A mouse model of Angelman syndrome imprinting defects

Abstract

Angelman syndrome, Prader-Will syndrome and Dup15q syndrome map to a cluster of imprinted genes located at 15q11-q13. Imprinting at this domain is regulated by an imprinting control region consisting of two distinct elements, the Angelman syndrome imprinting center (AS-IC) and the Prader-Willi syndrome imprinting center (PWS-IC). Individuals inheriting deletions of the AS-IC exhibit reduced expression of the maternally expressed UBE3A gene and biallelic expression of paternal-only genes. We have previously demonstrated that AS-IC activity partly consists of providing transcription across the PWS-IC in oocytes, and that these transcripts are necessary for maternal imprinting of Snrpn. Here we report a novel mouse mutation that truncates transcripts prior to transiting the PWS-IC and results in a domain-wide imprinting defect. These results confirm a transcription-based model for imprint setting at this domain. The imprinting defect can be preempted by removal of the transcriptional block in oocytes, but not by its removal in early embryos. Imprinting defect mice exhibit several traits often found in individuals with Angelman syndrome imprinting defects.

Figure 1

Map of the Snrpn U exons. (Top) Selected gene products of the mouse AS-PWS locus are shown. Paternally expressed transcripts are shown above the line. Ube3a is predominantly maternally expressed and shown below the line. Arrows indicate the direction of transcription. The PWS-IC is represented by an open circle spanning Snrpn exon 1. (Bottom) Realignment of the mouse U exons. The locations of U exons upstream of Snrpn exon 1 are shown to approximate scale. Internal Greek exons that are present in some transcripts are shown below the line. U exons with promoter activity documented in the UCSC Genome Browser are marked with an asterisk. The filled triangle indicates the location of the transcriptional terminator insertion. More precise information about the sequence and location of U and Greek exons is available in Supplementary Material, Figure S1 and Supplementary Material, Table S1.

Figure 2

The transcriptional terminator prevents methylation of the PWS-IC in oocytes. Oocytes were isolated from 3- to 6-week-old wild-type and AST^erm/AS^Term females. (A) BGS for the PWS-IC was performed using nested bisulfite primers indicated in Supplementary Material, Table S3. Three separate amplifications of each bisulfite converted DNA were performed to ensure that the same genome was not sampled multiple times. Three results from each amplification are shown. Open and closed circles indicate unmethylated and methylated CpG residues respectively. (B) BGS for an element of the Peg3 DMR was performed on AS^Term/AS^Term oocytes as a positive control for DNA methylation in this sample (26). Two separate amplifications were performed.

Figure 3

Maternal transmission of the transcriptional terminator insertion generates a maternal imprinting defect. (A) BGS of the PWS-IC in wild-type and m^ASTerm / p⁺ newborn brain. Open and closed circles indicate unmethylated and methylated CpGs, respectively. The maternal and paternal alleles were distinguished by polymorphisms arising from the paternal DBAJ2 within the sequenced region. (B) Quantitative RT-PCR of several RNAs from the locus in wild-type and m^ASTerm / p⁺ newborn brain. All differences from wild-type were significant at (P < 0.05) with the exception of Ndn in female (P = 0.061). (C) Allelic expression of RNAs in newborn brain. Dams bearing the AS^Term allele on the C57BL/6 (B6) background were mated with DBAJ2 (DBA) sires. Newborn brain cDNA was amplified with gene specific primers and digested with indicated enzymes sensitive to strain specific polymorphisms. The expected sizes of bands for each strain are indicated to the right. In each case the maternal allele is listed first. D. For Ube3a-ats and Ube3a, the available enzyme cut too poorly to generate reproducible results. Sequencing chromatograms primed with one of the PCR primers are shown instead. The polymorphic base is indicated by an arrow. The polymorphisms used in this and other figures are shown in Supplementary Material, Table S2.

Figure 4

Deletion of the transcriptional terminator during the oocyte growth phase prevents an imprinting defect. Analysis of offspring of matings of dams bearing the AS^Term allele and Zp3-Cre transgene with DBAJ2 sires. (A) BGS shows that the gDMR is hypermethylated in a newborn. (B) RT-PCR of the newborn brain shows that offspring exhibit correct allele specific expression of several genes across the AS-PWS locus as in Figure 3C. Allelic expression of Ube3a-ats and Ube3a in the same cDNAs as in Figure 4C. The first three panels of both Ube3a-ats and Ube3a are reproduced from Figure 3D as a reference.

Figure 5

Postzygotic removal of the terminator does not prevent an imprinting defect. Newborn brains of offpring from matings m⁺/p^ASTerm females with CMV-Cre, DBAJ2 males were analyzed. Procedures are as in Figure 3. (A) BGS shows the maternal PWS-IC is hypomethylated. (B) Several genes across the locus are expressed biallelically when the terminator is deleted early in development. Deletion of the terminator occurs only in females because CMV-Cre is X-linked (68). (C) Both Ube3a-ats and Ube3a exhibit biallelic expression. The first three panels of both Ube3a-ats and Ube3a are reproduced from Figure 3D as reference.

Figure 6

Weight gain in m^ASTerm/p⁺ mice. Mice were maintained on regular chow ad libitum. Females: m⁺/p⁺ n = 13, m^ASTerm/p⁺ n = 5. Males m⁺/p⁺ n = 9, m^ASTerm/p⁺ n = 5. Asterisks denote value differences that are statistically significant (P < 0.05 by unpaired t test).

Figure 7

Complementation by the AS^Term and Δ35kb PWS-IC mutations. Dams bearing the AS^Term allele were mated with sires bearing the Δ35kb PWS-IC mutation (ΔPWS-IC). (A) The number of animals of each genotype among 42 total weanlings. The maternal (Mat) and paternal (Pat) genotypes of the pups are indicated below the graph. (B) Pictures of littermates at 10 weeks. For both female and male, an m⁺/p⁺ animal is shown above an m^ASTerm/p^{Δ35kb PWS-IC} littermate. C. RT-PCR analysis of newborn brain RNA from pups of the same matings.