Abstract: In this paper, we describe how, in a model embryonic system, cyclin-dependent kinase (Cdk) activity controls the efficiency of DNA replication by determining the frequency of origin activation. Using independent approaches of protein depletion and selective chemical inhibition of a single Cdk, we find that both Cdk1 and Cdk2 are necessary for efficient DNA replication in Xenopus egg extracts. Eliminating Cdk1, Cdk2 or their associated cyclins changes replication origin spacing, mainly by decreasing frequency of activation of origin clusters. Although there is no absolute requirement for a specific Cdk or cyclin, Cdk2 and cyclin E contribute more to origin cluster efficiency than Cdk1 and cyclin A. Relative Cdk activity required for DNA replication is very low, and even when both Cdk1 and Cdk2 are strongly inhibited, some origins are activated. However, at low levels, Cdk activity is limiting for the pre-replication complex to pre-initiation complex transition, origin activation and replication efficiency. As such, unlike mitosis, initiation of DNA replication responds progressively to changes in Cdk activity at low activity levels.
Abstract: Topoisomerase II (topo II) is required for chromosome segregation and for reprogramming replicons. Here, we show that topo II couples DNA replication termination with the clearing of replication complexes for resetting replicons at mitosis. Topo II inhibition impairs completion of DNA replication, accounting for replication protein A (RPA) stabilization onto ssDNA. Topo II inhibition does not affect the caffeine-sensitive ORC1 degradation found upon origin firing, but it impairs the cdk-dependent degradation/chromatin dissociation of an ORC1/2 reservoir at mitosis. Our results show that ORC1 degradation is rescued by Pin1 depletion and that this topo II-dependent clearing of ORC1/2 from chromatin involves the APC.
Abstract: Previous observations led to the conclusion that in Xenopus eggs and during early development, DNA replication initiates at regular intervals but with no apparent sequence specificity. Conversely, here, we present evidence for site-specific DNA replication origins in Xenopus egg extracts. Using lambda DNA, we show that DNA replication origins are activated in clusters in regions that contain closely spaced adenine or thymine asymmetric tracks used as preferential initiation sites. In agreement with these data, AT-rich asymmetric sequences added as competitors preferentially recruit origin recognition complexes and inhibit sperm chromatin replication by increasing interorigin spacing. We also show that the assembly of a transcription complex favors origin activity at the corresponding site without necessarily eliminating the other origins. Thus, although Xenopus eggs have the ability to replicate any kind of DNA, AT-rich domains or transcription factors favor the selection of DNA replication origins without increasing the overall efficiency of DNA synthesis. These results suggest that asymmetric AT-rich regions might be default elements that favor the selection of a DNA replication origin in a transcriptionally silent complex, whereas other epigenetic elements linked to the organization of domains for transcription may have further evolved over this basal layer of regulation.
Abstract: Animal cloning by nuclear-transfer experiments frequently fails due to the inability of transplanted nuclei to support normal embryonic development. We show here that the formation of mitotic chromosomes in the egg context is crucial for adapting differentiated nuclei for early development. Differentiated erythrocyte nuclei replicate inefficiently in Xenopus eggs but do so as rapidly as sperm nuclei if a prior single mitosis is permitted. This mitotic remodeling involves a topoisomerase II-dependent shortening of chromatin loop domains and an increased recruitment of replication initiation factors onto chromatin, leading to a short interorigin spacing characteristic of early developmental stages. It also occurs within each early embryonic cell cycle and dominantly regulates initiation of DNA replication for the subsequent S phase. These results indicate that mitotic conditioning is crucial to reset the chromatin structure of differentiated adult donor cells for embryonic DNA replication and suggest that it is an important step in nuclear cloning.
Abstract: DNA replication licensing is an important step in the cell cycle at which cells become competent for DNA replication. When the cell cycle is arrested for long periods of time, this competence is lost. This is the case for somatic cells arrested in G0 or vertebrate oocytes arrested in G2. CDC6 is a factor involved in replication initiation competence which is necessary for the recruitment of the MCM helicase complex to DNA replication origins. In Xenopus, we have previously shown that CDC6 is the only missing replication factor in the oocyte whose translation during meiotic maturation is necessary and sufficient to confer DNA replication competence to the egg before fertilization (Lemaitre et al., 2002: Mol Biol Cell 13:435-444; Whitmire et al., 2002: Nature 419:722-725). Here, we report that this oogenesis control has been acquired by metazoans during evolution and conserved up to mammals. We also show that, contrary to eukaryotic metazoans, in S. pombe cdc18 (the S. pombe CDC6 homologue), CDC6 protein synthesis is down regulated during meiosis. As such, the lack of cdc18 prevents DNA replication from occurring in spores, whereas the presence of cdc6 makes eggs competent for DNA replication.
Abstract: Replication protein A (RPA) is a three subunit single-stranded DNA-binding protein required for DNA replication. In Xenopus, RPA assembles in nuclear foci that form before DNA synthesis, but their significance in the assembly of replication initiation complexes has been questioned. Here we show that the RPA34 regulatory subunit is dephosphorylated at the exit of mitosis and binds to chromatin at detergent-resistant replication foci that co-localize with the catalytic RPA70 subunit, at both the initiation and elongation stages of DNA replication. By contrast, the RPA34 phosphorylated form present at mitosis is not chromatin bound. We further demonstrate that RPA foci assemble on chromatin before initiation of DNA replication at sites functionally defined as initiation replication sites. Association of RPA with these sites does not require nuclear membrane formation, and is sensitive to the S-CDK inhibitor p21. We also provide evidence that RPA34 is present at initiation complexes formed in the absence of MCM3, but which contain MCM4. In such conditions, replication foci can form, and short RNA-primed nascent DNAs of discrete size are synthesized. These data show that in Xenopus, the hypophosphorylated form of RPA34 is a component of the pre-initiation complex.
Abstract: Meiotic maturation, the final step of oogenesis, is a crucial stage of development in which an immature oocyte becomes a fertilizable egg. In Xenopus, the ability to replicate DNA is acquired during maturation at breakdown of the nuclear envelope by translation of a DNA synthesis inducer that is not present in the oocyte. Here we identify Cdc6, which is essential for recruiting the minichromosome maintenance (MCM) helicase to the pre-replication complex, as this inducer of DNA synthesis. We show that maternal cdc6 mRNA but not protein is stored in the oocyte. Cdc6 protein is synthesized during maturation, but this process can be blocked by degrading the maternal cdc6 mRNA by oligonucleotide antisense injections or by translation inhibition. Rescue experiments using recombinant Cdc6 protein show that Cdc6 is the only missing replication factor whose translation is necessary and sufficient to confer DNA replication competence to the egg before fertilization. The licence to replicate is given by Cdc6 at the end of meiosis I, but the cytostatic factor (CSF) pathway, which maintains large amounts of active Cdc2/Cyclin B2, prevents the entry into S phase until fertilization.
Abstract: We have characterized Xenopus ISWI, a catalytic subunit of a family of chromatin-remodeling complexes. We show that ISWI is expressed constitutively during development but poorly expressed in adult tissues except oocytes which contain a large store of maternal protein. We further analyzed its localization both in vivo and in vitro in Xenopus cell cycle extracts and identified that ISWI binds to chromatin at the G1-S period. However, its association to chromatin does not require ongoing DNA replication. Immunodepletion of ISWI has no effect on either sperm chromatin decondensation or the kinetics and efficiency of DNA replication. Nucleosome assembly also occurs in ISWI-depleted extracts, but nucleosome spacing is disturbed. From these results, we conclude that ISWI is not necessary for sperm chromatin decondensation and the accelerated rates of DNA replication that characterize early development.
Abstract: To investigate the molecular mechanisms regulating c-myc RNA stability during late amphibian oogenesis, a heterologous system was used in which synthetic Xenopus laevis c-myc transcripts, progressively deleted from their 3' end, were injected into the cytoplasm of two different host axolotl (Ambystoma mexicanum) cells: stage VI oocytes and progesterone-matured oocytes (unfertilized eggs; UFE). This in vivo strategy allowed the behavior of the exogenous c-myc transcripts to be followed and different regions involved in the stability of each intermediate deleted molecule to be identified. Interestingly, these specific regions differ in the two cellular contexts. In oocytes, two stabilizing regions are located in the 3' untranslated region (UTR) and two in the coding sequence (exons II and III) of the RNA. In UFE, the stabilizing regions correspond to the first part of the 3' UTR and to the first part of exon II. However, in UFE, the majority of synthetic transcripts are degraded. This degradation is a consequence of nuclear factors delivered after germinal vesicle breakdown and specifically acting on targeted regions of the RNA. To test the direct implication of these nuclear factors in c-myc RNA degradation, an in vitro system was set up using axolotl germinal vesicle extracts that mimic the in vivo results and confirm the existence of specific destabilizing factors. In vitro analysis revealed that two populations of nuclear molecules are implicated: one of 4.4-5S (50-65 kDa) and the second of 5.4-6S (90-110 kDa). These degrading nuclear factors act preferentially on the coding region of the c-myc RNA and appear to be conserved between axolotl and Xenopus. Thus, this experimental approach has allowed the identification of specific stabilizing sequences in c-myc RNA and the temporal identification of the different factors (cytoplasmic and/or nuclear) involved in post-transcriptional regulation of this RNA during oogenesis.
Abstract: Acquisition of the competence to replicate requires the assembly of the MCM2-7 (minichromosome maintenance) protein complex onto pre-replicative chromatin, a step of the licensing reaction. This step is thought to occur through binding of a heterohexameric MCM complex containing the six related MCM subunits. Here we show that assembly of the MCM complex onto pre-replicative chromatin occurs through sequential stabilization of specific MCM subunits. Inhibition of licensing with 6-dimethylaminopurine results in chromatin containing specifically bound MCM4 and MCM6. A similar result was obtained by interference of the assembly reaction with an MCM3 antibody. The presence of chromatin-bound MCM intermediates was confirmed by reconstitution experiments in vitro with purified proteins and by the observation of an ordered association of MCM subunits with chromatin. These results indicate that the assembly of the MCM complex onto pre-replicative chromatin is regulated at the level of distinct subunits, suggesting an additional regulatory step in the formation of pre-replication complexes.
Abstract: It is becoming increasingly clear that transcription control is carried out at several interconnecting levels. Besides nucleosomal organization and interaction between transcription factors and gene promoters and other regulatory elements, long-range organization of chromatin in loops or domains seems to play a role in transcriptional regulation. A similar organization is likely to be crucial in the control of the timing and selection of origins of DNA replication. This review considers the implications of domain organization of eukaryotic genome in developmental control of transcription and replication.
Abstract: During Xenopus laevis early development, the genome is replicated in less than 15 min every 30 min. We show that during this period, DNA replication proceeds in an atypical manner. Chromosomes become surrounded by a nuclear membrane lamina forming micronuclei or karyomeres. This genomic organization permits that prereplication centers gather on condensed chromosomes during anaphase and that DNA replication initiates autonomously in karyomeres at early telophase before nuclear reconstruction and mitosis completion. The formation of karyomeres is not dependent on DNA replication but requires mitotic spindle formation and the normal segregation of chromosomes. Thus, during early development, chromosomes behave as structurally and functionally independent units. The formation of a nuclear envelope around each chromosome provides an in vivo validation of its role in regulating initiation of DNA replication, enabling the rate of replication to accelerate and S phase to overlap M phase without illegitimate reinitiation. The abrupt disappearance of this atypical organization within one cell cycle after thirteen divisions defines a novel developmental transition at the blastula stage, which may affect both the replication and the transcription programs of development.
Abstract: We report here unusual features of c-Myc specific to early embryonic development in Xenopus laevis, a period characterized by generalized transcriptional quiescence and rapid biphasic cell cycles. Two c-Myc protein forms, p61 and p64, are present in large amounts in the oocyte as well as during early development. In contrast, only p64 c-Myc is present in Xenopus somatic cells. p61 c-Myc is the direct translation product from both endogenous c-myc mRNAs and c-myc recombinant DNA. It is converted to the p64 c-Myc form after introduction into an egg extract, in the presence of phosphatase inhibitors. p61 and p64 belong to two distinct complexes localized in the cytoplasm of the oocyte. A 15S complex contains p64 c-Myc, and a 17.4S complex contains p61 c-Myc. Fertilization triggers the selective and total entry of only p64 c-Myc into the nucleus. This translocation occurs in a nonprogressive manner and is completed during the first cell cycles. This phenomenon results in an exceptionally high level of c-Myc in the nucleus, which returns to a somatic cell-like level only at the end of the blastulation period. During early development, when the entire embryonic genome is transcriptionally inactive, c-Myc does not exhibit a DNA binding activity with Max. Moreover, embryonic nuclei not only prevent the formation of c-Myc/Max complexes but also dissociate such preformed complexes. These peculiar aspects of c-Myc behavior suggest a function that could be linked to the rapid DNA replication cycles occurring during the early cell cycles rather than a function involving transcriptional activity.
Abstract: C-Myc is a nuclear phosphoprotein whose normal cellular function has not yet been clearly defined. Studies with this protein have always been constrained by the difficulty of obtaining full-length c-Myc in an active form, whatever the expression system used. We report here experimental conditions optimized to increase the solubility and the purification of c-Myc in a baculovirus expression system. Such conditions allow the production of both soluble and active full-length c-Myc. Interestingly, soluble c-Myc is found associated with a 500-kDa high-molecular-mass complex comparable to that found in human and Xenopus laevis embryos, and which may be required for its function in vivo.
Abstract: In eukaryotic cells, nucleus-cytoplasm exchanges play an important role in genomic regulation. We have analyzed the localization of four nuclear antigens in different growth conditions: two replicative proteins, DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), and two oncogenic regulatory proteins, c-Myc and c-Fos. A kinetic study of subcellular localization of these proteins has been done. In cultures in which cells were sparse, these proteins were detected in the nucleus. When proliferation was stopped by the high density of culture cells or by serum starvation, these proteins left the nucleus for the cytoplasm with different kinetics. DNA polymerase alpha is the first protein to leave the nucleus, with the PCNA protein, c-Fos, and c-Myc leaving the nucleus later. In contrast, during serum stimulation c-Fos and c-Myc relocalize into the nucleus before the replicative proteins. We also noticed that in sparse cell cultures, 10% of the cells exhibit a perinuclear staining for the DNA polymerase alpha, PCNA, and c-Myc proteins but not for c-Fos. This peculiar staining was also observed as an initial step to nuclear localization after serum stimulation and in vivo in Xenopus embryos when the G1 phase is reintroduced in the embryonic cell cycle at the mid-blastula stage. We suggest that such staining could reflect specific structures involved in the initiation of the S phase.
Abstract: The consequences of denervation on the expression of c-myc protein have been analyzed on the regenerating forelimb of young froglets of Xenopus laevis. The level of c-myc expression, low in control limbs and enhanced in the regenerate, is transiently increased after a three-hour total denervation. For this protein, the level of expression is not a function of the quantity of nerve in the regenerate. Four days after denervation, c-myc signal is back to its base level observed in the regenerate. A different pattern of expression is obtained for an S phase marker (PCNA protein) taken as a control in the same experimental conditions. The data presented here show that the nervous system normally exerts a negative control on the expression of c-myc and PCNA proteins in the limb regenerate of Xenopus.
