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Developmental regulation of MCM replication factors in Xenopus laevis Jill C. Sible*, Eleanor Erikson*, Mary Hendrickson†, James L. Maller* and Jean Gautier†
At the midblastula transition (MBT) during Xenopus Results and discussion laevis development, zygotic transcription begins [1], and The Xenopus genome encodes distinct forms of MCM6 the rapid, early cleavage cycles are replaced by cell- In a screen of a stage 17 X. laevis head cDNA library, a clone division cycles that lengthen and acquire G (gap) phases was fortuitously purified that exhibited high sequence [2] and checkpoints [3–5]. This cell-cycle remodeling homology to mammalian mcm6 genes. This clone (zmcm6a) may result from either a loss of maternal products, the was used to rescreen the library, and the longest clone transcription of zygotic genes, or the replacement of obtained was subjected to 5′ rapid amplification of cDNA maternal proteins by zygotic gene products. We have ends (5′RACE) PCR to yield a second, full-length cDNA identified an example of the third possibility: distinct (zmcm6b). On the basis of their near sequence identity maternal and zygotic genes encoding a member of the (Figure 1a) and their indistinguishable patterns of gene minichromosome maintenance (MCM) protein family. expression assessed by RNase protection assays (data not The mcm genes were identified in yeast by mutations shown), zmcm6a and zmcm6b are likely to be pseudo- that blocked replication of artificial chromosomes or alloploid alleles of the X. laevis allotetraploid genome. Fur- perturbed the G1/S transition in the cell cycle [6,7]. In thermore, a full-length PCR clone of zmcm6a cDNA was Xenopus eggs, the MCM2–MCM7 proteins assemble as more than 90% identical to zmcm6b at the nucleotide level multimeric complexes at chromosomal origins of (data not shown). The zmcm6a and zmcm6b cDNAs will not replication [8–14]. The sequential, cell-cycle-dependent be distinguished further in this paper. In contrast, both assembly of the origin replication complex (ORC), CDC6 zMCM6a and zMCM6b exhibit only 73% amino-acid iden- protein and the MCM complex at origins of replication tity with a published Xenopus MCM6 sequence ([14] and ensures that DNA replicates only once per cell cycle Figure 1a), suggesting that mMCM6 and zMCM6 are evo- [15,16]. The periodic association of the MCM complex lutionarily distinct. Specifically, zMCM6 contains an addi- with chromatin may be regulated via phosphorylation by tional 21 amino acids at the carboxyl terminus and a cyclin-dependent kinases (Cdks) [11]. We have cloned consensus phosphorylation site for cyclin–Cdk complexes the first example of a developmentally regulated mcm (Figure 1a, boxed) that is missing from mMCM6. Given gene, zygotic mcm6 (zmcm6), expressed only after that Cdks have both positive and negative effects on DNA gastrulation when the cell cycle is remodeled. The replication [17–21] and, more specifically, that the dissocia- zMCM6 protein assembles into MCM complexes and tion of MCMs from chromatin during S phase may be due differs from maternal MCM6 (mMCM6) in having a to cyclin B–Cdc2 phosphorylation of MCMs [11], the carboxy-terminal extension and a consensus cyclin–Cdk cyclin–Cdk phosphorylation site in zMCM6 suggests that phosphorylation site. There may also be differential regulation of mMCM6 and zMCM6 may occur. maternal–zygotic pairs of other MCMs. These data suggest that MCMs are critical for cell-cycle remodeling The mmcm6 and zmcm6 genes are expressed maternally during early Xenopus development. and zygotically, respectively To elucidate differences in the expression of mmcm6 and Addresses: *Howard Hughes Medical Institute and Department of Pharmacology, University of Colorado School of Medicine, Denver, zmcm6, probes were generated specific for the dissimilar Colorado 80262, USA. †Department of Genetics and Development regions of the cDNAs, which spanned the 3′-coding and 3′- and Department of Dermatology, College of Physicians and Surgeons untranslated regions (Figure 1b), and used for developmen- of Columbia University, New York, New York 10032, USA. tal northern blot analysis under high-stringency conditions Correspondence: James L. Maller (Figure 2). A single band of the predicted molecular weight E-mail: [email protected] (approximately 3 kb) was detected with each probe. The Received: 24 November 1997 mmcm6 probe detected RNA in the egg, which remained at Revised: 22 December 1997 a constant level until the gastrula stage (stage 10.5), and Accepted: 22 January 1998 declined drastically at the neurula stage (stage 13); this RNA is therefore a maternally supplied mcm6 (mmcm6). In Published: 2 March 1998 contrast, the zmcm6 probe did not detect RNA in early Current Biology 1998, 8:347–350 embryos, but a signal appeared after the MBT at the http://biomednet.com/elecref/0960982200800347 neurula stage; thus, this RNA is a zygotically transcribed mcm6 (zmcm6). This is the first example in Xenopus of a pair © Current Biology Ltd ISSN 0960-9822 of maternal and zygotic genes that encode distinct forms of 348 Current Biology, Vol 8 No 6
Figure 1
Two cDNAs encoding novel forms of MCM6 (a) were isolated from a X. laevis stage 17 cDNA zMCM6a ------ 100 library. (a) Predicted amino-acid sequences of zMCM6b MDLVDPSQS-AAAAAGTQLVKDEVAEKCQKLFQDFLEEFRGSDGELKYQSDAEELIRPERNTLLVSFVDLEQFNQQLATTIQEEFYRVYPYLCRAVKAFA mMCM6 MELGGPAAAGDTDIAGQQLFKDELSDKCQKLFLEFFEECKGKDGSNLYVSAAEELIRPERNTLAVNFTDIEYYNQQLATTIQEEYYRVYPHLCRAVRSFA proteins encoded by zygotic mcm6a zMCM6a ------ 200 (zMCM6a, partial sequence) and zygotic zMCM6b RDHGNIPQNKEFYVAFQELPTRHKIRELTTPRIGSLLRISGQVVRTHPVHPELVSGTFLCLDCQTLVRDVEQQFKYTQPSICRNPVCANRKRFMLDTNKS mMCM6 RQMGNIPANKEFYIAFSDFPARQKIRELSSAKIGTLLRISGQVVRTHPVHPELVSGTFLCMDCQSIVKDVEQQFRYTQPTICKNPVCANRRRFTLDTNKS mcm6b (zMCM6b, complete sequence) zMCM6a ------ 300 aligned with that of maternal mcm6 (mMCM6) zMCM6b RFVDFQKVRIQETQAELPRGSIPRSVEVILRAEAVESCQAGDRCDFTGSLIVVPDISQLSTPGVRAETSSRVGGREGYEAEGVQGLRALGVRDLSYKLVF [14]. Identical amino acids are shaded gray. mMCM6 RFVDFQKVRIQETQAELPRGAIPRSVEIILRAEAVESAMAGDRCDFTGTLIVVPDVSAFAAGDARMETGAKVTGGEGFNSEGVQGLKALGVRDLSYRLAF zMCM6a ------ 400 The peptide from zMCM6a that was used to zMCM6b LACYVCPTNPRFGGKDLHEEDMTAESIKNQMSVKEWEKVFEMSQDKNLYHNLCTSLFPTVHGNDEVKRGILLMLFGGVPKSTMEGTSLRGDINVCVVGDP generate polyclonal antiserum is shaded blue. mMCM6 LACYVGATNPRFGGKDLREEDQTAESIKNQMTVQEWEKVFEMSQDKNLYHNLCTSLFPTIHGNDEIKRGVLLMLFGGVPKTTMEGTSLRGDINVCIVGDP The portion of zMCM6b that was obtained by zMCM6a ------ 500 zMCM6b STAKSQFLKHVEEFSPRAVYTSGKASTAAGLTAAVVRDEESHEFVIEAGALMLADNGVCCIDEFDKMDTKDQVAIHEAMEQQTISITKAGVKATLNARTS 5′RACE is shown in italics. A Cdk consensus mMCM6 STSKSQFLKHVEEFSPRAVYTSGKASSAAGLTAAVVKDEESHEFVIEAGALMLADNGVCCIDEFDKMDLKDQVAIHEAMEQQTISITKAGVKATLNARTS phosphorylation site in zMCM6b is boxed. zMCM6a ------ 600 zMCM6b ILAAANPVGGRYDRAKSLKQNVNLSAPIMSRFDLFFILVDECNEVTDYAITRRIVDLHSRIEESIDRVYTLDEVRRYLLFAKQFKPKISKESEDFIVEQY (b) Alignment of zmcm6b and mmcm6 in the mMCM6 ILAAANPVGGRYERSKSLKHNVNLSAPIMSRFDLFFILVDECNEVTDYAIARRIVDLHARNEESIERVYSIEDIQRYLLFARQFQPKITKEAEEFIVEQY 3′ region of the cDNAs. The entire sequences zMCM6a ------PDVNLDQ-DDEHEPEDETQEGTNGDAEVPNGVNGHVN 700 zMCM6b KRLRQRDGSGVTKSAWRITVRQLESMIRLSEGMARMHCSDEVQPKHVKEAFRLLNKSIIRVETPDVNLDQ-EDEHEVE-EPQEGINGDADVPNGVNGHIN shown were used as probes for northern mMCM6 RRLRQRDGSGVAKSSWRITVRQLESLIRLSESMARMHCSDEVQPKHVKEAFRLLSKSIIRVDTPDVSFDQGEDEKNIE-----GENN----GNLNNGEEA blotting. Identical nucleotides are shaded; the zMCM6a GINGHSQESNAAAAK-PSLRLNFAEYKRISNLLVQQLRKMEDED---ETSQRRSELMNWYLKEIESEIDSEEELINRKQIIDKVIHRLVHYDQILIELTQ 800 zMCM6b GINGHAEETNAAPPK-PSLRLNFAEYKRISNLLVLQLRKIEDEDDENETSQRKSELINWYLKEIESEIDSEEELVNRKQIIDKVIHRLVHYDQILIALIQ translation stop codons are boxed. mMCM6 METNQDEPINEKPSSNAGLKMSFAEYKQISNLLVLYMQKMEETEE--ECHLTTTDLVNWYLKEMEAEIETETELILKKRLIEKVIHRLIYYDHILIRTEQ
zMCM6a TELKGTGDEVVAKEEDPYLVVNPNYILED zMCM6b TGLKGTEDENVAKE-DPYLVVNPNYILED mMCM6 IGIKNNG------(b) zmcm6b GAGGATGAACATGAGGTAGAGGAACCTCAGGAGGGCATTAATGGAGATGCTGATGTTCCCAATGGTGTTAATGGCCACATAAATGGAATTAATGGGCATG mmcm6 GGCGAGG------ATGAAAAGAACATTGAAGGAGAGAATAATGGCAATTTGAA------CAATGGAGAGGA-AGCAATGGAAACA-AATCA--GGATGAA
zmcm6b CTGAGGAGACGAATGCTGCTCCCCCAAAGCCATCTTTGCGTCTTAATTTTGCTGAATACAAAAGGATCTCCAACTTGCTGGTCCTGCAGTTGAGGAAGAT mmcm6 CCAATAAATGAAAAGCCAT--CCTCAAATGCTGGATTAAAAATGTCTTTTGCAGAGTATAAGCAAATCTCCAACTTGTTGGTTTTGTACATGCAGAAGAT
zmcm6b TGAAGATGAAGATGATGAAAATGAAACTTCTCAGAGAAAGAGTGAACTCATTAACTGGTACCTGAAGGAGATTGAGTCAGAAATTGACTCGGAAGAGGAG mmcm6 GGAAGA------AACTGAAGAAGAGTGCCATCTGACAACAACTGACTTGGTTAACTGGTATCTTAAAGAAATGGAAGCTGAAATAGAGACTGAAACAGAA
zmcm6b CTCGTTAATAGGAAGCAAATCATTGACAAAGTTATTCACAGACTTGTGCACTATGACCAAATTTTGATTGCGCTCATACAAACTGGATTAAAAGGCACTG mmcm6 CTAATCCTTAAAAAGCGTTTAATTGAAAA GTGATTCACAGATTAATTTATTATGATCATATTCTGATTAGAACTGAACAAATCGGAATTAAAAACAATG
zmcm6b AA-GACGAGAACGTAGCAAAAGAGGATCCTTACCTTGTGGTTAATCCCAACTACATACTGGAAGATTAATAGGCTGCTTTGTGCCTCACAGACTTCATTT mmcm6 GATGATACAAAGGAGACGGGTGAAGATGCT-GCTGAAGATCGGATTTTAGTTGTGAATCCTAATTATATGTTGGAAGATTAAACGTATGCAGGCAAACTT
zmcm6b GCAAGACTTGCGCTTTTCCTATTTGGAAATGTACA-CTGATTCTCATTTAGAGATTTTTTTTACCCAAGGATATACAGTATCTGCCATCTGG mmcm6 GTTTGGTAGAAATTTTTCTTGTGTGTAATTATGGAATTGCTCCTCTGTCCTAAAGTATGCAAG Current Biology
the same protein. Strikingly, these genes encode a DNA- region of zMCM6a (Figure 1a; blue shading). This region replication factor, and the switch from maternal to zygotic differs by only one amino acid between zMCM6a and forms occurs at a time of extensive cell-cycle remodeling. zMCM6b, so the antibody should recognize both forms, but the region is only 55% identical to mMCM6 and thus the The identification of other maternal–zygotic gene pairs antibody should cross-react poorly with mMCM6. Western encoding cell-cycle regulatory molecules would suggest a analysis using this antibody indicated the appearance of a paradigm for effecting changes in the cell-cycle after the 97 kDa zMCM6 protein during neurulation (Figure 3a; MBT. To explore this possibility further for the mcm genes, stage 15), consistent with the RNA profile (Figure 2). In the northern blot was reprobed for mcm3 RNA, and a dupli- contrast, antisera against MCM3 and MCM4 demonstrated cate blot was probed for mcm7 RNA [9] (Figure 2). The expression of these MCMs throughout development, indi- expression of mcm3 was similar to that of mmcm6, declining cating either that there is only a single form of these MCMs drastically at the gastrula stage; it therefore seems likely or that the antisera cross-react with two forms. The latter that there is a zygotic counterpart of mcm3 because the possibility is most likely for MCM3, as northern analysis protein was detected long after the MBT (Figure 3a,b) and suggested distinct maternal and zygotic forms (Figure 2). in cultured XTC cells (data not shown). In contrast, mcm7 RNA was present at constant levels throughout develop- The association of zMCM6 into higher molecular weight ment suggesting that there may be only a single form of complexes was examined by western blotting extracts that MCM7 that is not developmentally regulated. had been resolved on non-denaturing gradient gels (Figure 3b) [8]. Complexes containing zMCM6 resolved as Zygotic MCM6 protein accumulates after the MBT and two distinct high molecular weight species. Consistent with associates into complexes with MCM3 previous findings [14,22], MCM4 was also present in both Maternal MCM6 associates with MCMs 2,3,4,5 and 7 in a species, whereas MCM3 was present in only the slower high molecular weight complex required for the initiation of migrating species. This could reflect the existence of sub- DNA replication [14]. To explore whether zygotic MCM6 is complexes, one containing MCMs 2, 4, 6 and 7 and the likely to replace this function, antibodies were raised against other containing MCM3 and MCM5 in addition [14,22]. an 18-residue peptide corresponding to a carboxy-terminal Thus, the faster-migrating complex detected in the MCM4 Brief Communication 349
Figure 2 against MCM3, resolved by SDS–PAGE, and then blotted for zMCM6 (Figure 3c). These studies indicate that MBT MCM3 and zMCM6 begin to associate in vivo at the Stage: neurula stage. Similar results were obtained using an anti- 42 kb Egg 4–5 6–6.5 8 9 10.5 13 18 22–23 25 32–35 body against MCM7 to immunoprecipitate complexes 7.5– throughout development, followed by western blotting to 4.4– mmcm6 detect zMCM6 (data not shown). The protein and RNA 2.4– 1.4– analyses together suggest that zMCM6 may replace mMCM6 in the complex that functions during the initia- 0.2– tion of DNA replication. We cannot exclude the possibil- 9.5– ity that mMCM6 persists in a subset of these complexes, 7.5– however. Why would Xenopus need different MCMs for 4.4– zmcm6 early versus late embryonic cell cycles? There are several 2.4– 1.4– possibilities. Firstly, chromatin structure and the usage of origins of replication change after initiation of transcrip- 0.2– tion [23,24]. A different complement of MCMs might therefore be required after initiation of transcription, and 9.5– 7.5– zMCM6 could function to restrict the number of origins. 4.4– Secondly, the acquisition of checkpoints after the MBT 2.4– mcm3 that make cell-cycle progression dependent upon the 1.4– completion of prior DNA replication [3,5] might depend 0.2– upon MCMs that link these processes. Thirdly, as sug- gested by Kubota et al. [14], the entry of specific MCMs 9.5– into the nucleus might be regulated by selective perme- 7.5– 4.4– ability during early embryonic cycles — a stage when these proteins are very abundant — but this regulation 2.4– mcm7 1.4– may not occur during later somatic cycles. Any of these possible functions could be regulated through phosphory- 0.2– lation by Cdks, which would differentiate maternal and Current Biology zygotic MCM6. Studies to identify additional maternal–zygotic gene pairs involved in DNA replication Expression of zmcm6 and mmcm6 is developmentally distinct. A and to assess functional differences between the gene northern blot of total RNA (20 µg) isolated from staged Xenopus embryos was probed for zmcm6 RNA, and then stripped and reprobed products should provide insights into the cell-cycle sequentially for mmcm6 and mcm3 RNAs. A separate northern blot remodeling that occurs during early Xenopus development. using duplicate samples of the same RNAs was probed for mcm7 RNA. RNA preparation and northern analysis were performed as described [3]. Materials and methods To generate the probes, fragments of mmcm6 were amplified from a Xenopus oocyte cDNA library by reverse transcriptase PCR using Generation and maintenance of embryos primers based on the published sequence [14]. Analogous regions of Eggs from X. laevis (Xenopus I, Ann Arbor, MI) were fertilized in vitro, × zmcm6b were also amplified by PCR. These PCR products were cloned dejellied in 2% cysteine, and maintained in 0.1 MMR (10 mM NaCl, into pGEMT-easy (Promega). Inserts isolated from these plasmids were 0.2 mM KCl, 0.1 mM MgSO4, 0.2 mM CaCl2, 0.5 mM Hepes, pH 7.8). labeled using α-[32P]dCTP (Amersham, 3000 Ci/mmol) and the Random Embryos were staged according to Nieuwkoop and Faber [25] and Primed DNA kit (Boehringer Mannheim) according to the manufacturer’s snap-frozen on dry ice at the appropriate stage. protocol. The probes for mcm3 and mcm7 were generated from plasmids provided by M. Madine and P. Romanowski, respectively. Cloning of zygotic mcm6 Zygotic mcm6 clone a (zmcm6a) was isolated from a stage 17 embry- onic cDNA library in λgt10 (kindly provided by D. Melton, Harvard uni- versity) as a 783 bp cDNA that fortuitously copurified with another and MCM6 western blots could lack MCM3 and MCM5. cDNA. The zmcm6a clone was used to rescreen the same library, and These experiments demonstrate that zMCM6 appears in zygotic mcm6 clone b (zmcm6b) was isolated. An additional 327 bp of ′ ′ high molecular weight complexes at the neurula stage. In 5 coding sequence and 51 bp of 5 untranslated region were obtained for zmcm6b by 5′ RACE PCR using the Clontech Marathon cDNA contrast, MCM3 and MCM4 are found in such complexes system and poly A+ RNA from stage 18–20 embryos, and the full- throughout development. These data are consistent with length cDNA was generated by subcloning into pUC19. The zmcm6a the northern analysis demonstrating that MCM6 exists as and zmcm6b sequences have been submitted to GenBank under distinct maternal and zygotic forms and also suggest that accession numbers AF031140 and AF031139, respectively. zMCM6 associates with other MCMs once it is expressed. Acknowledgements This work was supported by a grant from the NIH to J.L.M. (GM26743). To explore the co-association of MCMs more directly, J.C.S. is an Associate and J.L.M. is an Investigator of the Howard Hughes extracts were immunoprecipitated with an antibody Medical Institute. 350 Current Biology, Vol 8 No 6
Figure 3
Accumulation of zMCM6 protein during (a) (b) neurulation and its assembly into MCM complexes. (a) Lysates (100 µg protein) of Stage : 1 7 9 11 15 19 23 28 31 37 Stage : 1 7 9 111519 232831 staged embryos were resolved by SDS–PAGE, kDa kDa 180– and a western blot [11] was probed with 669– affinity-purified anti-zMCM6 antiserum then 116– 440– MCM3 stripped and reprobed for MCM3 and MCM4. MCM3 232– (b) Lysates (200 µg protein) of staged embryos 84– 140– 67– were resolved on nondenaturing gradient 180– acrylamide gels as described [11], except that batches of 50 staged embryos were first 116– MCM4 crushed in EB buffer [11] containing 0.2% 84– 669– NP40 (5 µl per embryo), and western blots 440– MCM4 were probed for the indicated MCMs. 232– 180– 140– (c) Lysates (1 mg protein) were precipitated overnight with 4 µg affinity purified MCM3 116– 67– zMCM6 immunoglobulin G in EB containing 0.5% 84– NP40. Protein-A–Sepharose was added for 30 min, then immunoprecipitates were washed 669– five times with EB/NP40, resolved by SDS— 440– zMCM6 (c) 232– PAGE and western blotted for zMCM6. Stage : 1 7 9 11 15 19 23 28 31 37 140– Polyclonal antiserum against zMCM6 was kDa 67– raised in rabbits (Charles River Pharmaceutical Services) by injection of the peptide 180– CKEIESEIDSEEELINRKQ, in single-letter 116– zMCM6 amino-acid code, coupled to KLH (Pierce). The 84– antiserum was affinity purified on a column Current Biology containing the peptide conjugated to thiopropyl Sepharose 6B (Pharmacia). Affinity purified antibodies against MCM3 and MCM4 were characterized as described [11].
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