Transcription-dependent DNA methylation at the imprinted Zrsr1-DMR

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. In this study, we found Commed1 transcribed through the DMR in the growing oocyte. Zrsr1-DMR methylation was abolished by the prevention of Commd1 transcription. This result indicated that methylation at the Zrsr1-DMR was transcription-dependent.


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 ! %! 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 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 (Smith et al., 2011, 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 ! '! 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 (Wang et al., 2004). 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.

Results and Discussion
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 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  (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. The results 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.

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 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

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).

DNA preparation and methylation analysis
Approximately 200 growing oocytes and MII oocytes were lysed in lysis buffer (0.5% SDS, 250 ng/"l proteinase K, 100 ng/µl yeast tRNA) at 37 °C for 60 min, and DNA in the lysate was bisulfite-converted by treating the lysate with the EpiTect Bisulfite Kit (QIAGEN, #59104).
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 S2). DNA from 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

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