Transcription is required to establish maternal imprinting at the Prader-Willi syndrome and Angelman syndrome locus

Abstract

The Prader-Willi syndrome (PWS [MIM 17620]) and Angelman syndrome (AS [MIM 105830]) locus is controlled by a bipartite imprinting center (IC) consisting of the PWS-IC and the AS-IC. The most widely accepted model of IC function proposes that the PWS-IC activates gene expression from the paternal allele, while the AS-IC acts to epigenetically inactivate the PWS-IC on the maternal allele, thus silencing the paternally expressed genes. Gene order and imprinting patterns at the PWS/AS locus are well conserved from human to mouse; however, a murine AS-IC has yet to be identified. We investigated a potential regulatory role for transcription from the Snrpn alternative upstream exons in silencing the maternal allele using a murine transgene containing Snrpn and three upstream exons. This transgene displayed appropriate imprinted expression and epigenetic marks, demonstrating the presence of a functional AS-IC. Transcription of the upstream exons from the endogenous locus correlates with imprint establishment in oocytes, and this upstream exon expression pattern was conserved on the transgene. A transgene bearing targeted deletions of each of the three upstream exons exhibited loss of imprinting upon maternal transmission. These results support a model in which transcription from the Snrpn upstream exons directs the maternal imprint at the PWS-IC.

Figure 1. The PWS/AS imprinted domain and corresponding regions covered by the BAC transgenes.

(A) Schematic diagram of the murine PWS/AS locus. Genes expressed from the paternal allele are shown above the locus (blue rectangles) and maternally expressed gene are shown below (red rectangles)(not to scale). (B) Schematic diagram of the Snrpn locus and sequences included in the 380J10 and 425D18 BACs. The nine identified Snrpn U exons (filled red boxes) are shown as well as five additional exons, termed α-ε (open boxes) . For each BAC, the extent of genomic sequence relative to Snrpn is indicated (not to scale).

Figure 2. 380J10 BAC transgene expression analysis.

Northern blot analysis for Snrpn expression in the 380J10 transgenic lines. Two independent P1 brain samples were analyzed for each line and parental transmission. The PWS-IC^Δ35kb (KO) samples demonstrate the absence of endogenous Snrpn expression upon paternal transmission of this allele. The 380 transgenic samples all bear a paternal PWS-IC^Δ35kb allele. β-actin was assayed as a loading control.

Figure 3. Analysis of imprinting for the 425Δ5-7 BAC transgene.

(A) Schematic diagram of the strategy for Snrpn expression analysis by RT-PCR. The Snrpn gene contains ten exons (numbered boxes). The 425Δ5-7 transgene bears a deletion between exons 5 and 7 (dashed lines). PCR primers (arrows) in exons flanking the deletion distinguish the endogenous Snrpn product (513 bp) from transgenic Snrpn expression (350 bp). (B) RT-PCR analysis of P1 brain RNA from the 425Δ5-7A, 425Δ5-7H, and 425Δ5-7I (from left to right) transgenic lines. Non-transgenic littermates (-/-) were included as negative controls. NT indicates a control PCR reaction with no cDNA template added. Hprt amplification was performed to demonstrate cDNA integrity. The minus sign in RT-PCR analyses indicates control samples in which reverse transcriptase was omitted during cDNA synthesis. (C) Analysis of the DNA methylation imprint at the 425Δ5-7A Snrpn DMR. Genomic bisulfite sequencing was performed on P1 brain samples after both maternal and paternal transmission of the transgene. A 364 bp region of the Snrpn DMR (spanning from −175 to +189 relative to Snrpn exon 1) that contains 14 CpG dinucleotides was analyzed in the 425Δ5-7A line. A SNP located within the sequenced region distinguishes the transgenic from the endogenous alleles. Each row represents an individually sequenced clone, filled circles indicate methylated CpG dinucleotides, and open circles represent unmethylated CpG dinucleotides. DNA methylation patterns at the endogenous locus are shown in Figure S2.

Figure 4. U exon expression from the 425Δ5-7A transgene during maternal imprint establishment.

(A) Schematic diagram of the RT-PCR strategy used to analyze U exon transcription from the 425Δ5-7A transgene. The loxP site, unique to the transgene exon 5 to exon 7 deletion, is depicted as an open triangle. PCR primers (arrows) were designed to anneal to U1 or U2 and the loxP site. (B) RT-PCR analysis of U exon expression in ovary RNA. Two wild type (−/−) and four transgenic (Tg) sets of ovaries were analyzed from females at three weeks of age. Transgenes were maternally transmitted. Stella amplification was performed to verify the presence of oocyte RNA.

Figure 5. U exon expression in the developing germ line.

(A) Schematic diagram of the RT-PCR strategy used to analyze U exon transcription. PCR primers (arrows) were designed to anneal to U1 or U2 and Snrpn exon 3 . (B) RT-PCR analysis of U exon expression in 13.5 dpc PGCs from female (F) and male (M) embryos. Testis and adult ovary provide negative and positive controls, respectively. The RT-PCR reactions were Southern blotted and probed with verified product to confirm identity. Snrpn expression was also analyzed using primers spanning exons 4 to 8. (C) U exon expression was analyzed by RT-PCR in wild type ovaries spanning from P1 to adult. The identity of the products was confirmed by DNA sequencing. Stella expression was examined to verify the presence of oocyte RNA.

Figure 6. Analysis of imprinting of the 425ΔU1-U3D BAC transgene.

(A) Schematic representation of the 425ΔU1-U3 transgene. The three upstream exons (gray boxes) were deleted (dashed lines) from the 425Δ5-7 BAC to create this transgene. PCR primers (arrows) in exons flanking the deletion distinguish the endogenous Snrpn product (513 bp) from transgenic Snrpn expression (350 bp). (B) Expression analysis of the 425ΔU1-U3D transgene. RT-PCR was performed on P1 brain RNA after both maternal and paternal transmission of the transgene. (C) Analysis of the DNA methylation imprint at the 425ΔU1-U3D Snrpn DMR. Genomic bisulfite sequencing was performed on P1 brain samples after both maternal and paternal transmission of the transgene. A SNP located within the sequenced region distinguishes the transgenic from the endogenous alleles, only the transgenic alleles are shown.

Figure 7. Expression analysis of the imprinted 425ΔU1-U3H transgene.

(A) Schematic representation of the 425ΔU1-U3 transgene. PCR primers (black arrows) in exons flanking the deletion distinguish the endogenous Snrpn product (513 bp) from transgenic Snrpn expression (350 bp). (B) Expression analysis of the 425ΔU1-U3H transgene. RT-PCR analysis of P1 brain RNA after both maternal and paternal transmission of the transgene. (C) Analysis of transcription across the PWS-IC in transgenic ovaries during imprint establishment. P15 ovaries were isolated from 425Δ5-7A, 425ΔU1-U3D, and 425ΔU1-U3H females and analyzed by RT-PCR for Snrpn γ exon-containing transcripts. Primers anneal to the Snrpn γ exon and the loxP site (gray arrows in (A)). The RT-PCR product was Southern blotted and probed with verified product to confirm identity. Stella expression was examined to demonstrate the presence of oocyte RNA.

Figure 8. A working model for the establishment of the maternal imprint at the PWS-IC.

Schematic diagrams of the locus around Snrpn at various stages of development. The U exons are indicated by red boxes and the PWS-IC is depicted as an orange oval overlying Snrpn exon 1. Imprints are erased in fetal germ cells and the Snrpn upstream exons are not expressed. In the neonatal period, transcription is initiated from the Snrpn upstream exons prior to the appearance of the DNA methylation imprint in growing oocytes. These transcripts proceed through the PWS-IC, triggering repressive epigenetic modification of this element, indicated by black lollipops. The PWS-IC on the future maternal allele is thereby inactivated, rendering the paternally expressed genes silent.