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Human Molecular Genetics Advance Access originally published online on December 20, 2005
Human Molecular Genetics 2006 15(3):393-404; doi:10.1093/hmg/ddi456
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© The Author 2005. Published by Oxford University Press. All rights reserved.
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A human imprinting centre demonstrates conserved acquisition but diverged maintenance of imprinting in a mouse model for Angelman syndrome imprinting defects

Karen A. Johnstone*, Amanda J. DuBose, Christopher R. Futtner, Michael D. Elmore{dagger}, Camilynn I. Brannan{ddagger} and James L. Resnick

Department of Molecular Genetics and Microbiology, Center for Mammalian Genetics, University of Florida College of Medicine, Gainesville, FL 32610-0266, USA

* To whom correspondence should be addressed at: Department of Molecular Genetics and Microbiology, PO Box 100266, University of Florida, Gainesville, FL 32610, USA. Tel: +1 352 392 3296; Fax: +1 352 392 3133; Email: karenj{at}ufl.edu

Received November 16, 2005; Accepted December 9, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Prader–Willi syndrome (PWS) and Angelman syndrome (AS) are caused by the loss of imprinted gene expression from chromosome 15q11–q13. Imprinted gene expression in the region is regulated by a bipartite imprinting centre (IC), comprising the PWS-IC and the AS-IC. The PWS-IC is a positive regulatory element required for bidirectional activation of a number of paternally expressed genes. The function of the AS-IC appears to be to suppress PWS-IC function on the maternal chromosome through a methylation imprint acquired during female gametogenesis. Here we have placed the entire mouse locus under the control of a human PWS-IC by targeted replacement of the mouse PWS-IC with the equivalent human region. Paternal inheritance of the human PWS-IC demonstrates for the first time that a positive regulatory element in the PWS-IC has diverged. These mice show postnatal lethality and growth deficiency, phenotypes not previously attributed directly to the affected genes. Following maternal inheritance, the human PWS-IC is able to acquire a methylation imprint in mouse oocytes, suggesting that acquisition of the methylation imprint is conserved. However, the imprint is lost in somatic cells, showing that maintenance has diverged. This maternal imprinting defect results in expression of maternal Ube3a-as and repression of Ube3a in cis, providing evidence that Ube3a is regulated by its antisense and creating the first reported mouse model for AS imprinting defects.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although most autosomal genes are biallelically expressed, a small subset shows differential expression dependent on the parent of origin, a phenomenon called genomic imprinting. Most imprinted genes are located in clusters and are coordinately regulated by imprinting control regions (ICRs) (1Go). Within clusters, there is extensive conservation between mouse and human in terms of the order, structure and transcriptional orientation of genes (2Go–4Go).

Prader–Willi syndrome (PWS; 176270) and Angelman syndrome (AS; 105830) are two clinically distinct neurological syndromes caused by defects in an imprinted cluster on chromosome 15q11–q13. Loss of paternal contribution from 15q11–q13 results in PWS, whereas AS is caused by loss of maternal gene expression (5Go). The cluster is under the bidirectional control of an ICR, in this region designated as the imprinting centre (IC). The IC has a bipartite structure comprising the PWS-IC and the AS-IC. The PWS-IC is a positive regulatory element responsible for the establishment and maintenance of paternal gene expression (6Go). The shortest region of deletion overlap (PWS-SRO) of microdeletions of the PWS-IC is 4.3 kb and includes the promoter and exon 1 of SNRPN (7Go). The AS-IC lies around 35 kb upstream of SNRPN exon 1 and has been mapped to a region of just 0.88 kb (8Go). The AS-IC is thought to function in the female germline to negatively regulate the PWS-IC on the maternal chromosome (6Go).

The region contains several paternally expressed genes and transcripts, including NDN, MAGEL2, MKRN3, SNRPN, a number of snoRNAs and an antisense transcript to UBE3A (UBE3A-AS), and two maternally expressed genes, UBE3A and ATP10A (5Go,9Go,10Go). SNRPN, the snoRNAs and UBE3A-AS are components of a very long transcript that is extensively processed, and a number of upstream exons of SNRPN have also been identified (11Go). Absence of patients with single-gene defects suggests that PWS is a contiguous gene syndrome requiring loss of two or more paternal genes (12Go). AS on the other hand, can result from mutations in UBE3A (13Go,14Go).

Imprinted expression of Ndn, Magel2, Mkrn3, Snrpn, several snoRNAs, Ube3a-as and Ube3a is conserved in the orthologous region on mouse chromosome 7C (5Go,15Go). As in human, Snrpn–Ube3a-as is transcriptionally complex and also includes a number of upstream exons thought to be involved in regulation of Ube3a-as expression (16Go). We have previously shown that the location and function of the PWS-IC are also conserved in mouse (17Go), mapping the murine PWS-IC to a 35 kb region. Attempts to delineate it further have resulted in incomplete imprinting phenotypes, suggesting that it contains multiple elements (18Go). An AS-IC is yet to be identified in mouse. The PWS-IC contains a germline methylation imprint acquired during female gametogenesis in both mouse and human (19Go,20Go). In both species, differential methylation is associated with a CpG island surrounding the promoter and exon 1 of SNRPN.

A number of important differences have also been observed between imprinted regions in human and mouse, suggesting that the regions are rapidly evolving. Some regions contain genes unique to one species. For instance, Frat3 recently transposed to the mouse PWS/AS region and acquired the imprinted status of the insertion site (21Go). Also, some imprinted loci show divergent imprinting between the two species (4Go,22Go–24Go). Finally, functional evidence for the evolution of imprinting is provided by the failure of human transgenes to imprint in mouse, including an SNRPN transgene (25Go) and an H19 transgene (26Go). A human transgene containing both the PWS-SRO and AS-SRO expressed SNRPN following maternal inheritance (25Go), suggesting that a negative regulatory element is species-specific.

In this study, we have placed the murine PWS/AS region under the control of a human PWS-IC by targeted exchange in ES cells. The results show that a positive regulatory element within the PWS-IC required for paternal gene expression is also species-specific. Mice with a paternal imprinting defect show postnatal lethality and growth deficiency, despite expressing normal levels of Snrpn–Ube3a-as. Surprisingly, we found that the human PWS-IC acquired methylation in the female mouse germline, indicating that negative regulation of the PWS-IC by the AS-IC is, in fact, conserved. However, we conclude that the factors responsible for postzygotic maintenance of the imprint have diverged, as the human PWS-IC fails to maintain methylation in somatic cells. We also show that Ube3a-as is expressed from the maternal chromosome and Ube3a is downregulated in cis, providing further evidence that Ube3a is regulated by its antisense RNA and suggesting a disease mechanism for AS imprinting defects.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of mice with a human PWS-IC
The targeting vector (Fig. 1) was constructed to replace 6 kb spanning exon 1 of Snrpn with a 6.9 kb region from the human locus which includes exon 1 of SNRPN and the entire PWS-SRO (7Go). Screening for homologous recombinants identified eight clones that had undergone recombination. A methylation-sensitive restriction enzyme screen suggested that the maternal and paternal alleles were targeted with similar frequencies (data not shown). The neomycin resistance gene (neo) was deleted in two targeted clones by transient transfection with a Cre recombinase expression plasmid. Cre-mediated deletion left a single residual loxP site at the 3' junction. Two independently targeted clones were used to derive chimeras. Both lines transmitted the PWS-ICHs (Homo sapiens) allele through the germline with identical results.


Figure 4561
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  		Figure 1. Generation of mice with a targeted replacement of the PWS-IC. (A) A simplified schematic representation of gene content and imprinting in the PWS/AS orthologous region on chromosome 7C in mouse (not to scale). Paternally expressed genes are in blue, maternally expressed genes are in red and DMRs are indicated with black circles. (B) A map of the human locus around SNRPN exon 1 showing the 6.9 kb SmaI fragment used in the targeting construct. (C) The targeting construct was designed to replace 6 kb of sequence spanning exon 1 of the Snrpn gene at the mouse locus with an equivalent region from the human locus. The construct included positive (neo) and negative (tk) selection cassettes. (D) The mouse locus showing the 5' and 3' homology arms and the location of probes used to identify homologous recombinants. (E) The targeted mouse locus following Cre-induced deletion of the floxed neo gene. The human sequences introduce ApaI and HindIII restriction sites used to distinguish homologous recombinants with 5' and 3' probes, respectively. Restriction enzymes: A, ApaI; B, BamHI; E, EcoRI; H, HindIII; Hp, HpaI; S, SmaI. (F) Southern blot analysis of a targeted ES cell clone. The 5' probe detects a novel 8.3 kb ApaI fragment in the targeted allele (+/Hs-neo) and the 3' probe detects a novel 12.8 kb HindIII fragment (+/Hs-neo) and an 11.0 kb fragment following deletion of the neo cassette (+/Hs). (G) Sequence comparison of the 6.9 kb human sequence with the 6.0 kb mouse sequence it replaced, represented as a percent identity plot (Pip), shows that there is little sequence identity between the two regions outside coding sequence.

 
Paternal inheritance of a human PWS-IC is associated with neonatal lethality and growth deficiency
Mice inheriting the human PWS-IC paternally (PWS-IC+/Hs) showed significant postnatal lethality. In first generation matings with C57BL/6J females, 15% of mutant offspring died by the second postnatal day (P2) and a further 16% died by P16 (n=39; Fig. 2A). In second generation matings with C57BL/6J females, the N2 offspring showed greater postnatal lethality (10 litters, 47% lethality). Postnatal lethality was reduced in 129S1/Sv offspring (four litters, 16% lethality).


Figure 4562
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  		Figure 2. Mice inheriting the human PWS-IC paternally show postnatal lethality and growth deficiency. (A) Paternal inheritance of the PWS-ICHs allele resulted in 30% postnatal lethality in a first generation cross with C57BL/6J females. (B) Wild-type (+/+) and PWS-IC+/Hs (+/Hs) littermates at 7 days of age. (C) Weights of (C57BL/6x129S1/Sv)F1 PWS-IC+/Hs (+/Hs) mice and wild-type (+/+) littermates at 3 weeks of age expressed as the mean±s.e.m. (n=9–20 mice). Male and female PWS-IC+/Hs mice weigh on average 54% of the weights of wild-type mice.

 
In the first postnatal day (P1), PWS-IC+/Hs mice had less milk in their stomachs than wild-type littermates. A growth deficiency was evident soon after birth and persisted throughout postnatal development (Fig. 2B), suggestive of failure to thrive. By 3 weeks of age, surviving PWS-IC+/Hs mice had considerable growth retardation, with both sexes weighing on average 54% of the weight of wild-type littermates (Fig. 2C). They remain smaller than their wild-type littermates but have no other obvious defects. Both sexes are fertile.

SNRPN promoter function is conserved but IC function has diverged
In order to determine the cause of postnatal lethality and to determine which functions of the human PWS-IC are not conserved in mouse, we analysed gene expression in newborn brain following paternal inheritance of a human PWS-IC (PWS-IC+/Hs). Snrpn expression was retained in PWS-IC+/Hs mice (Fig. 3A). We used 5' RACE to determine whether transcription initiated at the human promoter or at previously reported upstream promoters of Snrpn (18Go). The majority of transcripts were products of the human promoter (20/21 sequenced; one clone initiated in intron 1 of the human sequence) and human exon 1 spliced to mouse exon 2 as expected (data not shown). Next we analysed expression of three snoRNA genes, MBII-13, MBII-85 and MBII-52. In all cases, wild-type expression levels were observed (Fig. 3A), suggesting that long-range activity of the human promoter is also conserved. In addition, the Ube3a-as was expressed in mutant animals (Fig. 3A), further demonstrating that SNRPN promoter function is conserved.


Figure 4563
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  		Figure 3. Analysis of gene expression following paternal and maternal inheritance of the PWS-ICHs allele. Gene expression was analysed in brain RNA from wild-type (+/+), PWS-IC+/del, PWS-IC+/Hs and PWS-ICHs/del P1 mice. (A) Expression of the Snrpn–Ube3a-as transcript. Expression of Snrpn and the snoRNA genes was analysed by northern blot. Ube3a-as expression was detected by RT–PCR. (B) Expression of genes in the upstream cluster. Ndn expression was analysed by northern blot. Other genes in the cluster were analysed by RT–PCR. ß-actin and 5.8s rRNA were used as loading controls.+and–represent +RT and –RT reactions.

 
We next analysed expression of the genes in the upstream cluster. Northern analysis of Ndn showed a complete absence of expression (Fig. 3B) in PWS-IC+/Hs mice. RT–PCR analysis of Ndn, Mkrn3, Magel2 and Frat3 showed that there was little or no expression of any of the currently identified imprinted genes in the upstream cluster (Fig. 3B).

We next showed that loss of expression of these genes correlates with increased methylation of their promoters. The methylation status of Ndn and Mkrn3 was assayed by methylation-sensitive restriction digest (Fig. 4). The methylation defect in PWS-IC+/Hs mice was comparable to that seen in mice with a 35 kb deletion of the PWS-IC (PWS-IC+/del), which shows complete loss of IC function (17Go).


Figure 4564
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  		Figure 4. Methylation analysis of the Ndn and Mkrn3 DMRs. DNA from brains of wild-type (+/+), PWS-IC+/del, PWS-IC+/Hs and PWS-ICHs/+ was digested with the restriction enzymes indicated (the methylation-sensitive enzyme is listed second), then Southern-blotted and probed. (A) An Ndn probe detects a methylated (Me) fragment of 4.8 kb and an unmethylated (UnMe) fragment of 3.3 kb. (B) An Mkrn3 probe detects methylated and unmethylated fragments of 23  and 17 kb, respectively.

 
The SNRPN promoter is not silenced following maternal inheritance
In order to detect gene expression from the maternal chromosome following maternal inheritance of the PWS-ICHs allele (PWS-ICHs/+), we took advantage of PWS-IC deletion mice (PWS-IC+/del) (17Go) which have no paternal gene expression. Females heterozygous for the PWS-ICHs allele were bred with carrier males for the PWS-ICdel allele. Following maternal inheritance onto a null paternal background (PWS-ICHs/del), the expression pattern observed was identical to that observed following paternal inheritance of the PWS-ICHs allele (PWS-IC+/Hs). Expression levels of Snrpn, MBII-13, MBII-85, MBII-52 and Ube3a-as were comparable to normal paternal levels of gene expression (Fig. 3A). This suggests that the PWS-ICHs allele is not silenced following maternal inheritance and the human SNRPN promoter therefore remains active. We confirmed by 5' RACE that transcription of Snrpn was initiated at the human promoter (21/21 sequenced; one clone contained a 26 base deletion of the exon including the ATG).

Northern and RT–PCR analysis of Ndn and RT–PCR analysis of Mkrn3, Magel2 and Frat3 showed that these genes were not expressed following maternal inheritance (Fig. 3B). This result is consistent with the loss of function of a positive regulatory element observed following paternal inheritance of the human PWS-IC.

Acquisition of a germline methylation imprint is conserved but maintenance has diverged
Expression of the Snrpn–Ube3a-as transcript from the maternal chromosome suggests that there is a failure to silence the human PWS-IC. A methylation sensitive restriction enzyme digestion was used to indicate the methylation status of the human differentially methylated region (DMR) in brains of PWS-ICHs/+ mice. Methylation analysis of mouse Snrpn confirmed that the methylation status of the remaining mouse PWS-IC allele was as expected (Fig. 5A). Using a human SNRPN probe, the human PWS-IC DMR was shown to be unmethylated following paternal inheritance (PWS-IC+/Hs) as would be expected (Fig. 5B). However, the human PWS-IC DMR also remained unmethylated following maternal inheritance (PWS-ICHs/+), suggesting that the factors involved in methylation of the IC have diverged.


Figure 4565
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  		Figure 5. Methylation analysis of the human SNRPN DMR. (A) DNA from brains of wild-type (+/+), PWS-IC+/del, PWS-IC+/Hs and PWS-ICHs/+ was digested with TaqI and SacII. A murine Snrpn probe detects a methylated maternal (Me) fragment of 5.3 kb and an unmethylated paternal (UnMe) fragment of 1.9 kb. The unmethylated allele is absent when the paternal allele is targeted (+/del and +/Hs) and the methylated allele is absent when the maternal allele is targeted (Hs/+) as expected. (B) A human SNRPN probe detects an unmethylated 0.9 kb XbaI/NotI fragment following both paternal and maternal inheritance of the PWS-ICHs allele and shows no evidence of a methylated fragment (4.0 kb) following maternal inheritance (Hs/+). (C) Methylation of the human SNRPN DMR was analysed by bisulphite sequencing in brain and oocytes from PWS-IC+/Hs and PWS-ICHs/+ females. Bisulphite analysis of the mouse Snrpn locus was used as a control. Filled circles are used to indicate methylated CpGs, whereas open circles represent unmethylated CpGs. Each line represents an individual clone. Three sets of oocytes were amplified. +/Hs, paternally inherited PWS-ICHs allele; Hs/+, maternally inherited PWS-ICHs allele.

 
Methylation of the PWS-IC DMR is acquired during female gametogenesis. The murine DMR is fully methylated in mature oocytes (19Go,27Go). The human DMR was originally reported to be unmethylated in oocytes (19Go) but a recent study showed it to be fully methylated in mature oocytes (20Go). To determine whether the imprinting defect lies in the acquisition of the methylation imprint or in imprint maintenance, we analysed the methylation status of the DMR in oocytes. Oocytes were isolated from superovulated heterozygous PWS-ICHs females. DNA isolated from brain and oocytes was bisulphite-converted and the exon 1 region of the DMR was amplified using both human SNRPN and mouse Snrpn specific primers. Sequence analysis confirmed that the mouse DMR was methylated in brain when maternally inherited (PWS-IC+/Hs) but not when paternally inherited (PWS-ICHs/+) and that it was methylated in oocytes (Fig. 5C). The SNRPN exon 1 region of the human PWS-IC was found to be completely unmethylated in brain regardless of parental inheritance (Fig. 5C), confirming the results of methylation-sensitive restriction enzyme analysis. Surprisingly, the same region of the human PWS-IC was shown to be fully methylated in oocytes (Fig. 5C). The results suggest that the imprinting machinery involved in the acquisition of the imprint is conserved but the machinery involved in its maintenance has diverged, suggesting different factors are involved in each process.

Maternal expression of Ube3a-as represses maternal Ube3a
Imprinted expression of UBE3A has been proposed to be regulated in cis by UBE3A-AS (28Go). In support of this model, we have previously shown that the PWS-IC positively regulates Ube3a-as and negatively regulates Ube3a (29Go). To assess the effect of maternal expression of Snrpn–Ube3a-as in PWS-ICHs/+ mice on maternal expression of Ube3a, we took advantage of an expressed polymorphism between Mus musculus domesticus and Mus musculus castaneus to distinguish allele-specific expression of Ube3a (29Go). A heterozygous PWS-ICHs female was crossed with a male mouse congenic for a region of castaneus chromosome 7 (B6.CAST.c7) and Ube3a expression was analysed in the F1 offspring by RT–PCR and restriction digest. In wild-type mice, expression of Ube3a was both maternal and paternal in origin (Fig. 6A). Following maternal inheritance of the PWS-ICHs allele, expression from the maternal domesticus allele was reduced, showing that maternal Ube3a expression is repressed in these mice. The maternal chromosome expresses Ube3a-as in cis (Fig. 3A), providing further evidence that antisense transcription regulates Ube3a imprinting.


Figure 4566
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  		Figure 6. Maternal expression of the Snrpn–Ube3a-as transcript represses maternal Ube3a and causes overgrowth. (A) Brain RNA was isolated from (PWS-IC+/HsxB6.CAST.c7)F1 mice at P1. Ube3a was amplified from two PWS-ICHs/+ mice and one wild-type littermate (+/+) by RT–PCR with Ube3a 5F and 6R and products were digested with Tsp509I. Fragment sizes for the castaneus (paternal) and domesticus (maternal) alleles are indicated. (B) Male and female (129S1/SvxC57BL/6)F1 mice inheriting the PWS-ICHs allele maternally were weighed at 3 week intervals. Weights are expressed as the mean±s.e.m. (n=5–6 mice). (C) Female PWS-ICHs/+ and PWS-IC+/+ littermates at 56 weeks of age when they weighed 79 and 47 g, respectively.

 
A maternal imprinting defect causes obesity
It became apparent that mice with maternal inheritance of the PWS-ICHs allele could be easily distinguished from their wild-type littermates at weaning by increased size. We followed weight gain in (129S1/SvxC57BL/6)F1 PWS-ICHs/+ mice from weaning until 18 weeks of age (n=5–6 mice). Both sexes gained weight more rapidly than their wild-type littermates (Fig. 6B). By 18 weeks of age, female and male PWS-ICHs/+ mice were, respectively, 63.7±8.3 and 26.7±4.3% heavier than wild-type littermates. There was no increase in morbidity up to 1 year of age although the increased growth trend continued. PWS-ICHs/+ and wild-type female littermates at 56 weeks of age are shown in Figure 6C. Preliminary analysis suggested that increased body weight was in part due to increased visceral fat deposition, particularly in females where the growth effect is more pronounced.

More than one locus contributes to PWS phenotypes in mice
In infants, PWS is characterized by neonatal hypotonia and poor suckle, which contribute to an overall ‘failure to thrive’ (30Go). Mice with paternal inheritance of a PWS-IC deletion have severe failure to thrive, resulting in 100% postnatal lethality on some strain backgrounds (17Go). Paternal deletion from Snrpn exon 2 to Ube3a exon 2 causes 80% postnatal lethality and growth deficiency in surviving mice (31Go), implicating a transcript in this region in failure to thrive. Paternal inheritance of the PWS-ICHs allele results in 30% postnatal lethality and growth deficiency, suggesting a locus outside the Snrpn-Ube3a-as transcript is also involved in failure to thrive. The existence of two independent failure to thrive loci is further supported by the results of complementation experiments. We crossed (C57BL/6Jx129S1/Sv)F1 or 129S1/Sv heterozygous PWS-ICHs females with C57BL/6 heterozygous PWS-ICdel males and 65 mice survived to weaning from 12 litters. Of these, 21 mice had inherited the human PWS-IC on the maternal chromosome in conjunction with a paternal PWS-ICdel allele (PWS-ICHs/del; observed/expected=98.4%). Only one PWS-IC+/del mouse survived to weaning (observed/expected=4.6%). PWS-IC+/del offspring from mothers of the F1 or 129S1/Sv background generally only survive when the wild-type littermates are removed shortly after birth (32Go). We conclude that maternal expression of the Snrpn–Ube3a-as transcript complements one failure to thrive locus and thereby rescues postnatal lethality caused by paternal inheritance of a PWS-IC deletion. PWS-ICHs/del mice still have a growth deficiency, suggesting failure to complement a second failure to thrive locus. (129S1/SvxC57BL/6)F1 PWS-ICHs/del male and female mice weigh 58 and 61%, respectively, of the weight of wild-type littermates by 3 weeks of age (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The failure of human SNRPN (25Go) and H19 (26Go) transgenes to imprint in mouse has led to the suggestion that the imprinting machinery has diverged. In previous studies, we found that mice carrying a human transgene expressed SNRPN following maternal inheritance, suggesting that a silencing element is species-specific (25Go). In this study, we placed an entire imprinted domain in the mouse under the control of a human ICR. This demonstrates that a positive regulatory element in the PWS-IC has also diverged, separating paternal IC function from promoter function. We now show that acquisition of silencing is conserved but maintenance of the silenced state has diverged. Expression of the Snrpn–Ube3a-as transcript from the unsilenced maternal chromosome leads to repression of maternal Ube3a and suggests a mechanism by which AS imprinting defects cause disease.

The PWS-IC functions to activate the paternal pattern of gene expression (Fig. 7A) (6Go). Paternal inheritance of the PWS-ICHs allele results in the loss of expression of genes in the upstream cluster (Frat3, Mkrn3, Magel2 and Ndn), confirming a proposed positive role for the PWS-IC in activating expression of these transcripts. The loss of IC function seen in human PWS-IC mice suggests that the sequences and trans-acting factors involved in activation have diverged. We have previously shown that the PWS-IC is required postzygotically (33Go), suggesting that it protects the upstream genes during de novo methylation in early embryogenesis or has a maintenance role in somatic cells. Chromatin looping could facilitate an interaction between the upstream genes and their distant regulatory element. Similar epigenetically regulated chromatin interactions have been demonstrated at the imprinted H19/Igf2 locus (34Go) where DMRs interact to protect each other from methylation in a hierarchical fashion (35Go).

In the prevailing model, the AS-IC functions to prevent the activation of a paternal programme of gene expression on the maternal chromosome. The AS-IC is thought to function in the female germline by silencing the PWS-IC through a methylation imprint (Fig. 7A) (6Go). We previously reported that an unmodified human transgene containing both the AS-SRO and PWS-SRO expressed SNRPN following both maternal and paternal inheritance (25Go), suggesting that the human AS-SRO is unable to silence the human PWS-SRO in mouse. Studies using fusion transgenes suggested that the human AS-SRO functions to silence the Snrpn minimal promoter in mouse (36Go) and, more recently, that it can functionally interact in mouse with human PWS-IC sequences (37Go). The PWS-IC contains a germline DMR established during oogenesis (19Go,20Go) likely through a mechanism involving the AS-IC. In this study, we have shown that the human PWS-IC acquires a methylation imprint during oogenesis, suggesting that the interaction between the AS-IC and the PWS-IC is conserved. However, the human PWS-IC is unmethylated in somatic tissue, showing that maintenance of methylation at the PWS-IC has diverged (Fig. 7B). A more detailed analysis of methylation during embryonic development will be required to establish the dynamics of demethylation. Unfortunately, the mouse strain carrying a human transgene (25Go) is no longer maintained, preventing analysis of the methylation status in oocytes of a human PWS-IC at a transgenic locus. It is interesting to note that human H19 transgenes were able to assemble partial methylation in the male germline, but only at high copy number, and methylation was unstable during early development (26Go). In stark contrast to the results presented here, a high copy number-modified transgene containing human AS-SRO and PWS-SRO sequences was unmethylated in oocytes but acquired methylation during development (37Go). The lack of a germline methylation imprint suggests that the human AS-IC does not function appropriately, supporting the notion that the imprinting machinery has diverged.


Figure 4567
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  		Figure 7. Gene regulation and function in the PWS/AS region in mouse. (A) Normal regulation of gene expression. The PWS-IC (green oval) activates expression of paternal genes. Expression of Ube3a-as in some tissues represses paternal Ube3a and, possibly, Atp10a. The AS-IC (red circle) negatively regulates the PWS-IC preventing activation of a paternal programme of gene expression on the maternal chromosome. The AS-IC is thought to function in the female germline. The PWS-IC contains a germline methylation imprint (filled lollipop). (B) A model for the establishment and maintenance of methylation of a human PWS-IC in mouse. The human PWS-IC (orange oval) acquires methylation in oocytes (filled lollipop) possibly through a mechanism involving the mouse AS-IC. Methylation is lost in somatic tissue possibly through the inability of murine trans-acting factors (yellow) to recognize the human sequence. Expression of Snrpn–Ube3a-as on the maternal chromosome leads to repression of maternal Ube3a expression. (C) Failure-to-thrive phenotypes in PWS mouse models. Paternal gene expression is lost following paternal inheritance of a PWS-IC deletion (PWS-IC+/del) resulting in failure to thrive and 100% postnatal lethality (17Go). Paternal inheritance of a deletion between Snrpn exon 2 and Ube3a exon 2 causes growth deficiency and 80% postnatal lethality (31Go), indicating a gene in this region is involved in failure to thrive. As reported here, paternal inheritance of a human PWS-IC (PWS-IC+/Hs) causes growth deficiency and 30% postnatal lethality, suggesting that a gene outside the Snrpn–Ube3a-as transcript is also involved in failure to thrive.

 
Identifying the sequence-specific factors involved in the regulation of methylation within the PWS-IC will be essential in determining how the imprinting machinery has evolved between mouse and human. Relatively little is known about the trans-acting factors involved in the establishment and maintenance of methylation imprints. Members of the DNA cytosine methyltransferase (Dnmt) family have essential roles in the establishment and maintenance of methylation at many imprinted loci, but little is known about other proteins involved in sequence-specific methylation. Dnmt3L (38Go) and Dnmt3a (39Go) have been shown to be required for the establishment of methylation at the PWS-IC DMR, and Snrpn expression is perturbed in Dnmt1–/– embryos (40Go), suggesting that Dnmt1 is required for the maintenance of methylation at the Snrpn locus. A small number of proteins that regulate the establishment or maintenance of methylation at only a few imprinted loci (41Go–44Go) have been identified. The ability of the human PWS-IC to assemble methylation in the germline but failure to maintain it during development indicates that different factors are involved in the two processes. Comparative analysis of the two loci in mice will assist in the identification of trans-acting factors involved in imprinted regulation in the region.

Expression analysis indicates that SNRPN promoter function is conserved. Expression of Snrpn, the snoRNAs and Ube3a-as suggests that these transcripts are regulated by promoter function and not by IC function per se and transcription of the entire locus supports a model in which transcription is initiated at Snrpn, as suggested for the human locus (9Go). On the maternal chromosome, loss of methylation at the human PWS-IC results in expression of the entire Snrpn–Ube3a-as locus, including Ube3a-as. Repression of Ube3a on the maternal chromosome supports a model in which Ube3a is imprinted by its antisense RNA, although we cannot yet exclude a role for one of the other components of the Snrpn–Ube3a-as transcript.

In the postnatal period, PWS is characterized by neonatal hypotonia and feeding difficulties resulting in failure to thrive (30Go). Mouse models of PWS demonstrate early growth restriction and postnatal lethality indicative of a failure to thrive phenotype. Paternal inheritance of a PWS-IC deletion allele results in the loss of paternal gene expression and is associated with severe failure to thrive, leading to 100% postnatal lethality in (C57BL/6Jx129S1/Sv)F1 offspring (17Go). A deletion spanning from Snrpn to Ube3a is associated with ~80% postnatal lethality and growth deficiency on a similar strain background (31Go). Here we show that paternal inheritance of a human PWS-IC leads to growth deficiency and 30% postnatal lethality among (C57BL/6Jx129S1/Sv)F1 offspring. Expression of Snrpn–Ube3a-as is unaltered in these mice but upstream gene expression is lost. This suggests that more than one locus is involved in growth deficiency and postnatal lethality in mouse models of PWS (Fig. 7C). This is further supported by the fact that maternal expression of the Snrpn–Ube3a-as transcript rescues postnatal lethality associated with paternal inheritance of a PWS-ICdel allele. These mice still have growth deficiency, presumably owing to loss of upstream gene expression.

Available evidence argues against involvement of several single-gene loci in failure to thrive. Mutations affecting either of the products of the bicistronic Snrpn transcript, Snurf (18Go,31Go) and SmN (17Go), have no overt phenotype. Postnatal lethality due to Ndn-deficiency is reported to occur within the first 30 h following birth (45Go,46Go). Loss of Ndn expression could therefore contribute to early postnatal lethality in PWS-IC+/Hs mice. However, 50% of postnatal lethality in PWS-IC+/Hs mice occurs more than 48 h after birth and growth deficiency has not been reported in surviving Ndn-deficient mice (45Go–47Go). Frat3-deficient mice have also been described recently and have no obvious phenotype (48Go). The phenotypes could require the loss of more than one gene or could be due to the loss of an as-yet-uncharacterized transcript.

AS-IC deletions in humans result in failure to silence the PWS-IC, as indicated by the presence of a paternal epigenotype on the maternal chromosome and biparental expression of SNRPN and IPW (49Go). In these individuals, AS is thought to be caused by the repression of maternal UBE3A expression, through a mechanism proposed to involve UBE3A-AS (28Go). In this study, the human PWS-IC fails to maintain the methylation imprint acquired during oogenesis, leading to expression of the Snrpn–Ube3a-as transcript. We have shown that maternal Ube3a expression is reduced in these mice, supporting a role for the Snrpn–Ube3a-as transcript in Ube3a repression and generating a mouse model for AS imprinting defects.

Phenotypically, these mice serve as a model for increased body weight in AS. Of the five classes of mutations which cause AS in humans, three are associated with increased body weight (50Go). The most striking increase is seen in uniparental disomy (UPD) and IC mutation individuals, but body weight is also increased in individuals with a UBE3A mutation. Although this suggests that obesity could be due to loss of imprinted UBE3A expression, increased body weight has not been described in Ube3a-deficient mice (51Go,52Go). However, UPD and deletion mouse models of AS (5Go,53Go) are associated with obesity. A maternally inherited obesity phenotype of radiation-induced deletions mapping to Atp10a (54Go) suggests that increased body weight in UPD, deletion and PWS-ICHs/+ mice could be due to loss of maternal Atp10a expression and supports tissue-specific imprinting of Atp10a (55Go). Although we have not shown that maternal Atp10a is repressed in PWS-ICHs/+ mice, the phenotypic overlap supports this possibility and suggests that Atp10a imprinting could also be regulated by Ube3a-as.

More extensive phenotype analysis will be required to assess the merit of these mice as an animal model of AS imprinting defects. Maternally deficient Ube3a mutant mice are indistinguishable from their wild-type littermates (51Go,52Go). More detailed phenotypic analysis reveals impaired motor function, inducible seizures, abnormal electroencephalograph (EEG) and a defect in long-term potentiation. Mice with paternal duplication for the region also show mild gait ataxia and abnormal EEG, but no seizures were reported (53Go). Seizures occur with lower incidence and later onset in AS individuals with UPD or imprinting defects (50Go). This suggests that seizures could be due to haploinsufficiency of UBE3A in other brain regions rather than loss of imprinted UBE3A expression. In this case, the frequency of seizures would be expected to be lower in UPD mice and the model reported here.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
ES cells and mice
The 5' and 3' arms of homology were cloned from a 129S1/Sv phage library as a 4.3 kb BamHI–HpaI fragment and a 3.5 kb EcoRI fragment, respectively (Fig. 1). A 6.9 kb SmaI fragment spanning SNRPN exon 1 was isolated from an SNRPN-containing P1 clone. A floxed PGK-neo gene and a PGK-tk gene were used for positive and negative selections. CJ.7 ES cells were grown under standard conditions on mitomycin C-treated MEFs (17Go) and selected in 200 µg/ml G418 and 0.7 µM FIAU. Homologous recombinants were identified using a HindIII digest and a 5' probe and targeting was confirmed using an ApaI digest and a 3' probe. The floxed neo gene was deleted in two independent targeted lines by transient transfection with a Cre recombinase expression plasmid (56Go).

ES cells were injected into C57BL/6J blastocysts and transferred into pseudopregnant B6D2F1 recipients. Chimeric males were mated with 129S1/Sv and C57BL/6J females. All weight analyses and oocyte collections were performed on F1 hybrids of 129S1/Sv and C57BL/6. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Florida.

Sequence analysis
The human and mouse sequences were compared using PipMaker (http://pipmaker.bx.psu.edu/pipmaker), with default settings. The human sequence was used as the base sequence and repeat regions were identified using Repeat Masker (http://www.repeatmasker.org).

Analysis of gene expression
RNA was isolated from brains of P1 mice, using RNazol (TelTech). Total RNA was separated on 1% agarose formaldehyde gels and transferred to Hybond N+ membranes. To detect snoRNAs, total RNA was separated on 8% acrylamide-7 M urea gels and electroblotted onto Hybond N+ membranes. Membranes were hybridized overnight at 58°C with oligonucleotides for MBII-13, MBII-85, MBII-52 and 5.8S rRNA (57Go) end-labeled with {propto}32P-ATP (PerkinElmer) and T4 polynucleotide kinase (Invitrogen).

RNA was reverse-transcribed with Superscript II (Invitrogen) and PCR reactions were performed under standard conditions. Primer sequences are available on request.

5' RACE was performed using the 5' RACE ready kit (Invitrogen) according to the manufacturer's instructions, with gene specific primers Gsp1 (5'-CCTTGAAGTTGCAATACTGC-3') and Gsp2 (5'-CGTGGGTACAAGTGACACTCTTGG-3').

Allele-specific expression of Ube3a was analysed as previously described (29Go) using a polymorphic Tsp509I site present in M. domesticus and absent in M. castaneus. Females heterozygous for the PWS-ICHs allele were crossed with B6.CAST.c7 males (recombinant congenic for a region of castaneus chromosome 7 on a C57BL/6 background). Brain RNA was isolated from the resulting offspring at postnatal day 1 (P1). Random primed cDNA was amplified with Ube3a 5F (5'-CACATATGATGAAGCTACGA-3') and 6R (5'-CACACTCCCTTCATATTCC-3'). PCR products were gel-purified, digested with Tsp509I and electophoresed on 4.8% agarose gels.

Methylation analysis
DNA was isolated from brain, digested with the appropriate restriction enzymes and analysed by Southern blot. Probe details are available on request.

For bisulphite analysis, groups of 3 or 4 PWS-ICHs/+ or PWS-IC+/Hs females were superovulated at 7–8 weeks of age. Oocytes were collected in M-2 medium (Specialty Media) and cumulus cells dispersed using 1 mg/ml hyaluronidase (Sigma). Oocytes were washed four or five times to remove somatic cells and cells were lysed in 1 mM SDS in the presence of 280 µg/ml proteinase K.

Bisulphite conversion of DNA was performed as described (58Go). Briefly, DNA was denatured in 0.3 M NaOH. Conversion was performed in 1.55 M sodium metabisulphite and 0.5 mM hydroquinone at 55°C in the dark for 16–20 h. Free bisulphite was removed on a desalting column (Promega Wizard DNA Clean-Up). Samples were desulphonated by alkali treatment and precipitated prior to PCR.

Brain samples were amplified for 35 cycles with Bi-SNP-F1 and Bi-SNP-R1 (19Go) and with mouse specific primers Snrpn inside forward and inside reverse (27Go). Half of the converted DNA from pools of 70–130 oocytes was amplified for 45 cycles with SNIF and SNIIR (20Go), using SNIIF and SNIIR in second round amplifications where necessary. The remaining half was amplified for 45 cycles, with the mouse primers Snrpn inside forward and inside reverse. PCR products were cloned directly into the TOPO TA cloning kit (Invitrogen) or were gel-purified and cloned into the pGEM-T Easy vector system (Promega) prior to sequencing. Human PCR products were also directly sequenced with SNIIR. Sequences with <95% bisulphite conversion efficiency were not included in the analysis. Incomplete conversion enabled us to confirm that individual clones were derived from independent oocyte genomes.


   ACKNOWLEDGEMENTS
 
We thank Marisa Bartolomei for support, advice and critical reading of the manuscript; Edwin Peery for critical reading of the manuscript; Daniel Driscoll and Stormy Chamberlain for helpful discussion; Danielle Maatouk for advice on bisulphite sequencing and Jingda Shi and the Center for Mammalian Genetics for sequencing. This work was supported by NIH grants GM55272 and HD37872 awarded to C.I.B. This work is dedicated to the memory of Camilynn Brannan.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} Present address: Stower's Institute for Medical Research, 1000 E 50th Street, Kansas City, MO 64110, USA. Back

{ddagger} Deceased. Back


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