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

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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 CELL BIOLOGY 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. www.pnas.org/cgi/doi/10.1073/pnas.1000587107 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 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 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.
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