Roughly 4% of individuals with AS have an imprinting defect, characterized by a paternal epigenotype at the PWS/AS locus on the maternally inherited chromosome. Of these individuals, 10–15% have a microdeletion that includes the AS-IC 
. The AS-IC is thought to operate in the female germ line to epigenetically inactivate the PWS-IC and thereby silence the paternally expressed genes on the future maternal allele 
. Among these maternally silenced genes is UBE3A-ATS
, a transcript antisense to UBE3A
is thought to silence UBE3A
expression by an unknown mechanism 
. In individuals lacking AS-IC function, UBE3A-ATS
is expressed from the maternal allele in addition to the paternal allele and the consequent lack of UBE3A
results in AS. Hypomethylation of the maternal SNRPN
DMR is diagnostic of an AS imprinting defect 
The AS-IC functions in the female germ line, complicating investigations into imprinting mechanisms at the PWS/AS locus. Deletion analysis mapped the human AS-IC to 35 kb upstream of the PWS-IC but deletion of similarly positioned sequences in the mouse had no detectable consequence 
. Here we used BAC transgenes to identify murine AS-IC activity. We found that 15 kb of sequence upstream of Snrpn
exon 1 was insufficient to direct imprinted expression of Snrpn
. Transgenes containing 100 kb of sequence 5′ to Snrpn
exon 1 directed the appropriate imprinted expression of Snrpn
. Furthermore, this sequence was sufficient to direct appropriate hypermethylation of the Snrpn
DMR following maternal but not paternal transmission. We conclude that an AS-IC activity is contained within 15 to 100 kb of the PWS-IC. This region contains three alternative exons, at least two of which are expressed in oocytes and splice into Snrpn
exon 2. Deletion of these U exons from the BAC transgene resulted in biparental Snrpn
expression and DNA hypomethylation at the PWS-IC, characteristic of an imprinting defect. Surprisingly, a second single copy line lacking the U exons displayed appropriate maternal silencing. In contrast to the biparentally expressed transgene but similar to imprinted transgenes with the U exons intact, this line exhibited transcription through the PWS-IC, consistent with the notion that transcription is an essential element of AS-IC activity. Lastly, we demonstrated that U exon transcription is undetectable in fetal oogonia as they are entering meiotic prophase, a time when imprinting is absent, but becomes evident after birth and prior to the application of the DNA methylation imprint at the PWS-IC. We conclude that the U exons exhibit AS-IC activity as previously proposed 
transcription becomes biallelic in PGCs as they colonize the gonad at midgestation 
. This stage of imprinting erasure in the germ line coincides with the removal of the DNA methylation imprint at the PWS-IC 
. The appearance of the DNA methylation imprint is tied to postnatal oocyte growth 
. Our data support a model in which growing oocytes direct transcription from the U exons through the PWS-IC. This transcription is necessary to epigenetically modify the PWS-IC leading to the maternal imprint (). Importantly, our results do not distinguish whether the RNA transcript or the act of transcription is essential for AS-IC activity. However, since transcription presumably arising from sequences flanking the BAC insertion site appears to be sufficient for imprinting in the 425ΔU1-U3H line, we suggest that the U exon sequences per se
are not required.
A working model for the establishment of the maternal imprint at the PWS-IC.
Our data support a model in which U exon promoter activity supplies AS-IC function in the mouse. The human AS-IC, however, includes two upstream exons that are internal to transcripts encoded in brain RNA. If the human AS-IC does not possess promoter activity in oocytes, we speculate that the AS-IC may serve an RNA antitermination or stabilization role.
In both human and mouse, splicing of the upstream exons to Snrpn
exon 2 can encode the Snurf polypeptide 
. It is unknown whether Snurf
is expressed in oocytes but the absence of an overt phenotype in Snurf
knockout mice indicates that it is not involved in the imprinting process 
Our BAC transgene system allows simultaneous assessment of both the transgene and endogenous locus. U exon transcription arising from the endogenous locus does not impose imprinting upon a maternally transmitted transgene harboring a PWS-IC but lacking U exons ( and ). This observation suggests that if the RNA transcript is an essential element of the imprinting machinery, then it does not function in trans.
Wu et al. 
described two mutants with a maternal imprinting defect. Maternal transmission of an 80 kb deletion spanning from 10 to 90 kb upstream of Snrpn
exon 1 resulted in an extensive loss of DNA methylation at the PWS-IC in only three of eight offspring. This deletion removes only U1 and U3, leaving the remaining seven U exons intact. Our model suggests that extensive imprinting defects were not observed in the remaining offspring due to compensatory transcription arising from the remaining U exons. The second mutant described by these investigators exhibited a highly penetrant imprinting defect leading to expression of paternal genes from the maternal allele and reduced expression of the Ube3a
gene product, E6AP. This mutant carried an insertion 10 kb upstream of Snrpn
exon 1 that was comprised of a puromycin
resistance cassette in the opposite transcriptional orientation to Snrpn
, exons 3–9 of an Hprt
minigene, and a duplication of 6 kb of endogenous sequence. Our findings support a model in which the insertion interferes with transcription from the U exons in oocytes.
The imprinting activity of the human AS-IC element has been tested in mice. Five independent lines of a human P1 phage transgene that included both the human AS-IC and PWS-IC failed to show evidence of imprinted gene expression in the brain 
. This lack of imprinting may be explained by our more recent observation that a human PWS-IC substituted for the endogenous murine PWS-IC can acquire a DNA methylation imprint in oocytes but this imprint is subsequently lost during development 
. Shemer et al. 
characterized transgenes containing the human AS-IC linked to the murine PWS-IC and observed preferential Snrpn
expression following paternal inheritance. Transcription through the PWS-IC during oogenesis was not examined in these transgenes but our model suggests that the partial imprinting seen in these transgenes resulted from transcription of the human upstream exons.
Zogel et al. 
reported that a maternally inherited polymorphism at the AS-IC increases the risk of imprinting defects leading to AS. Our observations strongly support these investigators′ hypothesis that this polymorphism may affect a transcription factor recognition site. Our model suggests that this polymorphism affects transcription through the PWS-IC during oogenesis, decreasing the efficiency of epigenetic modification at the PWS-IC and thereby raising the risk of AS imprinting defects.
Transcription is implicated in allelic silencing at several imprinted gene clusters (See Peters and Robson 
for a review). Our results at the PWS/AS locus are consistent with the observation that in growing oocytes, transcripts traverse maternal germ line DMRs at eight locations, suggesting that transcription is commonly associated with the establishment of maternal imprints 
. A recent report indicates that 35% of CpG islands that are methylated in oocytes are downstream of active promoters 
. The role of transcription through a germ line DMR in establishing a DNA methylation imprint in oocytes has been tested at the Gnas
. At this locus, Nesp
transcription is active in oocytes and traverses two maternal DMRs. Similar to the U transcripts at the PWS/AS locus, appearance of the Nesp
transcript at the Gnas
locus temporally precedes the establishment of the maternal methylation imprint in growing oocytes. Furthermore, truncation of the Nesp
transcript led to a loss of the DNA methylation imprint at several maternal DMRs within the Gnas
locus and activation of normally silent maternal transcripts. Chotalia et al. 
suggested several mechanisms by which transcription might contribute to DNA methylation including an interaction of the transcription complex with the methylation machinery, creating an open chromatin environment allowing DNA methylation machinery access to the locus, or recruiting factors necessary for epigenetic modification. These same mechanisms could operate to set the methylation imprint at the PWS/AS locus. Similar to the Gnas
locus, truncation of the U exon transcripts upstream of the PWS-IC may confirm their role in establishing imprinting throughout the PWS/AS locus.