Initiation of DNA Replication After Fertilization Is Regulated by P90rsk at Pre-RC/Pre-IC Transition in Starﬁsh Eggs

Initiation of DNA replication after fertilization is regulated by p90Rsk at pre-RC/pre-IC transition in starﬁsh eggs

Kazunori Tachibana, Masashi Mori1, Takashi Matsuhira, Tomotake Karino, Takuro Inagaki, Ai Nagayama, Atsuya Nishiyama2, Masatoshi Hara, and Takeo Kishimoto3

Laboratory of Cell and Developmental Biology, Graduate School of Bioscience, Tokyo Institute of Technology, Yokohama 226-8501, Japan

Communicated by Joan Ruderman, Harvard Medical School, Boston, MA, January 17, 2010 (received for review August 31, 2009) Initiation of DNA replication in eukaryotic cells is controlled through ase, p90Rsk) pathway causes the G1-phase arrest at the pronu- an ordered assembly of protein complexes at replication origins. The cleus stage (6–10). Fertilization induces degradation of Mos to molecules involved in this process are well conserved but diversely shutdown this pathway, leading to the first S phase with no regulated. Typically, initiation of DNA replication is regulated in requirement of new protein synthesis. However, it remains response to developmental events in multicellular organisms. Here, unclear how p90Rsk negatively controls the G1/S-phase tran- we elucidate the regulation of the first S phase of the embryonic cell sition, or to which stage the initiation complex for DNA repli- cycle after fertilization. Unless fertilization occurs, the Mos-MAPK- cation is assembled in unfertilized G1-phase eggs. Here we show p90Rsk pathway causes the G1-phase arrest after completion of that the p90Rsk-dependent G1-phase arrest of unfertilized meiosis in starfish eggs. Fertilization shuts down this pathway, starfish eggs occurs at the pre-RC stage, and that in the absence leading to the first S phase with no requirement of new protein of Cdk1 and Cdk2 activities and Cdc7 accumulation, inactivation synthesis. However, how and in which stage the initiation complex of p90Rsk is necessary and sufficient for further loading of Cdc45 fi for DNA replication is arrested by p90Rsk remains unclear. We nd and the subsequent initiation of DNA replication. that in G1-arrested eggs, chromatin is loaded with the Mcm complex to form the prereplicative complex (pre-RC). Inactivation of p90Rsk Results fi is necessary and suf cient for further loading of Cdc45 onto Female Pronuclei in Unfertilized Eggs Are Licensed for DNA chromatin to form the preinitiation complex (pre-IC) and the Replication. We first determined the timing of S phase. A 5-min subsequent initiation of DNA replication. However, cyclin A-, B-, pulse incorporation of BrdU to DNA indicates that the first S and E-Cdk’s activity and Cdc7 accumulation are dispensable for ∼ ∼ fi phase begins 30 min and ends 45 min after insemination of these processes. These observations de ne the stage of G1 arrest eggs arrested at G1 phase (Fig. 1A and Fig. S1) (11). In parallel, in unfertilized eggs at transition point from pre-RC to pre-IC, and congression and fusion between female and male pronuclei reveal a unique role of p90Rsk for a negative regulator of this tran- occurred (12), while DNA replication appeared to start sepa- sition. Thus, initiation of DNA replication in the meiosis-to-mitosis rately and almost simultaneously in each pronucleus and then to transition is regulated at the pre-RC stage as like in the G1 check- continue for a little longer period in male pronuclei. Thereafter, point, but in a manner different from the checkpoint. M phase started at ∼60 min (Fig. 1A and Fig. S1). To investigate assembly of the initiation complex for DNA Cdc45 | G1 arrest | Mcm complex | Mos-MAPK pathway | oocyte-to-embryo transition replication before and after fertilization, we isolated cDNAs of starfish orthologs of Cdc6, Mcm2, Mcm7, and Cdc45 and raised antibodies to these proteins (Figs. S2 A–D and S3 A–D). All of NA replication in eukaryotic cells is initiated through an these proteins were detectable in unfertilized eggs, and their Dordered assembly of protein complexes at replication origins E fi protein levels remained constant during cleavage cycles (Fig. 1 (1, 2). Replication origins are rst recognized and bound by the for Cdc45 and Fig. S3H for Cdc6 and Mcm2). Both Cdc6 and origin recognition complex (ORC). During late M or early G1 Mcm2 were already detectable on chromatin of female pronuclei phase, Cdc6 associates onto ORC-containing DNA. Then, MCM in unfertilized eggs arrested at G1 phase (Fig. 1B and C; 0 min). (minichromosome maintenance) proteins associate with the After fertilization, they remained localized on chromatin at ORC- and Cdc6-containing replication origins, requiring Cdt1 to almost constant levels until S phase. As DNA replication pro- form a prereplicative complex (pre-RC). At the onset of S phase, gressed, the Mcm2 signal decreased in mid-S phase (Fig. 1C; Cdc45 associates with the pre-RC to form a preinitiation com- 35 min) and disappeared in late S phase (Fig. 1C; 40 min), plex (pre-IC) that is capable of origin unwinding and of pro- whereas the Cdc6 signal remained until early G2 phase (Fig. 1B; moting assembly of replication forks at replication origin. Thus, Cdc45 plays a crucial role in activation of replication origins. Although the mechanism of initiation of DNA replication is Author contributions: K.T., M.M., M.H., and T. Kishimoto designed research; K.T., M.M., T.M., well conserved, its control is diverse (3). In addition to evolu- T. Karino, T.I., A. Nagayama, A. Nishiyama, and M.H. performed research; K.T., M.M., T.M., tionary variation, DNA replication is regulated in response to M.H., and T. Kishimoto analyzed data; and K.T. and T. Kishimoto wrote the paper. developmental events in multicellular organisms. Fertilization is The authors declare no conflict of interest. the first major event in development and is necessary for both Data deposition: The sequences reported in this paper have been deposited in the DNA releasing meiotic arrest and restarting the cell cycle with ini- Data Bank of Japan (DDBJ) (accession nos. AB474909, AB474910, AB474911, AB474912, AB481214, AB481376, and AB530248). tiation of the first round of DNA replication. In some organisms, Drosophila – 1Present address: Gene Expression Unit, European Molecular Biology Laboratory (EMBL), including and echinoderms (4 6), fertilization is not a Meyerhofstrasse 1, D-69117 Heidelberg, Germany. prerequisite for the completion of meiosis, but required to trig- 2Present address: Laboratory of Replication and Genome Dynamics, Institute of Human ger entry into the first S phase and the subsequent cleavage Genetics, CNRS, 34396 Montpellier Cedex 5, France. fi Asterina pectinifera Patiria pecti- cycles. In star sh (renamed to 3To whom correspondence should be addressed. E-mail: [email protected]. nifera in 2007 at the NCBI Taxonomy Browser), the Mos-MAPK This article contains supporting information online at www.pnas.org/cgi/content/full/ (mitogen-activated protein kinase)-Rsk (p90 ribosomal S6 kin- 1000587107/DCSupplemental.

5006–5011 | PNAS | March 16, 2010 | vol. 107 | no. 11 www.pnas.org/cgi/doi/10.1073/pnas.1000587107 Downloaded by guest on September 27, 2021 A G1 S G2 M BrdU DNA Merge

B Cdc6 DNA Merge

C Mcm2 DNA Merge

D Cdc45 DNA

Merge 10 µm 0 5 10 15 20 25 30 35 40 45 50 55 60 Time after fertilization (min)

E Insemination Pro-I MI IK MII G1 S G2 M Cdc45 0 30 60 90 120 150 180 210 Time after 1-MeAde addition (min)

Fig. 1. In female pronuclei of starfish eggs, chromatin is loaded with Cdc6 and Mcm2 before fertilization, and then with Cdc45 after fertilization at G1/S- phase transition. (A) Mature eggs, which were arrested at G1 phase after completion of meiosis II, were inseminated. To monitor DNA replication, every 5 min, fertilized eggs were pulse labeled for 5 min with BrdU, extracted, and immunostained with anti-BrdU antibody (green). DNA was stained with DAPI (red). Insets indicate male chromatin, and main figures indicate female chromatin or fused chromatin. (B–D) Every 5 min, isolated eggs were extensively extracted and then immunostained with anti-starfish Cdc6 antibody (B, green), anti-starfish Mcm2 antibody (C, green), and anti-starfish Cdc45 antibody (D, green). (Scale bar, 10 μm.) (E) Cdc45 protein is detectable in immature oocytes and its levels remain constant during meiotic and cleavage cycles.

45 min), declined after mid-G2 phase (Fig. 1B; 50 min), and In male pronuclei, both Cdc6 and Mcm2 were undetectable ﬁnally disappeared at M phase (Fig. 1B; 60 min). Similar immediately after fertilization, and then soon became detecte- behavior of Mcm was observed with other antibodies against able (Fig. 1 B and C Insets;0∼20 min), indicating that male human Mcm2 (BM28) and starﬁsh Mcm7 (Fig. S4). pronulei become licensed after fertilization. Indeed, undegrad- Before the G1 arrest, Mcm2 was detectable on chromatin at able form of geminin prevented DNA replication in male pro-

each end of meiosis I and II during meiotic cycle, coincidentally nuclei (but not in female pronuclei) after fertilization, even when CELL BIOLOGY with decreased levels of cyclin B-Cdk1 activity (Fig. 2A). The it was introduced into G1-phase eggs (Fig. 2D, arrowhead). After Mcm2 loading was dependent on geminin degradation, sup- the loading onto chromatin, Cdc6 and Mcm2 behaved similarly porting that it was mediated by Cdt1 (Fig. 2 B and C) (13). to those in female pronuclei. Consistently, undegradable form of geminin prevented DNA replication in female pronuclei after fertilization, when it was Replication Origins Are Not Activated in Female Pronuclei of introduced into oocytes before, but not after, meiotic maturation Unfertilized Eggs. In contrast to Cdc6 and Mcm2, Cdc45 was (Fig. 2D). Taken together, pre-RC is already formed in female undetectable on chromatin in female pronuclei of unfertilized pronuclei of unfertilized eggs; that is, female pronuclei are G1-phase eggs (Fig. 1D; 0 min). During meiotic maturation licensed for DNA replication before fertilization. before the G1 arrest, Cdc45 protein was already present in the

Tachibana et al. PNAS | March 16, 2010 | vol. 107 | no. 11 | 5007 Downloaded by guest on September 27, 2021 A H1K activity Mcm2 DNA Merge Cdc45 DNA

Merge 20 µm 50 55 60 65 70 75 80 85 90 95 100 105 Time after 1-MeAde addition (min)

Injection B C D stage GV PN Control Geminin DEL (12) Control GemininDEL Control GemininDEL 40 60 150 40 60 150 100 (10) 80 DNA

DNA 60 40 BrdU

Mcm2 20 µm 20 (11) (10) (12) (8) 0 Mcm2 positive eggs (%) Cont Gem Cont Gem Cont Gem 40 60 150 Merge Merge Time after 1-MeAde addition (min) 20 µm

Fig. 2. Loading of Mcm2 onto chromatin during meiosis is blocked by geminin. (A) At each end of meiosis I and II, Mcm2, but not Cdc45, is loaded onto chromatin. Immature oocytes were treated with 1-MeAde to undergo meiotic maturation. Beginning at 50 min, every 5 min, oocytes were either ﬁxed for immunostaining with anti-Mcm2 (green) or anti-Cdc45 antibodies along with DAPI (red) staining, or extracted for histone H1 kinase assay to monitor meiotic cell cycle progression (Top). Note that Mcm2 was undetectable on chromatin until 55 min (metaphase I) and at 90 min (metaphase II). Arrow indicates the second polar body. (B–D) Nondegradable geminin inhibits loading of Mcm2 onto chromatin during meiosis, and hence prevents DNA replication after fertilization. Immature oocytes were injected with nondegradable form of geminin (gemininDEL, Gem), wild-type (degradable) geminin (B, control; C, Cont), or control buffer (D, control) and then treated with 1-MeAde to undergo meiotic maturation. At 40 min (metaphase I), 60 min (anaphase I), or 150 min (G1 phase after completion of meiosis II), oocytes were ﬁxed for staining with anti-Mcm2 antibody or with DAPI (B). Number of eggs examined for loading of Mcm2 onto chromatin is indicated in parentheses (C). Alternatively, 150 min after 1-MeAde addition, mature eggs were inseminated in the presence of BrdU, and 40 min later, DNA replication was examined (D, GV). For reference, after meiotic maturation, G1-phase eggs were injected with nondegradable form of geminin and then inseminated (D, PN). Note that BrdU incorporation (red) was undetectable both in female and male (arrowhead) pronuclei (D, GV), though detectable only in female pronucleus (D, PN), showing that only the female, but not the male, pronucleus is licensed for DNA replication before fertilization.

oocyte but undetectable on the chromatin, even though the began to accumulate after meiotic reinitiation, but their levels did Mcm2 loading occurred at each end of meiosis I and II (Figs. 1E not fluctuate during the cleavage cycles (Fig. 3A and Fig. S3I). and 2A). After fertilization, however, Cdc45 became detectable Cdk2 was predominantly associated with cyclin E, whereas Cdk1 on chromatin both in female and male pronuclei just before the was associated with each of cyclin A and cyclin B (Fig. 3A; see also start of S phase (Fig. 1D; 25 min). Thereafter, the signal of ref. 15). Neither a single knockdown of Cdk2 nor a triple Cdc45 on chromatin increased to peak at the beginning of S knockdown of cyclins A, B, and E with respective morpholino phase, decreased along with the progression of S phase, and oligonucleotides prevented initiation of DNA replication after finally disappeared at the end of S phase (Fig. 1D;30∼45 min). fertilization (Fig. 3 B–E). Consistently, in the presence of Cdk Fertilization thus caused the loading of Cdc45 onto chromatin, inhibitor roscovitine, Cdc45 became detectable both on maternal indicating that the female pronuclei in unfertilized eggs are and paternal chromatin almost at normal timing (Fig. 3F). Thus, licensed but that their replication origins are not activated. This whereas the G1-phase eggs of starfish are arrested at pre-RC implies that in G1 phase, unfertilized eggs arrest at a stage stage, cyclin A-Cdk1, cyclin B-Cdk1, and cyclin E-Cdk2 are all not before pre-IC and most likely at the stage of pre-RC. essential for further loading of Cdc45 and the subsequent initiation of DNA replication after fertilization. Cdk Activity and Cdc7 Synthesis Are Dispensable for Cdc45 Loading We then isolated cDNA of starfish ortholog of Cdc7 and After Fertilization. S-Cdk and Cdc7 (Dbf4-dependent kinase, raised its antibody (Figs. S2G and S3G). Cdc7 protein was DDK) are implicated in the Cdc45 loading onto chromatin in detectable at low level in immature oocytes, accumulated during yeast, frog eggs, and mammalian cultured cells (1, 2, 14). To meiotic maturation, and remained at elevated levels during the investigate whether this is the case in fertilized starfish eggs, we first cleavage cycle (Fig. 4A). However, when accumulation of isolated cDNAs of starfish orthologs of cyclin E and Cdk2, and Cdc7 was prevented with its morpholino oligonucleotide, Cdc45 raised antibodies against these proteins (Figs. S2 E and F and S3 E became detectable on chromatin after fertilization with ∼10-min and F). Both proteins were undetectable in immature oocytes and delay compared with control eggs, followed by BrdU incorpo-

5008 | www.pnas.org/cgi/doi/10.1073/pnas.1000587107 Tachibana et al. Downloaded by guest on September 27, 2021 A Cdk2 IP Cdk1 IP WCE A Meiotic cell cycle 0 Time (min) 0 0 30 60 30 60 30 60 Cdc7 120 160 120 160 120 160 Cyclin A 0 10 20 30 40 50 60 70 80 90 100 110 120 130 150140 Time after 1-MeAde addition (min) Cyclin B Mitotic cell cycle B GV PN Cdc7 Cyclin E MO ––Cont Cdc7 0 1020304050607080 Time after insemination (min) Cdk1 Cdc7 * * Cdk2 MAPK

C MO Cdc7 B C MO Cdk2 MO control F MO Cont MO Cdc7 Roscovitine MO Cdc7 Roscovitine 30 30 40 30 40 50 (min) MO Cdk2 Uninjected MO control BrdU – Cdk2 + 0330 0(min) MAPK DNA DNA DNA

D EMO CycABE MO control BrdU Cdc45 Uninjected MO control MO CycABE 10 µm Cyclin A BrdU

Cyclin B 10 µm Cdc45 DNA Fig. 4. Cdc45 is loaded onto chromatin and DNA replication is initiated in Cyclin E the absence of new synthesis and accumulation of Cdc7 when G1-phase eggs MAPK DNA are fertilized. (A) On immunoblots, Cdc7 protein was slightly detectable in immature oocytes. Its levels increased during meiotic maturation and Fig. 3. Cdc45 is loaded onto chromatin and DNA replication is initiated in remained elevated in the first cleavage cycle. (B and C) Delayed loading of the absence of Cdk activity when G1-phase eggs are fertilized. (A) In starfish Cdc45 occurs in Cdc7-knocked-down eggs after fertilization. Immature eggs, Cdk1 associates with cyclin A and cyclin B, and Cdk2 associates pre- oocytes (GV) were injected with morpholino oligonucleotide against Cdc7 dominantly with cyclin E. Immature oocytes were treated with 1-MeAde to and then treated with 1-MeAde to resume meiosis. Female pronucleus stage resume meiosis, and then at 50 min, meta-I oocytes were inseminated to eggs (PN) were either processed to examine Cdc7 protein levels (B)or initiate embryonic cycles after completion of meiosis II. Oocyte or egg inseminated in the presence or absence of 10 μM roscovitine to examine extracts were prepared at 0 min (immature), 30 min (GVBD), 60 min (inter- Cdc45 loading at 30 and 40 min or BrdU incorporation at 50 min (C). As kinesis), 120 min (the first M phase in cleavage cycle), and 160 min (the controls, control morpholino oligonucleotide was injected (MO Cont) or not second M phase) (Fig. S3I). Whole-cell extracts (WCE), and their immuno- (−). Note that paternal DNA is discerned as a bright spot within zygotic precipitates with anti-Cdk2 (Cdk2 IP) or anti-Cdk1 (Cdk1 IP) were immuno- nucleus in the absence of roscovitine, whereas male (Inset) and female blotted with each of cyclins A, B, and E, and Cdk1 and Cdk2. Lower band of pronuclei are separated in the presence of roscovitine. Asterisks in the Cdc7 Cdk1 corresponds to its active form. (B–E) A single knockdown of Cdk2 (B blots indicate nonspecific bands, and upper and lower bands of MAPK cor- and C) or a triple knockdown of cyclins A, B, and E (D and E) does not respond to active and inactive form, respectively (B). prevent DNA replication induced by fertilization. Immature oocytes were injected with morpholino oligonucleotides against Cdk2 or cyclins A and E, and then treated with 1-MeAde to resume meiosis. At 120–150 min after 1- MeAde addition, mature eggs that had been injected with cyclins A and E timing if new synthesis and accumulation of Cdc7 occur during morpholino oligonucleotides were further injected with morpholino oligo- meiotic maturation. nucleotide against cyclin B. At 180 min, all eggs were inseminated in the presence of BrdU. Successful insemination was confirmed by elevation of Loading of Cdc45 onto Chromatin Is Negatively Regulated by p90Rsk. fertilization envelope. Fifty (B and C)or80(D and E) min later, eggs were How then is the loading of Cdc45 onto chromatin regulated recovered to confirm knockdown of Cdk2 (B, MO Cdk2) or cyclins A, B, and E upon fertilization? Because the Cdc45 loading coincided with (D, MO CycABE) with immunoblots and to examine BrdU incorporation (C inactivation of p90Rsk after fertilization (Fig. S1A), we tested E and ). As controls, respective control morpholino oligonucleotides were how p90Rsk activity is related to Cdc45 loading by manipulating injected (MO Control). In the triple knockdown (E), significant delay was observed in BrdU incorporation into female pronucleus chromatins. MAPK the activity of p90Rsk in eggs. When unfertilized G1-phase eggs was used as a loading control for immunoblots (B and D). Knockdown of received an injection with a constitutively active mutant protein Cdk2 or cyclins A and E did not affect the meiotic cell-cycle progression. (F) of p90Rsk (CA-Rsk-EE) that can maintain Rsk activity (9), Inhibition of Cdk activity does not affect Cdc45 loading onto chromatin after neither the Cdc45 loading onto chromatin nor the subsequent fertilization. G1 eggs were inseminated in the presence or absence of 10 μM DNA replication occurred even after fertilization (Fig. 5 A and roscovitine, followed by examination of Cdc45 loading at 30 min. Note that B). Conversely, when unfertilized G1-phase eggs received an BrdU incorporation (E) or Cdc45 loading (F) was observed separately in injection with the neutralizing antibody against starfish p90Rsk female and male pronuclei. Such a failure in pronuclear congression is evi- that can inhibit its activity (9), Cdc45 was loaded onto chromatin dence for sufficient knockdown of cyclin B (E)orsufficient inhibition of Cdk F in female pronucleus, and the subsequent DNA replication was ( ), because pronuclear congression requires low levels of cyclin B-Cdk1 C D activity (12). induced in the absence of fertilization (Fig. 5 and ). Further, coinjection of CA-Rsk-EE with the neutralizing anti-p90Rsk antibody restored the arrest at G1 phase (Fig. 5 E–G). Thus, B C fi ration (Fig. 4 and ). No further delay in the Cdc45 loading inactivation of p90Rsk is necessary and suf cient for Cdc45 CELL BIOLOGY was detectable, even when fertilized eggs incurred both Cdk loading onto chromatin in unfertilized G1-phase eggs. inhibition and Cdc7 knockdown (Fig. 4C). Taken together, both Cdk and Cdc7 are likely to be dis- Discussion pensable for the Cdc45 loading onto chromatin after fertiliza- The present observations show that in unfertilized starfish eggs, tion. However, we cannot exclude the possibility that Cdk may G1-phase arrest occurs at the stage of pre-RC. Fertilization affect the efficiency of DNA replication (16), and that very low targets the pre-RC/pre-IC transition through loading of Cdc45 level of Cdc7 detectable in immature oocytes remained even onto chromatin, which requires p90Rsk inactivation but neither after Cdc7 knockdown and contributed to the delayed formation Cdk activity nor Cdc7 accumulation, resulting in initiation of of the pre-IC, whereas the pre-IC would be formed at normal DNA replication.

Tachibana et al. PNAS | March 16, 2010 | vol. 107 | no. 11 | 5009 Downloaded by guest on September 27, 2021 A Uninjected CA-Rsk-EE KD CA-Rsk-EE C Control IgG Anti-Rsk As for the initiation of DNA replication at fertilization, there are striking differences in the regulatory mechanisms used by different species. In Xenopus, Cdc6 is the missing factor in DNA DNA immature oocytes (17), and cyclin E is necessary for DNA replication in egg extracts (18), whereas the Mos-MAPK-Rsk pathway is unlikely to play any role for progression through G1 phase due to its preceding inactivation (19, 20). In sea urchin ﬂ

Cdc45 eggs, which also arrest at G1 phase until fertilization, con icting Cdc45 20 µm 20 µm reports have accumulated: MAPK inactivation is necessary for B D DNA replication (21); Cdk activity is not required for DNA (12)(45) (31) replication (22, 23); MAPK inactivation does not cause DNA 100 (8) (23) 100 (52) replication (24); and MAPK activation and cyclin E are required for DNA replication (25). No study, however, examined the 50 50 immediate downstream of MAPK or the behavior of Mcms and Cdc45, excluding further comparison with starﬁsh egg system. (6) (29) (21)(42) In eukaryotic cells, there is a major cell-cycle checkpoint at 0 0 late G1 phase called “START” in yeast or “restriction point” in mammalian cells, after which commitment to a new cell cycle is irreversible (26). As the G1 checkpoint occurs at the stage of pre- Anti-Rsk Cdc45 on chromatin (%)( ) Uninjected Cdc45 on chromatin (%)( ) RC (14), the arrest stage appears to be conserved in unfertilized CA-Rsk-EE Control IgG BrdU incorporated eggs (%)( ) BrdU incorporated eggs (%)( ) G1-phase eggs as well. However, both types of arrest are unlikely CA-Rsk-EE KD to be equivalent. In the G1 checkpoint, the pre-RC/pre-IC E F transition is intervened by transcriptional control (27) that leads to the activation of Cdk and Cdc7, which support the Cdc45 loading. In contrast, the Cdc45 loading by fertilization does not even require translation. Indeed, there is an apparent similarity Anti-Rsk +CA-Rsk-EE Anti-Rsk +CA-Rsk-EE KD Anti-Rsk +CA-Rsk-EE Anti-Rsk +CA-Rsk-EE KD Anti-Rsk Uninjected Control IgG between the G1 checkpoint arrest in budding yeast and the G1 Fertilization - + --- - arrest in unfertilized starﬁsh eggs because both require Fus3/ S6K activity MAPK, whereas Fus3 directly targets Far1, an CDK inhibitor,

MAPK DNA resulting in transcriptional repression toward prevention of DNA G replication (28). Such a different dependence on transcription may explain the different requirement both of Cdk and Cdc7 for the Cdc45 loading. It is plausible that in case of fertilization, BrdU 20 µm Cdk- and Cdc7-dependent processes could be accomplished Anti-Rsk +CA-Rsk-EE Anti-Rsk Anti-Rsk +CA-Rsk-EE KD during meiotic maturation. Control IgG 100 (15) So far, p90Rsk is a unique negative regulator for the pre-RC (20) stage. In this arrest, p90Rsk should ﬁnally affect replication 75 initiation proteins that are engaged in loading of Cdc45 onto chromatin, e.g., GINS, Dpb11/Cut5/Mus101/TopBP1, Mcm10, 50 Sld2/RecQL4, or Ctf4/And-1 (1, 2, 29, 30). Our preliminary

eggs (%) observations suggest that p90Rsk do not directly phosphorylate 25 Cdc45 in vitro, whereas starfish Cut5 contains consensus BrdU incorporated (22) (18) sequences for phosphorylation by p90Rsk. In somatic cell cycle, 0 however, MAPK-p90Rsk signaling positively regulates the G1 Fig. 5. Inactivation of p90Rsk is necessary and sufficient for loading of progression (31). If so, the direct target of p90Rsk in unfertil- Cdc45 onto chromatin and the subsequent initiation of DNA replication in ized starfish eggs is unlikely to be a replication initiation protein G1-phase-arrested starfish eggs. (A and B) Maintenance of p90Rsk activity itself but a specific mediator to the initiation complex for DNA prevents loading of Cdc45 onto chromatin and the subsequent DNA repli- replication, as in a manner that p90Rsk directly targets Erp1/ cation in fertilized eggs. Mature eggs after completion of meiosis II were Emi2, an APC/C inhibitor, but not the APC/C itself, in meta- uninjected or injected with 0.6 ng of either constitutively active p90Rsk (CA- phase arrest of meiosis II in Xenopus eggs (19, 20). Further Rsk-EE) or its control kinase-dead form (CA-Rsk-EE KD). Thereafter, these eggs were inseminated and incubated in the presence of BrdU and then studies will contribute to elucidating the rewiring of signal fixed either at 30 min for anti-Cdc45 immunostaining or at 50 min for anti- transduction pathways to different cell-cycle controls. BrdU immunostaining. Successful fertilization was confirmed by formation of fertilization membrane. (C and D) Inhibition of p90Rsk activity causes Materials and Methods loading of Cdc45 onto chromatin and the subsequent DNA replication in the Oocytes and Eggs. Fully grown, immature oocytes were isolated from the absence of fertilization. Unfertilized mature eggs were injected in the starfish Asterina pectinifera. Oocyte maturation was induced by 1 μM presence of BrdU with 3 ng of either neutralizing anti-p90Rsk antibody or 1-methyladenine (1-MeAde), the starfish maturation-inducing hormone (32). control preimmune IgG, followed by incubation. Eggs were fixed either at 30 Some 150 min later, mature eggs with a female pronucleus, which were min for anti-Cdc45 immunostaining or at 60 min for anti-BrdU immuno- arrested at G1 phase after completion of meiosis, were inseminated to start staining. (E–G) Constitutively active p90Rsk restores G1-phase arrest in eggs the embryonic mitotic cycle (7). Microinjection into eggs was performed as injected with neutralizing anti-p90Rsk antibody. Unfertilized mature eggs described (33). were injected with neutralizing anti-p90Rsk antibody along with CA-Rsk-EE or control CA-Rsk-EE KD. After 60 min incubation in the presence of BrdU, Immunofluorescence Staining. Immunofluorescent or DAPI staining was per- eggs were fixed for anti-BrdU immunostaining (F and G). Egg lysates were formed essentially as described (12). Additional information, including either immunoblotted with anti-MAPK antibody, or assayed for phosphor- antibody preparation, morpholino-mediated knockdown, and others, see SI ylation of GST-S6 (S6K activity) (E). DNA was stained with DAPI. Number of Materials and Methods. eggs examined for Cdc45 loading or BrdU incorporation are indicated in B D G parentheses ( , , and ). ACKNOWLEDGMENTS. We thank Thomas J. McGarry for geminin and gemininDEL constructs; Angel R. Nebreda for CA-Rsk-EE constructs; Yuichi

5010 | www.pnas.org/cgi/doi/10.1073/pnas.1000587107 Tachibana et al. Downloaded by guest on September 27, 2021 Kumaki and Masato Yoshizawa for help in preparing antibodies; Keita saki for reading the manuscript. This work was supported by grants from Ohsumi, Eiichi Okumura, and Mari Iwabuchi for discussion; and Mark Tera- MEXT Japan (to K.T. and T.K.).

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