(A) Total RNA samples from three homozygous (-/-) and three littermate heterozygous (-/+) embryos derived from the same null mother were probed with Dlk1 cDNA in Northern blot.
(B), (C), (D) Equal amount of polyA RNA samples from a homozygous (-/-) and two littermate heterozygous (-/+) embryos derived from the same null mother were hybridized with a probe derived from Rtl1, Dio3 or Gtl2, respectively. Numbers in (D) indicate the band intensity of Gtl2 transcripts in each lane based on phosphor image analysis. Similar band intensity of 18S ribosomal RNA shown in the bottom panels of all figures indicates equal loading of the RNA samples.
All embryos are in the pure 129 Sv/Ev background.

Genomic DNA samples from oocytes or embryos in the 129 Sv/Ev background were subjected to bisulphite sequencing. A total of 16 differentially methylated CpG sites at the Snrpn DMR are shown for Figures A-D. Filled oval, methylated CpG site. Open oval, unmethylated CpG sites. Line with ovals, a unique clone. A unique clone was assigned based on unconverted C residues in the sequences. The number of sequences is shown for a non-unique clone. The high conversion rate fails to distinguish whether these non-unique sequences represent clonal or individual products.
A, Two different pools of unfertilized oocytes isolated from heterozygous female mice.
B, Two different pools of unfertilized oocytes isolated from homozygous female mice.
C, pooled E3.5 embryos. -/+ (E3.5), 29 heterozygous E3.5 embryos from the crosses between homozygous female mice and wild-type male mice. -/- (E3.5), 10 homozygous E3.5 embryos from the crosses between homozygous female mice and homozygous male mice. +/- (E3.5), 16 heterozygous E3.5 embryos from the crosses between wild-type female mice and homozygous male mice. D, genomic DNA samples were made from littermate E13.5 embryos from the cross between a homozygous female mouse and a heterozygous male mouse.
E, Acquisition of DNA methylation imprint occurred on the maternally derived Snrpn DMR. Histograms are shown for the levels of methylation at the CpG sites of this DMR in E11.5-E13 embryos from the cross between a homozygous female mouse in the 129 Sv/Ev background and a wild-type male mouse in the DBA/2 background. Unfilled bars, eight littermate heterozygous embryos (1- 8) from this cross. Filled bar, seven littermate wild-type (WT) embryos from the cross between a wild-type 129 female mouse and a wild-type DBA/2 male mouse. Vertical axis, percentage of methylated CpG sites analyzed. Left panel, maternally derived 129 allele. Right panel, paternally derived DBA/2 allele.

Pre-implantation embryos were stained with affinity-purified polyclonal antibodies against ZFP57 (Figures A-H). They were also incubated with Hoechst dye to illuminate genomic DNA (Figures A’-H’).
A homozygous E1.5 embryo (A or A’) and a homozygous E3.5 embryo (D or D’) were derived from the cross between homozygous female mice and homozygous male mice. Littermate E1.5 embryos (B and C) and E3.5 embryos (F and G) were derived from the crosses between heterozygous female mice and homozygous male mice.
B (or G), a homozygote (-/-).
C (or F), a heterozygote (+/-).
The wild-type E3.5 embryo (E) was derived from the cross between a wild-type female mouse and a wild-type male mouse. The heterozygous (-/+) E3.5 embryo (H) was from the cross between a homozygous female mouse and a wild-type male mouse.

(A) Schematic diagram of the ZFP57 protein.
(B) Sequence alignment between the mouse ZFP57 and the human ZFP57. The identical amino acids are boxed in black and the similar residues are shaded.
(C) Co-IP assays were carried out for KAP-1 and myc epitope-tagged ZFP57. Three left lanes are the western blot of the total cell lysate (Input) from the KAP-1-transfected (lane 1), the KAP-1 and myc epitope-tagged ZFP57 co-transfected (lane 2) and the untransfected cos cells (lane 3). Lane 4, 5, 6 are the western blot of the immunoprecipitate (IP) derived from these three samples when the antibodies against myc epitope were used to pull down ZFP57-associated proteins. The rabbit polyclonal antibodies used for the western blot are Ab1 (anti-KAP1 RBCC) and Ab2 (anti-KAP1 CT) (Schultz et al., 2001).
(D) Adult mouse organ Northern blot of Zfp57. A 1.2kb cDNA fragment encompassing the entire open-reading-frame of Zfp57 was used to probe a Northern blot. Equal amount of polyA RNA was loaded on each well (Chester et al., 1998) and was prepared from the various organs as follows: (S) stomach; (LI) large intestine; (L) liver; (SP) spleen, (T) thymus; (K) kidney; (LG) lung; (C) cerebrum; (H) heart; (M) muscle; (MG) mammary gland; (O) ovary; (TE) testis; (SV) seminal vesicle.
(E) RNA in-situ hybridization reveals that Zfp57 is expressed specifically in the maturing oocytes (purple stain). Frozen sections of the wild-type ovary were probed with an antisense riboprobe derived from a 0.5kb fragment of the 5’ portion of the Zfp57 cDNA. Arrows point to the labelled oocytes inside the cavity of the follicles.

Differential DNA methylation was assessed with the methylation sensitive restriction enzyme HhaI. StuI-digested (Figure 3A) and PstI–digested (Figure 3B) genomic DNA samples from embryos were HhaI digested and hybridized with a probe derived from the differentially methylated regions using Southern blot analysis (left panel). Histograms represent relative differential methylation levels based on quantitative phosphor image analysis of the band intensity of the southern blots shown on the left. The numbers above each bar indicate the representative lane on the Southern blot. Numbers within the bars indicate percentage of differential DNA methylation relative to that of wild-type embryos. Zero indicates absence of methylation. Genotypes and numbers analyzed for each class of embryo are indicated along the horizontal axis. All embryos are E11.5-E13 on a 129 Sv/Ev background.
(A)The IG-DMR from the Dlk1-Dio3 region. Lane 1, wild-type embryos. Lane 2, heterozygous (+/-) embryos from a heterozygous (het) mother. Lane 3, homozygous (-/-) embryos from a het mother. Lane 4, heterozygous (-/+) embryos from a homozygous (null) mother. Lane 5, -/- embryos from a null mother.
(B) The Snrpn DMR. Lane 1, wild-type embryos. Lane 2, +/- embryos from a het mother. Lane 3, -/- embryos from a het mother. Lane 4 and lane 5, -/+ embryos from a null mother. Lane 6, -/- embryos from a null mother and a het father. Lane 7, -/- embryos from a null mother and a null father. This Snrpn Southern data was further confirmed by COBRA analysis on samples including a subset analyzed by Southern blotting. A total of 25 mid-gestation heterozygous embryos from null mothers were assessed with 14 exhibiting methylation acquisition and 11 remaining unmethylated (data not shown).

A, Western blot analysis of affinity purified anti-ZFP57 polyclonal antibodies with total cell lysate samples. Lane 1, ZFP57 was over-expressed in COS cells. Lane 2, an ES clone containing one floxed allele (F) and one targeted allele (T) at the Zfp57 locus. Lane 3, wild-type TC1 ES cells. Lanes 4 and 5, two independent Zfp57-null ES clones carrying two deleted alleles. Please see Supplemental Figure S2 and Supplemental Data for the description of the targeted, floxed and deleted alleles.
(B-E) Chromatin immunoprecipitation (ChIP) was performed with approximately one million ES cells. No ChIP PCR product was observed in the negative control samples without the addition of any antibodies (data not shown).
B, the first round PCR product at the Snrpn DMR region.
C, the second round PCR product at the Snrpn DMR region.
D, the second round PCR product at a region about 40kb upstream of the Snrpn DMR region.
E, the second round PCR product at the H19 DMR region.
ChIP, ChIP PCR product. Input, PCR product from the input/starting samples; ns-IgG, ChIP PCR product from the control samples when rabbit non-specific IgG (ns-IgG) antibodies were added during immunoprecipitation. (B-E) Lane 1, an ES clone containing one floxed allele (F) and one targeted allele (T) at the Zfp57 locus. Lane 2, wild-type TC1 ES cells. Lanes 3 and 4, two independent Zfp57-null ES clones carrying two deleted alleles.