Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR

Background Zrsr1 is a paternally expressed imprinted gene located in the first intron of Commd1, and the Zrsr1 promoter resides in a differentially methylated region (DMR) that is maternally methylated in the oocyte. However, a mechanism for the establishment of the methylation has remained obscure. Commd1 is transcribed in the opposite direction to Zrsr1 with predominant maternal expression, especially in the adult brain. Results We found Commed1 transcribed through the DMR in the growing oocyte. Zrsr1-DMR methylation was abolished by the prevention of Commd1 transcription. Furthermore, methylation did not occur at the artificially unmethylated maternal Zrsr1-DMR during embryonic development when transcription through the DMR was restored in the zygote. Loss of methylation at the maternal Zrsr1-DMR resulted in biallelic Zrsr1 expression and reduced the extent of the predominant maternal expression of Commd1. Conclusions These results indicate that the establishment of methylation at Zrsr1-DMR occurs in a transcription-dependent and oocyte-specific manner and caused Zrsr1 imprinting by repressing maternal expression. The predominant maternal expression of Commd1 is likely caused by transcriptional interference by paternal Zrsr1 expression. Electronic supplementary material The online version of this article (10.1186/s13072-018-0200-6) contains supplementary material, which is available to authorized users.


Introduction
Genomic imprinting is an epigenetic phenomenon of parent-of-origin-dependent expression that is observed in a subset of mammalian genes. Imprinted genes are expressed exclusively or predominantly from one of the two parental alleles, and are frequently located in clusters known as imprinted domains. The expression of genes in an imprinted domain is regulated by a discrete element called an imprinting center (IC) or an imprinting control region (ICR). Imprinted genes or imprinted domains have been found to link to differentially methylated regions (DMRs) that exhibit parent-of-origin-specific DNA methylation. Two classes of DMRs have been identified as follows: germline DMRs (gDMRs), or primary DMRs; and somatic DMRs (sDMRs), or secondary DMRs. gDMR methylation is established during gametogenesis, and sDMRs acquire methylation after fertilization under the direction of gDMRs. The ICs of the imprinted genes are located in their corresponding gDMRs. More than 20 gDMRs have been identified in mice, of which only three are paternally methylated (Arnaud, 2010, Barlow & Bartolomei, 2014, Ferguson-Smith, 2011, Kobayashi, Sakurai et al., 2012. Recent studies have identified an additional 11 new putative maternally methylated gDMRs (Wang, Zhang et al., 2014).
DNA methylation at gDMRs is the primary determinant of the allelic expression of imprinted genes, and the mechanisms of methylation establishment have been extensively investigated.
The specific recruitment of de novo methylation machineries to gDMR methylation sites via the recognition of sequence elements and/or chromatin structures has been considered as a potential mechanism of germline-specific gDMR methylation establishment (Arnaud, 2010, Bartolomei & Ferguson-Smith, 2011. However, efforts to identify sequence motifs for gDMR methylation have not been successful. Several trans-acting factors for maternally methylated not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/249524 doi: bioRxiv preprint first posted online  gDMRs have been found to be essential for the establishment of germline methylation in mice. For example, Dnmt3a has been identified as the enzyme responsible for de novo methylation of many maternal gDMRs (Hata, Okano et al., 2002, Kaneda, Okano et al., 2004. Dnmt3l, a DNA methyltransferase (DNMT)-like protein without enzymatic activity, is the likely co-factor of DNMTs (Bourc'his, Xu et al., 2001). Ablation of Kdm1b, a histone demethylase of H3K4 di-and trimethylation, in oocytes resulted in the failure of methylation establishment at some maternal gDMRs (Ciccone, Su et al., 2009). In addition, the deletion of Hira, which encodes a histone H3.3 chaperon (Hira), led to global hypomethylation in oocytes (Nashun, Hill et al., 2015).
Kelsey et al. proposed a model for the establishment of methylation at the maternal gDMRs in the oocyte (Kelsey & Feil, 2013, Veselovska, Smallwood et al., 2015, which suggests that maternal methylation of gDMRs is regulated by the same mechanisms of general gene-body methylation reported for active genes (Ball, Li et al., 2009, Maunakea, Nagarajan et al., 2010. This was based on the findings that most maternal gDMRs are located in actively transcribed regions (Chotalia, Smallwood et al., 2009), that transcription is a prerequisite for the establishment of methylation at four maternal gDMRs (Chotalia et al., 2009, Singh, Sribenja et al., 2017, Smith, Futtner et al., 2011, Veselovska et al., 2015, and regarding the characteristics of the methylome and transcriptome in growing oocytes (Smallwood, Tomizawa et al., 2011, Veselovska et al., 2015. Analyses of the methylome revealed that most methylation in the oocyte genome occurs within actively transcribed regions, and that maternal gDMRs are not specifically targeted for methylation, but are instead methylated along with other parts of the transcribed regions where the gDMRs reside. Unlike the rest of the transcribed regions, gDMRs not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/249524 doi: bioRxiv preprint first posted online  are likely protected against global demethylation during early embryonic development; only the gDMRs escape global demethylation and remain methylated for lifetime. Methylation failed at the gDMRs in the Gnas locus and the KvDMR in the Kcnq1 locus when a poly(A) signal-truncation cassette was inserted into these loci to prevent transcription from elongation through the gDMRs (Chotalia et al., 2009, Singh et al., 2017. Failure of methylation was also reported at PWS-IC and the Zac1-DMR when the promoter regions from which transcription originated and then proceeded through the maternal gDMRs were deleted , Veselovska et al., 2015. Furthermore, most of these maternal gDMRs were located within transcribed regions in the growing oocyte (Chotalia et al., 2009). On the other hand, the establishment of sDMR-and lineage-specific methylation during the post-implantation stage clearly indicates the de novo methylation potency of early embryonic cells, including naïve/primed pluripotent stem cells (Monk, 2015). It is unknown whether de novo methylation occurs at gDMRs during the post-implantation stage after failure to establish gDMR methylation in the oocyte.
Zrsr1 (U2af1-rs1) and Commd1 (Murr1) are imprinted genes located in mouse proximal chromosome 11. Zrsr1 is expressed ubiquitously in all adult tissues examined, and is expressed exclusively from the paternal allele. Zrsr1 resides in the first intron of the Commd1 gene and is transcribed in the opposite direction to the host gene. The Zrsr1 promoter is located in a maternally methylated gDMR, i.e., the Zrsr1-DMR (Nabetani, Hatada et al., 1997). It is likely that maternal methylation at the Zrsr1-DMR causes imprinted expression by repressing maternal expression of the gene. Commd1 is likewise expressed ubiquitously in adult mice, but is not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/249524 doi: bioRxiv preprint first posted online  expressed from both parental alleles. However, Commd1 expression from the maternal allele is stronger than expression from the paternal allele (i.e., predominant maternal expression), as exemplified in the adult brain. Although Zrsr1-DMR resides in the transcribed region of Commd1, the link between transcription and the establishment of methylation at this DMR has not been clarified.
In this study, we found that methylation at Zrsr1-DMR failed when transcription through the DMR was abolished by the insertion of a poly(A) signal cassette into the site between the Commd1 promoter and the Zrsr1 gene. Furthermore, upon deletion of the cassette in the zygote, Zrsr1-DMR transcription resumed, but methylation at the DMR during early development was not restored. These results indicate that transcription-dependent methylation at the DMR occurs specifically in the growing oocyte, but not during early development. We also found that interference with transcription likely caused predominant maternal expression of Commd1 in the adult brain.

Truncation of Commd1 transcription results in methylation failure at Zrsr1-DMR in the growing oocyte
Methylation of maternal gDMRs of imprinted genes is established asynchronously during postnatal oocyte growth, typically between 5 and 25 days postpartum (dpp) (Lucifero, Mann et al., 2004). To verify that de novo methylation at Zrsr1-DMR is dependent on transcription resulting not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/249524 doi: bioRxiv preprint first posted online  from Commd1 expression in the oocyte, we analyzed Commd1 expression and the methylation status of Zrsr1-DMR in growing oocytes from this period and in ovulated MII oocytes. Commd1 was expressed in all periods of oocyte maturation analyzed ( Fig 1B). De novo methylation at Zrsr1-DMR started after 10 dpp and was completed between 15 dpp and maturation ( Fig 1C).
Thus Zrsr1-DMR was transcribed before and during the establishment of methylation. Zrsr1 expression was not detected by RT-PCR during oocyte maturation ( Fig EV1).
To confirm that de novo methylation at Zrsr1-DMR was a prerequisite for Commd1 expression, we inserted a truncation cassette containing three tandem copies of SV40 poly(A) signal into intron 1 of Commd1 to generate the transcription-truncation allele Commd1 PA (Fig 1A), and obtained Commd1 +/PA -heterozygous mice in a C57BL/6J background. No Commd1 PA/PA mice were born from the intercrossing of heterozygous parents. Commd1 -/mice have been shown to be embryonically lethal at E9.5 to E10.5 (van de Sluis, Muller et al., 2007); the absence of homozygous pups for the truncation allele was thus likely attributable to embryonic lethality, which strongly suggests that a truncation occurred as expected and rendered the Commd1 PA allele functionally null.
To assess the truncation of the Commd1 PA allele and Zrsr1-DMR methylation in the MII oocyte, MII oocytes were obtained from adult F1 females generated from the cross between Commd1 +/PA B6 females and WT PWK males. Commd1 PA(B6)/+(PWK) mice, termed PA F1 mice, and Commd1 +(B)/+(PWK) mice, termed WT F1 mice, were obtained from F1 littermates. The allelic expression of Commd1 was analyzed by RFLP analysis of RT-PCR products with the primers Comm-F1 and Comm-R1, located at exon 1 and exon 2, respectively. Expression of the not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/249524 doi: bioRxiv preprint first posted online Jan. 17, 2018; Commd1 PA allele was not detected in MII oocytes prepared from the PA F1 females, although expression of the PWK allele was detected. In contrast, both alleles were expressed in oocytes from WT F1 female littermates (Fig 2A). The Zrsr1-DMR was completely unmethylated on the truncated allele (B6) in the MII oocytes from the PA F1 females, in contrast to the WT PWK allele, which was completely methylated. As expected, in oocytes from the WT F1 females, Zrsr1-DMR was completely methylated on both the B6 and PWK alleles ( Fig 2B). These results indicate that transcription termination occurred in intron 1, likely at the truncation cassette, and resulted in the loss of transcription through the DMR, which led to methylation failure at the DMR during oogenesis.

Maternal methylation at Zrsr1-DMR causes Zrsr1 imprinting
To investigate the causative link between the maternal methylation at Zrsr1-DMR and the imprinted expression of Zrsr1, we analyzed Zrsr1-DMR methylation and Zrsr1 allelic expression in the adult F1 mice described above. The truncation of Commd1 transcription was also observed in the brain and liver of the PA F1 mice ( Fig 3A). The Zrsr1-DMR on the maternal Commd1 PA(B6) allele of the PA F1 mice was completely unmethylated, whereas normal maternal methylation was observed in the WT F1 mice ( Fig 3B). These results indicate that the truncation cassette was also functional in the adult mice, and that the unmethylated status of maternal Zrsr1-DMR persisted throughout the embryo stage and postnatal growth to adulthood. Zrsr1 was biallelically expressed in the PA F1 mice, although the gene exhibited paternal expression in the WT F1 mice ( Fig 3C). Quantitative analysis of total Zrsr1 mRNA levels indicated that the PA F1 mice expressed twice as much Zrsr1 as the WT F1 mice (Fig 3D), which suggests that the not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/249524 doi: bioRxiv preprint first posted online Jan. 17, 2018; maternal allele in the PA F1 mice was completely de-repressed and was expressed at the same level as the paternal allele. Thus it is evident that the Zrsr1 gene is imprinted by suppression of maternal allele expression, and that maternal methylation at the DMR is the primary imprint mark.

Zygotic deletion of the truncation cassette does not result in the acquisition of methylation at Zrsr1-DMR during embryonic development and postnatal growth
We showed that Zrsr1-DMR acquires methylation in a transcription-dependent manner during oogenesis. However, Commd1 is expressed from both alleles and paternal Zrsr1-DMR is unmethylated in adult mice (Wang, Joh et al., 2004). This fact indicates that transcription-dependent methylation does not occur at paternal Zrsr1-DMR in differentiated somatic cells. However, it was not known that Commd1 is expressed in early embryo, in which genome-wide de novo methylation is about to occur (Howlett & Reik, 1991, Kafri, Ariel et al., 1992, Smallwood et al., 2011. Commd1 was expressed from both alleles in the WT blastocysts described below (WT in Fig 4A and B), which indicated that the paternal Zrsr1-DMR was also unmethylated, even during transcription in pluripotent cells. To confirm this in the maternal allele, the floxed truncation cassette (refer to Fig 1A) was deleted from the maternal Commd1 PA allele via zygotic expression of Cre recombinase. Then, methylation at the maternal Zrsr1-DMR, which was inherited in an unmethylated state, was analyzed. The resulting deleted allele was denoted The blastocysts were prepared via in vitro fertilization (IVF) with MII oocytes from the Commd1 +/PA B6 females and sperm from CAG-Cre transgenic males in a BALB/c background. not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/249524 doi: bioRxiv preprint first posted online Jan. 17, 2018; Deletion of the truncation cassette occurred in all blastocysts that inherited the Commd1 PA allele and the CAG-Cre transgene(s) (Fig EV2). Judging from the high incidence of deletion at the blastocyst stage, it is plausible that the deletion actually occurred much earlier. To determine whether deletion of the truncation cassette would restore expression of the maternal Commd1 allele, we analyzed the allelic expression of Commd1 in Commd1 +(B6)/+(BALB) blastocysts (WT), Commd1 PA(B6)/+(BALB) blastocysts (PA), and Commd1 ΔPA(B6)/+(BALB) blastocysts (ΔPA). Allelic expression was analyzed quantitatively by pyrosequencing of RT-PCR products with the primers CommExpre-F1-bio and CommExprePyro-R, which are located in exon 2. In the WT-blastocysts, Commd1 was equally expressed from both parental alleles, but only the paternal allele was expressed in the PA-blastocysts. This indicates that the truncation cassette was also functional in the blastocysts, and suggests that Commd1 was expressed from both alleles during the developmental period in which global de novo DNA methylation occurs. In ΔPA-blastocysts, in which the truncation cassette was deleted, the maternal ΔPA allele was expressed at levels comparable to the paternal allele ( Fig 4A). Furthermore, the unmethylated status of maternal Zrsr1-DMR was maintained, despite transcription of the DMR (Fig 4B). To determine whether this status was maintained while under transcription from the early embryonic stage to adulthood, we analyzed the brain of ΔPA adult F1 mice from the cross between Commd1 +/PA B6 females and CAG-Cre transgenic BALB/c males. The maternal Zrsr1-DMR in the Commd1 ΔPA allele was unmethylated, even though the maternal allele was expressed (Fig 4C and D). Thus, we concluded that maternal Zrsr1-DMR was not methylated after fertilization, irrespective of DMR transcription. not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

Zrsr1
We reported predominant maternal expression of Commd1, especially in the brains of adult mice (Wang et al., 2004) (WT in Fig 4D and Fig EV3). Based on the two observations (Wang et al., 2004) discussed below, we hypothesized that the expression of Zrsr1 decreased Commd1 expression from the paternal allele by interfering with the transcription elongation of Commd1 within the gene body and with transcription initiation in the Commd1 promoter. First, in the adult liver, Zrsr1 is expressed at extremely low levels (less than 10%) relative to Commd1, which exhibits less-pronounced predominant maternal expression ( Fig EV3). However, in the adult brain, Zrsr1 is expressed to the same extent as Commd1, which exhibits remarkable predominant maternal expression. Second, the antisense transcript of Commd1 was observed in the promoter region of Commd1 only in the paternal allele, and was observed at higher levels in the brain than in the liver (also refer to Fig 5B). The observed antisense Commd1 transcript is presumably generated by read-through transcription of Zrsr1, which may interfere with the initiation of Commd1 transcription.
According to this hypothesis, we expected that predominant maternal expression would not occur in the ΔPA mice, which express Zrsr1 equally from both parental alleles. Indeed, quantitative analysis by pyrosequencing demonstrated that the ratio of maternal to paternal expression was significantly decreased in these mice relative to WT mice ( Fig 4D). The total Commd1 expression levels were then quantitatively analyzed in both lines using the TaqMan assay ( Fig 4E). Expression of Commd1 in the ΔPA mice was 57% of that in the WT mice, which not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/249524 doi: bioRxiv preprint first posted online  is consistent with the notion of decreased maternal expression in the ΔPA mice. Moreover, the relative quantities of the overall expression were consistent with the quantitative allelic expression analysis shown in Fig 4D, in which the total expression levels were 5 (maternal = 4, paternal = 1) and 3 (maternal = 2, paternal = 1) in the WT mice and the ΔPA mice, respectively, given no change in the level of expression from the paternal allele. Collectively, these results indicate that expression of Zrsr1 reduces the expression of Commd1 from the paternal allele, and results in the predominant maternal expression of Commd1.
In the Commd1 promoter region, we analyzed antisense transcription of Commd1 in F1 mice generated from the cross between Commd1 +/PA B6 females and WT PWK males. Antisense transcripts were expressed at higher levels in the brain than in the liver of the WT mice, as reported previously (Wang et al., 2004), and the same transcript was observed in the PA mice ( Fig 5B). In addition, the antisense transcript was expressed only from the paternal allele in the WT mice, which is congruent with previous studies (Wang et al., 2004). However, it was expressed from both alleles in the PA mice (Fig 5C), which was consistent with the pattern of allelic expression of Zrsr1 in these mice. This consistency of allelic expression between the opposite transcripts and Zrsr1 strongly supports the idea that to some degree the transcription of Zrsr1 extends to the Commd1 promoter region beyond the poly(A) signal of the Zrsr1 gene. Such antisense transcription might interfere with the initiation of Commd1 transcription and thereby contribute to its predominant maternal expression, especially in the brain.
Discussion not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/249524 doi: bioRxiv preprint first posted online Jan. 17, 2018; The results of this study indicate that transcription through the Zrsr1-DMR is essential for the establishment of methylation at the DMR in the growing oocyte, as reported for four other imprinted loci (Chotalia et al., 2009, Singh et al., 2017, Veselovska et al., 2015. Our results also indicate that transcription-dependent methylation in the Zrsr1-DMR does not occur after fertilization, which suggests that the mechanism of de novo methylation of the Zrsr1-DMR is oocyte-specific. We also found that paternally imprinted expression of Zrsr1 resulted in predominant maternal expression of Commd1, which is likely caused by transcriptional interference between Zrsr1 and Commd1 on the paternal allele. The mouse embryo undergoes genome-wide de novo DNA methylation during the post-implantation period, at a level comparable to the growing oocyte (Howlett & Reik, 1991, Kafri et al., 1992, Smallwood et al., 2011. In fact, the early embryo expresses factors essential for de novo DNA methylation, such as Dnmt3a, Dnmt3b, and Dnmt3l (Okano, Bell et al., 1999, Uysal, Akkoyunlu et al., 2015, Watanabe, Suetake et al., 2002. In this respect, the de novo DNA methylation activity in the pluripotent cells of the early embryo is comparable to that in the growing oocyte. However, our results indicated that methylation at Zrsr1-DMR does not occur on either parental allele in the early embryo, despite Commd1 expression from both alleles (Fig 4A, B, C). This suggests some differences in the mechanisms of de novo methylation between the early embryo and the growing oocyte, at least in the gDMR. Two possible differences are considered: first, there may be factor(s) inhibiting de novo methylation at Zrsr1-DMR in the early embryo, e.g., histone modification(s) at the DMR, or a trans-acting factor(s) expressed in pluripotent cells; second, the early embryo may lack factor(s) required for de novo methylation at not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/249524 doi: bioRxiv preprint first posted online Jan. 17, 2018; the DMR, which could include epigenetic modification(s) at the DMR that are erased after fertilization, or trans-acting oocyte-specific factor(s). In contrast to the methylation at Zrsr1-DMR, transcription-dependent methylation occurs during embryonic development at sDMRs located upstream of Zdbf2, which is an imprinted gene on the paternal allele, during the period around implantation (Greenberg, Glaser et al., 2017). This suggests variation in DNA methylation mechanisms among DMRs in the imprinted loci, and it will be interesting to examine whether transcription-dependent methylation is also oocyte-specific at the four previously reported loci.
Methylation of a gene promoter usually represses expression of the respective gene (Bird, 2002, Esteller, 2007. Thus, the paternal allele-specific expression of Zrsr1 seems to be caused by maternal methylation at Zrsr1-DMR. This was confirmed by analyses of the expression and methylation of Zrsr1 in the PA mice, in which complete unmethylation at the maternal Zrsr1-DMR resulted in equal levels of gene expression from both alleles (Fig 3C and D). A decrease in maternal expression of Commd1 also occurred concomitantly with the activation of maternal Zrsr1, which strongly suggests that the predominant maternal expression of Commd1 results from Zrsr1-mediated reduction in the expression of Commd1 from the paternal allele. Studies on genetic organization in genomes have identified overlapping and antisense-oriented genes, and the mutual cis-acting effect on their expression, which is termed transcriptional interference (Bordoy & Chatterjee, 2015). The active transcription of Zrsr1 within Commd1 may cause collisions between the opposing Zrsr1 and Commd1 elongation complexes, which would result in the reduction in their expression. We observed an antisense-oriented transcript in the Commd1 promoter region, which was hypothesized to be an overshoot of Zrsr1 transcription (Fig 5) and not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/249524 doi: bioRxiv preprint first posted online Jan. 17, 2018; suggests that Zrsr1 transcription potentially interferes with the initiation of Commd1 transcription. However, complete biallelic Zrsr1 expression did not give rise to equal levels of Commd1 expression from both parental alleles (Fig 4D). Therefore, unidentified differences between the parental Commd1 alleles may exist, despite the fact that thus far we have found no differences in DNA methylation or some histone modifications in our examination of the Commd1 promoter Our results implicate the existence of unidentified growing-oocyte-specific factors for the establishment of methylation at maternal gDMRs. We have yet to determine whether these factors are proteins, RNAs, or chromatin structures. Future extensive proteomic, transcriptomic, and epigenetic analyses of the growing oocyte may elucidate the underlying mechanism for the primary establishment of methylation at the maternal gDMRs.

Generation of Commd1-PA mice
A truncation cassette was constructed by cloning three copies of the SV40 poly(A) signal from the expression vector pGFP-N1 (Clontech) into pT7Blue (Novagen). The truncation cassette was inserted at the genomic site 23 bp downstream of exon 1 using the gene-targeting method ( Fig   1A). The targeting construct was generated by homologous recombination with a truncation cassette clone and a mouse Commd1 BAC clone (BAC RP24-216A32, BACPAC Resources) in E. coli as previously described (Zhang, Muyrers et al., 2000). The targeting construct contained not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/249524 doi: bioRxiv preprint first posted online Jan. 17, 2018; the following mouse Commd1 genomic sequences: a 5.2-kb 5′ sequence containing exon 1, and a 5.1-kb 3′-sequence containing a part of intron 1. An embryonic stem cell (ES) clone with the truncation cassette inserted in the precise genomic position was identified by Southern blotting and PCR analyses of genomic structure, and was used to generate chimeric Commd1-PA mice.
The neo gene in the targeting vector was flanked by FRTs and removed from the Commd1-truncated mice via Flippase expression. Commd1-PA mice in a C57BL/6J (denoted B6) genetic background was obtained by backcrossing with wild type (WT) C57BL/6J mice for five generations. This study was approved by the Ethical Committee for Animal Experiment of Saga University.

Preparation of primordial germ cells, metaphase II oocytes, and blastocysts
Mouse female primordial germ cells (PGC) were prepared from Day 5, Day 10, and Day 15 female C57BL/6 neonates as previously described (Kobayashi, Sakurai et al., 2013). The metaphase II (MII) oocytes were obtained from superovulated six-to eight-week-old female mice using the procedure described by Nakagata et al. (Nakagata, Takeo et al., 2013). Blastocysts were prepared by in vitro fertilization (IVF) using MII oocytes from Commd1 PA/+ B6 mice and sperm from 12-to 14-week-old CAG-Cre transgenic BALB/c mice. After cumulus-oocyte complexes had been coincubated with sperm for 3 h, fertilized oocytes were cultured to the blastocyst stage at 37 °C and 5% CO 2 in humidified air for 96-120 h, as previously described (24). CAG-Cre BALB/c mice were obtained from the RIKEN BioResource Center (RBRC No. RBRC06155). The CAG promoter drives ubiquitous expression of Cre recombinase in mice.

DNA preparation and methylation analysis
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Recovered DNA was used for amplification of Zrsr1-DMR via nested PCR. Amplified DNA was cloned in pT7Blue T-vector (Novagen, #69820) and the resulting sequences were analyzed with BigDye Terminator v3.1 (Applied Biosystems, #4337455) on an ABI3130 sequencer (Applied Biosystems). Blastocyst DNA was prepared from the precipitate of ISOGEN II lysate during RNA preparation with ISOGENOME (NIPPON GENE, #318-08111). DNA from adult tissues was prepared using QIAamp DNA Mini Kit (QIAGEN, #51306). Blastocyst DNA was genotyped by genomic PCR, using primers on both sides of the truncation cassette (Fig EV2). DNA from blastocysts and tissues was also bisulfite-converted using the EZ DNA Methylation Kit (Zymo Research Corp., #D5001). PCR, cloning, and sequencing were performed as described for oocyte DNA.

RNA preparation
RNA was prepared from approximately 100 growing and MII oocytes using the RNeasy Micro Kit (QIAGEN, #74004). For RNA preparation from blastocysts and adult tissues, samples were lysed with ISOGEN II (NIPPON GENE, #311-07361), and the cleared lysates were recovered by centrifugation according to the manufacturer's instructions. Three blastocysts were pooled for RNA preparation, and the lysate was loaded onto a spin column from the RNeasy Mini Kit (QIAGEN, #74104). DNase I treatment, column wash, and RNA elution were performed according to the manufacturer's instructions. not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.

Expression analysis
To analyze Commd1 expression in oocyte and adult tissues, cDNA was synthesized with random primers (Takara, #3802) and reverse transcriptase (TOYOBO, TRT-101), and the Commd1 cDNA was amplified by PCR with the primers Comm-F1 (exon 1) and Comm-R1 (exon 2). To analyze allelic expression, restriction fragment length polymorphism (RFLP) analysis was performed via NlaIII digestion of the amplified cDNA. Allelic expression of Zrsr1 was analyzed by BigDye terminator sequencing of the cDNA amplified with the primers Zrsr-F1 and Zrsr-R1. No rs numbers were assigned to the single nucleotide polymorphisms (SNPs) that were used to discriminate between the C57BL/6J and PWK alleles. Quantitative allelic expression analysis of Commd1 in blastocyst and adult tissues was performed in triplicate by pyrosequencing with PyroMark Q24 (QIAGEN) in the AQ assay mode. The rs26846230 SNP was used to discriminate between the C57BL/6J and BALBc alleles. Total expression levels were quantitated in triplicate using the StepOnePlus real-time PCR (RT-PCR) system with TaqMan Gene Expression Assay (Applied Biosystems) Mm00495837_s1 and Mm01239669_m1 for Zrsr1 and Commd1, respectively. Actb (β-actin) mRNA was quantitated as an internal control using TaqMan Assay Mm00607939_s1.

Primers
All primers used in this study are listed in Table EV1.
Expanded View for this article is available online: http://emboj.embopress.org not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/249524 doi: bioRxiv preprint first posted online Jan. 17, 2018;    A Allelic expression of Commd1 was analyzed as in Figure 2A in the brain (Br) and liver (Lv) of B Methylation at Zrsr1-DMR was analyzed as in Figure 2B in the brain of adult F1 mice. not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/249524 doi: bioRxiv preprint first posted online Jan. 17, 2018 C Allelic expression of Zrsr1 was analyzed in the F1 adult brain by direct RT-PCR sequencing of the cDNA, which contains two SNP sites between B6 and PWK. D Total Zrsr1 expression was analyzed in triplicate in the adult F1 brain and liver via TaqMan RT-PCR. Total expression is presented relative to the WT expression level in each tissue. The differences in levels of expression between WT and ΔPA F1 mice were statistically significant in the brain and liver (two-sided Student's t test, * P < 0.01).      A Brains and livers from two WT adult F1 mice between B6 females and PWK males. not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/249524 doi: bioRxiv preprint first posted online Jan. 17, 2018; B Brains and livers from three WT adult F1 mice between B6 females and BALBc males. not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/249524 doi: bioRxiv preprint first posted online Jan. 17, 2018;  Figure EV1. RT-PCR analysis of Zrsr1 expression in growing oocytes RT-PCR was done with primers Zrsr-F1 and Zrsr-R1 using cDNAs in Fig 1B. Growing oocytes were prepared from B6 female neonates at Day 5 (D5), Day 10 (D10), and Day 15 (D15) postpartum, and fully grown MII oocytes (MII) from B6 adult females. PC: positive control for RT-PCR using adult brain cDNA. MW: molecular weight marker. Two different cDNA batches were used for D5 RNA. Figure EV2. Genotyping PCR of blastocysts carrying Commd1 PA and CAG-Cre transgene A Schematic representation of PCR for three Commd1 alleles, Commd1 PA (PA), Commd1 DPA (DPA) and Commd1 + (WT). B Electrophoresis of PCR products of nine blastocysts positive for Commd1 PA and CAG-Cre transgene among 24 blastocysts obtained from an IVF performed with oocytes from PA female mice and sperm from CAG-Cre male mice. Two blastocysts (#5, #9) contained small amount of undeleted truncation cassette. MW: molecular weight marker.