative to S12/APD1 in 5 day differentiated cells. Outside the Airn transcription unit the mean ratio is close to 1 (dashed line), indicating equal expression in the experimental (S12/+, S12/APD-PGK and S12/APD-TET) and reference cell lines (S12/APD1). S12/+ cells show elevated levels inside the Airn transcription unit that is reproduced by S12/APD-PGK, indicating no significant gain of splicing or premature termination in this replacement allele. Weak Airn expression from S12/APD-TET was not detected.

Figure 4

Igf2r promoter DNA methylation. (A) Map of an 8-kb region containing the Igf2r promoter (black arrow: transcription orientation). White box: CpG island; black box 1: Igf2r exon 1. Be2i: Southern blot probe. (B) Southern blot on genomic DNA of days 0 and 14 RA differentiated ES cells using EcoRI and the methyl-sensitive NotI enzyme and probe Be2i. One of two biological replicates is shown. S12/+ ES cells at day 14 show DNA methylation on NotI that is indicative of general methylation levels on the CpG island on the paternal allele (Stoger et al, 1993), whereas S12/APD1 and S12/APD-TET lack methylation. S12/APD-PGK cells show partial NotI methylation at day 0 (29–34%, asterisks) and full methylation at day 14 (48–55%, note that only the paternal allele is methylated (Stoger et al, 1993)). Numbers below indicate the relative intensities of methylated bands (ImageJ).

Figure 5

PGK-Airn and induced TET-Airn silence Igf2r in cis. (A) RNA FISH with the Igf2r intronic FP1 strand-specific probe in S12/APD ES cells. Representative examples of day 5 RA differentiated cells with double, single and no transcription spots are shown. (B) Left panel: quantification of Igf2r transcription by FISH using intronic probes FP1 (left bar) and FP3 (right bar). Double spots (2) indicate biallelic expression, single spots (1) monoallelic or stochastic biallelic expression. +: multiple spots indicate unspecific signals. Control S12/+ cells (dark grey bars) show mainly single spots. Airn promoter deletion S12/APD2 (white bars) shows an increase in cells with double spots, indicating a loss of imprinted expression. S12/APD-PGK (light grey bars) cells show mainly single spots consistent with imprinted Igf2r expression. Error bars represent means of two independent counts (one performed blind). Right panel: as left panel, but using an independently targeted APD allele (S12/APD1). (C) Left panel shows induced Airn expression levels assayed by QPCR as described in Figure 3 in APD-TET-Rolo ES clones carrying a transactivator (rtTA) gene targeted into the ROSA26 locus (Beard et al, 2006). Treatment of S12/APD-TET-Rolo cells with Doxycycline during 5d of ES cell differentiation leads to induction of the Airn ncRNA comparable to wild-type levels. Right panel shows an RNA FISH analysis of Igf2r transcription in S12/APD-TET-Rolo cells induced to express high levels of Airn. Uninduced S12/APD-TET-Rolo cells show a transcription pattern indistinguishable from S12/APD cells (double spots: 24%, −Dox). Induction of Airn ncRNA expression decreases biallelic Igf2r expression (double spots: 12%, +Dox). Error bars represent means of three independent counts (two were performed blind).

Figure 6

PGK promoter-driven Airn silences paternal Igf2r. (A) Allele-specific QPCR showing the ratio of maternal/paternal Igf2r expression in undifferentiated and differentiated wild-type and targeted ES cells. Numbers are ratios of maternal (S12) to paternal (+) allele. The ratio at day 0 in S12/+ cells was set to 1 (asterisk) and an increased ratio at day 5 indicates higher expression of the maternal relative to the paternal allele. The ratios of all targeted ES cell clones (S12/APD, S12/APD-PGK and S12/APD-TET) at day 5 are not significantly different from control cells (S12/+). Error bars represent standard deviation of three technical replicates (one biological replica). (B) As (A), but cells were cultured at day 5 for 10 h with or without (+/−) emetine. Mean values and standard deviations of three biological replicates are shown. ^*Set to 17.5, the value obtained from day 5 S12/+ cells shown in (A). A Student's t-test (two-tailed, equal variance) shows that S12/APD and S12/APD-TET cells show a significant lower ratio of maternal to paternal Igf2r under emetine treatment compared with S12/+ cells, whereas S12/APD-PGK cells do not (P>0.1). (C) QPCR showing changes in Airn and H19 expression after emetine treatment in targeted ES cells differentiated for 5 days. Mean values and standard deviations of 6 (Airn) or 15 (H19) biological replicates are shown. ^*Set to 100.

Figure 7

Airn expression protects the Airn CpG island from DNA methylation. (A) Map of a 20-kb region containing the Airn promoter. White box labelled CGI: CpG island; dark grey box: Airn promoter deletion or replacement cassette, which introduced a BamHI site. ICE, details in Figure 1A, Msi Southern blot probe, SfuI: methyl-sensitive site assayed in (B). (B) Left panel: Southern blot on genomic DNA of days 0 and 14 RA differentiated ES cells digested with BamHI+SfuI and probed with fragment Msi. Wild-type fragments from S12/+ are too large to separate and migrate together above 14 kb, all fragments below this arise from the paternally targeted allele. In S12/APD and S12/APD-TET cells, no or weak Airn transcription leads to gain of DNA methylation on the paternal Airn CpG island (met). DNA methylation is prominent in differentiated ES cells at day 14 (36–49% methylation); however, some DNA methylation is also present in undifferentiated ES cells (19–28% methylation). In contrast, S12/APD-PGK that shows strong Airn expression lacks DNA methylation on the Airn CpG island (unmet). Numbers below indicate the relative intensities of methylated bands (ImageJ). Right panel: the same methylation assay performed on S12/APD-TET-Rolo cells differentiated for 14 days minus (−) or plus (+) doxycycline (Dox), shows that in both conditions the Airn CpG island gains similar levels of DNA methylation.