Mechanisms of establishment and maintenance of germline-derived methylation imprints are not fully understood. In a screen for genes down-regulated upon ES cell differentiation, we identified a maternal-zygotic effect gene, Zfp57, that contributes to the stable maintenance of methylation imprints during development.
Consistent with its expression in the female germline, Zfp57 is required for the establishment of the germline methylation imprint at the Snrpn imprinted region. More detailed biochemical analysis needs to be undertaken to determine whether ZFP57 is directly involved in the acquisition or maintenance of this methylation in the female germline. In contrast, maternal Zfp57 is not essential for the establishment of the genomic imprints at other examined maternally methylated regions (Peg1, Peg3 and Nnat/Peg5) in oocytes, or in the male germline. Consistent with the maternal-zygotic effect evident from the genetic data, a role for Zfp57 in imprinting establishment appears to be specific to the female germline.
In contrast, maternal Zfp57
plays a much broader role in the maintenance of both paternal and maternal methylation imprints in embryos. Genomic imprints are not maintained at the paternally methylated IG-DMR of the Dlk1
domain and at maternally methylated Peg1
in the absence of both maternal and zygotic functions of Zfp57
in embryos. These data indicate that ZFP57 plays a role in the post-fertilization maintenance of genomic imprints at a large subset of the imprinted regions. Indeed, it was just published online that zygotic mutations in human Zfp57
also affect differential DNA methylation at a subset of imprinted regions (Mackay et al., 2008
). Variable effects of this mutation in different individuals are consistent with the defects we observe in the zygotic mutant mice. Another maternal effect gene, Stella/PGC7,
appears to play a partially protective role in the maintenance of the other two paternally-derived methylation imprints at H19
, and three maternally-derived methylation imprints at Peg1
(Nakamura et al., 2007
). In contrast to Zfp57, Stella/PGC7
does not affect Dlk1-Dio3, Snrpn
or play a role in the germline establishment of imprints. The overlapping effect of Zfp57
suggests that their maintenance functions may not be mutually exclusive.
Interestingly, differential DNA methylation at the Snrpn
DMR was established in the presence of the zygotic Zfp57
in post-implantation embryos even though germline methylation was either not established or lost in oocytes and remained absent at E3.5 (Supplemental Figure S13
). Since ZFP57 can bind to the Snrpn
DMR in ES cells, it is likely that ZFP57 can bind to the unmethylated Snrpn
DMR directly during methylation acquisition in embryos. This acquisition of methylation only occurs at the maternally derived Snrpn
DMR. This suggests that there may be a DNA methylation-independent genomic imprint that can act as imprinting memory at the Snrpn
imprinting control region. Previously it has been shown that the imprinting of some placental genes occurs in the absence of maintenance methylation, likely involving repressive histone modifications (Lewis et al., 2004
; Umlauf et al., 2004
). It will be interesting to determine whether repressive histone modifications play a role in the heritable methylation-independent germline mark at Snrpn
. Future experiments will address the many intriguing questions arising from this finding including the nature of the heritable memory and whether there is a bias for the grandmaternal over the grandpaternal maternal allele. Since some heterozygous embryos derived from null female mice had no or only partial DNA methylation around midgestation whereas others displayed a fully methylated maternal allele, it is possible that differential methylation at the Snrpn
DMR in the presence of the zygotic Zfp57
occurs over time in embryos and all progeny from this cross may acquire full differential methylation at the maternal allele in late stages of development.
The effects of ZFP57 on imprint methylation are consistent with the lethality of embryos described for the range of genetic crosses: (1) Loss of just zygotic Zfp57 causes partial neonatal lethality as well as partial loss of differential DNA methylation; (2) loss of only the maternal Zfp57 does not appear to cause any lethality or any loss of differential DNA methylation due to the rescue of the maternal effect by zygotic Zfp57; (3) eliminating both the maternal as well as zygotic Zfp57 results in complete loss of differential DNA methylation as well as a highly penetrant embryonic lethality around mid-gestation. This lethality is consistent with phenotypes expected from the cumulative effects of loss of imprinting of a subset of imprinted genes although Zfp57 may also play a role at some unidentified non-imprinted loci.
How might this KRAB zinc finger protein be influencing the epigenetic state of imprinted domains? Interestingly, it was recently reported that the KRAB domain can trigger heritable de novo
DNA methylation when targeted to a reporter transgene specifically in early mouse embryos (Wiznerowicz et al., 2007
). This is consistent with an endogenous role for the KRAB zinc finger protein ZFP57 in mediating DNA methylation at some imprinted domains. We therefore suggest a model in which ZFP57 might be directly involved in targeting methylation to certain imprinting control regions; in the female germline at Snrpn
and at several imprinted ICRs after fertilization. This implies that the maintenance of imprints during early development is a regulated event and more than a mere protection against active and passive demethylation. It is possible that ZFP57 can also bind to the methylated Snrpn
DMR and other imprinted regions. Future experiments focusing on the functions of ZFP57 and associated complexes may elucidate a novel pathway of epigenetic control involving zinc-finger proteins and lead to a better understanding of the underlying mechanisms of the establishment and maintenance of DNA methylation.