The Japanese Society of Developmental Biologists

Develop. Growth Differ. (2014) 56, 625–639

doi: 10.1111/dgd.12180

Original Article

Maturation-associated Dbf4 expression is essential for mouse zygotic DNA replication Shin Murai, 1 * Yukiko Katagiri 2 and Shigeru Yamashita 1 1

Department of Biochemistry, Toho University School of Medicine, 5-21-16 Omorinishi Otaku, 143-8540; and Department of Obstetrics and Gynecology Reproduction Center, Omori Medical Center, Toho University, 6-11-1, Omori-Nishi, Ota-ku, 143-8541 Tokyo, Japan

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Cdc7 is an S-phase-promoting kinase (SPK) that is required for the activation of replication initiation complex assembly because it phosphorylates the MCM protein complex serving as the replicative helicase in eukaryotic organisms. Cdc7 activity is undetectable in immature mouse GV oocytes, although Cdc7 protein is already expressed at the same level as in mature oocytes or early one-cell embryos at zygotic S-phase, in which Cdc7 kinase activity is clearly detectable. Dbf4 is a regulatory subunit of Cdc7 and is required for Cdc7 kinase activity. Dbf4 is not readily detectable in immature GV oocytes but accumulates to a level similar to that in one-cell embryos during oocyte maturation, suggesting that Cdc7 is already activated in unfertilized eggs (metaphase II). RNAi-mediated knockdown of maternal Dbf4 expression prevents the maturation-associated increase in Dbf4 protein, abolishes the activation of Cdc7, and leads to the failure of DNA replication in one-cell embryos, demonstrating that Dbf4 expression is the key regulator of Cdc7 activity in mouse oocytes. Dormant Dbf4 mRNA in immature GV oocytes is recruited by cytoplasmic polyadenylation during oocyte maturation and is dependent on MPF activity via its cytoplasmic polyadenylation element (CPE) upstream of the hexanucleotide (HEX) in the 30 untranslated region (30 UTR). Our results suggest that Cdc7 is inactivated in immature oocytes, preventing it from the unwanted phosphorylation of MCM proteins, and the oocyte is qualified by proper maturation to proceed following embryogenesis after fertilization through zygotic DNA replication. Key words: Cdc7/Dbf4 kinase, cytoplasmic polyadenylation, DNA replication, mouse zygote, oocyte maturation.

Introduction Eukaryotic DNA replication is initiated by multiple steps in which many replication-related proteins participate and interact with each other. In somatic cells, pre-replication complexes (preRCs) are assembled, which entails the assembly of an ORI that is composed of ORC1-6 at many sites in the genome during the M to G1 transition. CDC6 and CDT1 are next recruited to the ORC-binding chromatin sites on chromatin and, in turn, recruit an MCM complex composed of MCM2-7. Recruitment of MCM is termed replication licensing *Author to whom all correspondence should be addressed. Email: [email protected] Conflict of interest: The authors declare that they have no conflicts of interest. Received 4 June 2014; revised 24 August 2014; accepted 27 August 2014. ª 2014 The Authors Development, Growth & Differentiation ª 2014 Japanese Society of Developmental Biologists

because the origin recognition complex (ORC) is capable of supporting DNA replication (Tada & Blow 1998; Nishitani & Lygerou 2002). Protein phosphorylation is required for the progression of these steps, and it has been reported that Cdk2 and Cdc7 are major serine/ threonine kinases that play central roles in the initiation and progression of eukaryotic S-phase and thus are referred to as S-phase-promoting kinases (SPKs). SPKs phosphorylate replication-related proteins, such as ORC, MCMs, or GINS, and this phosphorylation is important for the DNA replication in somatic cells (Masai et al. 2006; Tanaka et al. 2007; Lee et al. 2012). Cdk2 requires a regulatory subunit for activation, which has been identified as A- or E-type cyclins in eukaryotic cells. Mouse cyclin A1, one of the subtypes of A-type cyclin, is already expressed in immature GV oocytes, but expression decreases during oocyte maturation. On the contrary, cyclin A2 protein, another A-type cyclin, is not expressed before fertilization but accumulates at the one-cell stage when zygotic DNA

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replication is already initiated (Fuchimoto et al. 2001). Therefore, it is suggested that mouse A-type cyclins are not expressed when the MCM complex is loaded onto chromatin (Swiech et al. 2007; Ortega et al. 2012). Although cyclin E is essential for the loading of the MCM complex onto chromatin in cultured cells, cyclin E-deficient mice are viable and have no reproductive defects, suggesting that embryogenesis progresses normally without cyclin E1/E2 expression (Geng et al. 2003). Therefore, it is suggested that Cdk2 kinase is significant not for zygotic DNA replication but for meiosis or zygotic gene activation, whereas Cdk2 activity is clearly required for DNA replication in Xenopus oocytes (Hua & Newport 1998; Ortega et al. 2003; Hara et al. 2005). From these reports, the molecular mechanism of zygotic DNA replication in mouse oocytes is suggested to be quite different from that in Xenopus oocytes. Cdc7 is another SPK that promotes DNA replication origin firing during S-phase through the phosphorylation of the MCM complex. In somatic cells, Cdc7 kinase is activated only upon initiation of DNA replication at the G1/S boundary, although the level of Cdc7 protein is constant throughout the cell cycle (Sclafani et al. 1988; Yoon et al. 1993; Lei et al. 1997; Jiang et al. 1999; Masai et al. 2000; Bell & Dutta 2002; Masai & Arai 2002). It has been reported that in eukaryotes, the binding of the regulatory protein DBF4 (ASK) is required for the activation of Cdc7 kinase (Jackson et al. 1993; Kumagai et al. 1999; Yamashita et al. 2005). Human DBF4 protein is synthesized prior to entry into S phase and is bound to nuclear chromatin with Cdc7, suggesting that Cdc7 kinase activity is regulated at the level of Dbf4 protein (Kumagai et al. 1999; Sato et al. 2003). DRF1 (ASKL1) has also been identified as another activator of Cdc7 kinase in human cells. DRF1 protein accumulates when cells are in S phase and decreases when cells exit mitosis, suggesting that DRF1 only interacts with and activates Cdc7 at early S-phase, as does Dbf4 (Montagnoli et al. 2002). It has been reported that Cdc7 knockout mice present early embryonic lethality, whereas Cdk2-deficient mice are normally viable, suggesting that Cdc7 kinase is more responsible for early embryogenesis than is Cdk2 (Masai & Arai 2002; Berthet et al. 2003). In Xenopus eggs, Cdc7 predominantly associates with Drf1, and only Drf1 is required for DNA replication in Xenopus egg extracts, although both Dbf4 and Drf1 are present in the early Xenopus embryo before MBT (mid-blastula transition) (Takahashi & Walter 2005; Silva et al. 2006). Therefore, Drf1 is considered to be the only activator that plays an essential role in the activation of Cdc7 kinase in Xenopus zygotes.

However, distinct from Xenopus or human cells, it has been reported that Dbf4 is a unique regulatory subunit of Cdc7 in mouse cells, as there are no expressing genes of any other regulatory factors of Cdc7, including Drf1, in rodent. Therefore, it has been suggested that Dbf4 is a major activator of Cdc7 kinase activity in mouse oocytes and embryos and regulates zygotic DNA replication and the following embryogenesis. In this study, we examined the regulatory mechanism of Cdc7 activity to investigate mouse zygotic DNA replication.

Materials and methods Isolation and culture of oocytes and embryos Fully grown, germinal vesicle intact oocytes (GV) were obtained from pregnant serum gonadotropin (PMSG)primed, 6–8-week-old, ddY female mice (Japan SLC) and dissociated from the attached cumulus cells as previously described (Schultz et al. 1983). Germinal vesicle breakdown (GVBD) was inhibited by adding 2.5 lmol/L milrinone to the isolation and culture media (Tsafriri et al. 1996). The collection medium was bicarbonate-free minimal essential medium (Earle’s salts) supplemented with 3 mg/mL of polyvinylpyrrolidone (PVP) and 25 mmol/L HEPES (pH 7.3) (MEM-PVP). After collection, the oocytes were cultured in MEM (Invitrogen, 12571) supplemented with 5% FCS and 10 ng/mL EGF containing milrinone. To mature oocytes in vitro, GV oocytes were washed with MEM-PVP 8–10 times and then cultured in MEM supplemented with FCS and EGF for 16 h. If necessary, the medium was supplemented with 60 lmol/L roscovitine (Calbiochem) or 10 lg/mL cycloheximide (Sigma) to inhibit Cdk1 activity or protein synthesis, respectively. For the isolation of metaphase II eggs (MII eggs), ddY female mice were superovulated with an injection of 5 IU of PMSG and an injection of 5 IU of human chorionic gonadotropin (hCG) 48 h later. MII eggs were collected 12–16 h post-hCG administration. In vitro fertilized (IVF) 1-cell embryos were prepared with the following protocol (Aoki & Schultz 1999). Sperm were prepared from the cauda epididymis of B6D2 F1 males aged 8–16 weeks old. The eggs were inseminated in the HTF medium with capacitated sperm that had been incubated for 1.5 h at 37°C. One hour after insemination, the eggs were washed with MEM-PVP and cultured in a humidified atmosphere of 5% CO2/95% air at 37°C. In some experiments, the prepared MII eggs were pre-incubated with 10 lg/mL of aphidicolin (Calbiochem) or 5 lmol/L PHA767491 (Tocris bioscience) and then treated with these agents continuously after

ª 2014 The Authors Development, Growth & Differentiation ª 2014 Japanese Society of Developmental Biologists

Dbf4 mRNA expression in mouse oocytes

IVF. For the generation of 1-cell embryos in vivo, the females were mated with B6C3F1 male mice (Japan SLC) and the embryos were collected 20–24 h after the hCG injection. The cumulus cells were removed by a brief hyaluronidase treatment (3 mg/mL). One-cell embryos were cultured in 10-lL drops of KSOM supplemented with amino acids (KSOM + AA) under mineral oil (Ho et al. 1995). To generate parthenogenetic embryos, MII eggs were activated with 10 mmol/ L SrCl2 in Ca2+- and Mg2+-free CZB for 2.5 h and further cultured in KSOM + AA. All oocytes and embryos were cultured at 37.5°C in a humidified atmosphere of 5% CO2 in air. Antibodies To produce the anti-Dbf4 antibody, full-length mouse Dbf4 cDNA was cloned into the pET28 plasmid (Novagen) and transformed into Escherichia coli. Bacterially expressed recombinant 6xHis-tagged Dbf4 was purified with a HisLink (Promega) column according to the manufacturer’s protocol. Antisera were raised against the recombinant protein (Operon Biotechnologies, Japan). The rabbit polyclonal antibody was affinitypurified against the antigen-conjugated HiTrap NHSactivated HP Columns (GE Healthcare). Anti-CDC7 (sc-13010, Santa Cruz), anti-bromodeoxyuridine (11170376001, Roche), anti-MCM2 (#2929, Epitomics), anti-MCM2 Phospho (pS53) (#3386, Epitomics), anti-HA (#2367S, Cell Signaling Technology) and anti-ß tubulin (cat # T4026, Sigma) were used.

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peroxidase-conjugated secondary antibody (1:20 000 dilution in blocking solution A, GE Healthcare) for 1 h at room temperature. After washing in PBST three times (15 min each), the membranes were developed using the ECL Advance Western Blotting System (GE Healthcare). As a loading control, the membranes were stripped according to the manufacturer’s protocol and reprobed with a monoclonal mouse anti ß-tubulin antibody at a 1:10 000 dilution. Immunofluorescence Oocytes and eggs were fixed in 3.7% paraformaldehyde for 1 h at room temperature for CDC7 detection or 2.5% paraformaldehyde for 20 min at room temperature for MCM2 detection. The cells were permeabilized for 15 min in PBS containing 0.2% Triton X-100 and blocked in PBS containing 0.1% bovine serum albumin (BSA) and 0.01% Tween-20 (blocking solution B). They were then incubated with the primary antibody for 1 h at room temperature (1:50 dilution in blocking solution B for CDC7 and 1:250 dilution for MCM2). After three washes in the blocking solution, the cells were incubated in Cy5-conjugated secondary antibody for 1 h (Jackson Immunoresearch Laboratories). The DNA was stained with 1 lmol/L DAPI (Sigma). The cells were then washed and mounted onto a slide in VectaShield antibleaching solution (Vector Laboratories). The fluorescence was detected on a laser-scanning confocal microscope. Preparation of double-stranded RNA (dsRNA)

Immunoblotting Oocytes, eggs and embryos were directly lysed in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer (100 mmol/L TrisHCl, pH 6.8, 200 mmol/L dithiothreitol, 4% SDS, 0.2% bromophenol blue and 20% glycerol) and stored at 20°C until use. The samples were boiled for 5 min prior to being loaded onto a 7.5% SDS–PAGE gel. The proteins were then transferred to an Immobilon-P membrane (Millipore), and the membranes were incubated in blocking solution A, 2% ECL Advance Blocking Agent (GE Healthcare) in PBST (phosphatebuffered saline with 0.2% Tween-20), for 1 h at room temperature. After blocking, the membranes were washed twice in PBST for 10 min each and then incubated with the primary antibody (1:10 000 dilution in blocking solution A, except for Dbf4, for which a 1:100 000 dilution was used) overnight at 4°C. Following the incubation with the primary antibody, the membranes were washed three times in PBST for 15 min each and then incubated with horseradish

Total RNA was isolated from 30 MII eggs using the Picopure RNA isolation kit (Arcturus) according to the manufacturer’s protocol. A reverse transcription (RT) reaction, primed with oligo dT, was performed using PrimeScript Reverse Transcriptase (TAKARA BIO) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was performed using this oocyte cDNA to generate the templates for in vitro transcription. dsDbf4 is the dsRNA to target mouse Dbf4 mRNA (accession number NM_013726.3). For the amplification of the coding region of mouse Dbf4, a pair of primers was designed based on the cDNA sequence. The sequence of the upstream primer was 50 -ATGAACCTCGAGACCATGAGGATC-30 , and the downstream primer was 50 -ACTCCCCATGATAA GGCATTTGATAA-30 . These primers generated a PCR product that was 500 bp in length and corresponded to the 50 terminal region of the Dbf4 coding region. The PCR product was cloned into the pGEM-T Easy vector (Promega), and the plasmid was digested and linearized with FspI and served as a template for

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in vitro transcription. Sense and antisense RNAs were transcribed in vitro using SP6 and T7 MEGAscript kits (Ambion) and mixed. After heating at 75°C for 5 min, the RNA was cooled and slowly annealed at room temperature. The sample was treated with DNase I and RNase A and then purified twice by phenol/chloroform extraction and ethanol precipitation. Gfp dsRNA, whose sequence was derived from EGFP, was prepared in the same way and used as a control dsRNA for injections (Murai et al. 2010). The purified dsRNAs (1 mg/mL) were stored at 20°C. Quantitative real-time PCR (qRT-PCR) Total RNAs were prepared from oocytes or embryos of ddY female mice and reverse transcribed using PrimeScript Reverse Transcriptase (Invitrogen) and oligo dT to detect the expression level of Dbf4 mRNA or random hexamer primers to detect the level of Dbf4 mRNA after dsRNA knockdown. Real-time PCR analysis was carried out using an ABI Prism 7000 sequence detection system (Applied Biosystems). TaqMan probes corresponding to Dbf4 (ABI assay ID Mm00516462_m1) and histone H2A (ABI assay ID Mm00501974_s1) were used. cDNA equivalent to the amount from a single oocyte was used in each reaction, and the reactions were performed in triplicate. The amount of Dbf4 cDNA was normalized by the comparative Ct method (http://www.ambion.com) using amplification of endogenous histone H2A in the same samples. Microinjection The injections were performed in 10-lL drops of modified Whitten’s medium containing 15 mmol/L HEPES, pH 7.2, 7 mmol/L Na2HCO3, 10 lg/mL gentamicin and 0.01% PVA containing 2.5 lmol/L milrinone. Approximately 10 pL of dsRNA was injected into the cytoplasm of GV oocytes using an IM300 microinjector (Narishige, Japan) on the stage of a microscope equipped with Hoffman optics and Narishige micromanipulators. BrdU incorporation assay To detect DNA replication, embryos with a pronucleus were labeled at 7 h after Sr2+ activation with 1 mmol/L BrdU in KSOM + AA for 30 min. The embryos were fixed in 2.5% paraformaldehyde in PBS containing 0.5 N NaOH for 15 min at room temperature, blocked in PBS containing 10% FBS and 0.2% Triton X-100 for 30 min at 37°C and then washed with washing solution (PBS containing 2% FBS and 0.1% Triton

X-100). BrdU incorporation into chromatin was detected with a mouse monoclonal antibody against bromodeoxyuridine (1:20 dilution in washing solution), followed by Alexa 546-conjugated goat anti-mouse IgG (1:100 dilution in washing solution; cat. # A-11003, Invitrogen). The DNA was detected with 0.02% SITOX Green (Invitrogen) for 15 min at room temperature. The embryos were mounted in Vectashield (Vector Laboratories) and observed with a laserscanning confocal microscope. DNA constructs, in vitro transcription, and site-directed mutagenesis The mouse Dbf4 30 UTR was amplified by RT–PCR from mouse GV oocyte cDNA using an upstream primer (50 -TATTAAATGTGCTTTTCAGAAGT-30 ) and a downstream primer (50 -TTCAGTGATAAA GCAAAT GATATT-30 ), which were designed based on the cDNA sequence (accession number NM_013726). The PCR product was cloned into the pGEM-T Easy vector. The pGEM-Dbf4 30 UTR was digested with EcoRI, and the DNA fragment was cloned into a unique site in pIVT replacing the b-globin 30 -UTR. Firefly luciferase was amplified from pGL3 (Promega) and cloned into the expression vector containing the 30 UTR of Dbf4. Renilla luciferase was amplified from pGL4.70 [hRluc] (Promega) and cloned into pIVT vector, which contained 33 A residues followed by 12 C residues at the 30 end (Igarashi et al. 2007; Murai et al. 2010). The deletion of CPEs in the 30 UTR of Dbf4 was performed by a procedure based on long PCR (Imai et al. 1991). Complementary RNAs (cRNAs) of all constructs were synthesized from linearized plasmid DNAs using T7 RNA polymerase and the mMESSAGE mMACHINE kit (Ambion) according to the manufacturer’s instructions. The cRNA was cleared and purified by LiCl precipitation. The final cRNA concentration was determined by spectrophotometry, and cRNA integrity was confirmed by analyzing a sample on a formaldehyde gel. Luciferase assay The cRNAs of luciferase with Dbf4 30 UTR (10 pg/ oocyte) and Renilla luciferase (1.5 pg/oocyte) were coinjected into GV-intact oocytes as described above and incubated in vitro for 16 h with or without maturation (Murai et al. 2010). Ten injected oocytes were collected and incubated with Passive Lysis Buffer (1 lL/ oocyte) for 15 min at room temperature with shaking. The samples were stored at 80°C until the luciferase activity was assayed using the Dual-Luciferase Reporter Assay System (Promega). This was carried out according to the manufacturer’s instructions, except

ª 2014 The Authors Development, Growth & Differentiation ª 2014 Japanese Society of Developmental Biologists

Dbf4 mRNA expression in mouse oocytes

that 3 lL of sample was used for each measurement. The signal intensities were measured using a Mithras LB940 luminometer (Berthold). The luciferase activities of the constructs with Dbf4 30 UTR were normalized to that of Renilla luciferase.

Results Cdc7 activity is required for the initiation of mouse zygotic S-phase After fertilization, the mouse meiotic cell cycle resumes, and the paternal and maternal pronuclei are formed from sperm nuclei and egg spindle chromatin, respectively, for 3 h after fertilization. To observe the replication of pronuclear chromatin in fertilized mouse eggs, the fertilized mouse eggs were prepared by in vitro fertilization (IVF), and DNA replication was detected with a bromodeoxyuridine (BrdU) incorporation assay 4 or 7 h after insemination (4 or 7 hpi in Fig. 1A). At 4 hpi, BrdU signals in either paternal or maternal pronuclei were not observed in mouse IVF eggs, although the resumed meiosis process was already exited, and pronuclei were observed. It is suggested that mouse one-cell embryos at 4 hpi are still at the pre-replicating stage, like the G1 phase of the somatic cells (Ortega et al. 2012). At 7 hpi, intense BrdU signals were observed in both paternal and maternal pronuclei in IVF eggs, suggesting that DNA replication was initiated at that point (Fig. 1A, 7 hpi). When IVF was performed in the presence of aphidicolin, which is an inhibitor of DNA polymerase a required for physiological DNA replication, BrdU signals were completely absent in all of the one-cell embryos (Fig. 1A, 7 hpi + APH), confirming that the BrdU signals detected in the pronuclear chromatin are highly specific for the initiation of DNA replication (Ikegami et al. 1978; Yamauchi et al. 2007). These results indicate that zygotic DNA replication is initiated at 7 hpi and are consistent with previous reports studying IVF and ICSI eggs (Aoki & Schultz 1999; Ajduk et al. 2006; Yamauchi et al. 2009). To confirm the presence of Cdc7 kinase activity in one-cell mouse embryos, we used an antibody that specifically detects MCM2 phosphorylation at Ser53 (pSer53 in Fig. 1B). In human cells, Ser53 on MCM2 is the specific phosphorylation site for Cdc7 kinase, and its phosphorylation level specifically reflects Cdc7 activity (Montagnoli et al. 2006; Hughes et al. 2010), although it is still unclear whether phosphorylation is required for eukaryotic DNA replication. Phosphorylation of this serine residue on mouse MCM2 was faintly detected in unfertilized eggs by immunoblotting analysis, and its level was gradually increased from 4 to

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7 hpi, suggesting that Cdc7 kinase is already active before fertilization and further activated after fertilization (Fig. 1B). To inhibit the Cdc7 kinase activity, we used PHA767491, the pyrimidylpyrrole derivatives (Montagnoli et al. 2008; Menichincheri et al. 2009). PHA767491 is designed as a specific inhibitor of Cdc7 kinase, and it was reported that PHA767491 inhibits Cdc7 and Cdk9 kinase activity efficiently but not Cdk2 or Cdk4, which the inhibitor displays