Developmental determinants in non-communicable chronic diseases and ageing.

Prenatal and peri-natal events play a fundamental role in health, development of diseases and ageing (Developmental Origins of Health and Disease (DOHaD)). Research on the determinants of active and healthy ageing is a priority to: (i) inform strategies for reducing societal and individual costs of an ageing population and (ii) develop effective novel prevention strategies. It is important to compare the trajectories of respiratory diseases with those of other chronic diseases.

Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin.

Eukaryotic replication origins are 'licensed' for replication early in the cell cycle by loading Mcm(2-7) proteins. As chromatin replicates, Mcm(2-7) are removed, thus preventing the origin from firing again. Here we report the purification of the RLF-B component of the licensing system and show that it corresponds to Cdt1. RLF-B/Cdt1 was inhibited by geminin, a protein that is degraded during late mitosis. Immunodepletion of geminin from metaphase extracts allowed them to assemble licensed replication origins. Inhibition of CDKs in metaphase stimulated origin assembly only after the depletion of geminin. These experiments suggest that geminin-mediated inhibition of RLF-B/Cdt1 is essential for repressing origin assembly late in the cell cycle of higher eukaryotes.

Dynamics of the genome during early Xenopus laevis development: karyomeres as independent units of replication.

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.

Transition in specification of embryonic metazoan DNA replication origins.

In early Xenopus embryos, in which ribosomal RNA genes (rDNA) are not transcribed, rDNA replication initiates and terminates at 9- to 12-kilobase pair intervals, with no detectable dependence on specific DNA sequences. Resumption of ribosomal RNA (rRNA) synthesis at late blastula and early gastrula is accompanied by a specific repression of replication initiation within transcription units; the frequency of initiation within intergenic spacers remains as high as in early blastula. These results demonstrate that for rRNA genes, circumscribed zones of replication initiation emerge in intergenic DNA during the time in metazoan development when the chromatin is remodeled to allow gene transcription.

Transcription complex disruption caused by a transition in chromatin structure.

Chromatin structure is known to influence class III gene expression in vitro. We describe the active transcription of Xenopus class III genes following replication and assembly into chromatin by using Xenopus egg extracts. Changes in the structure of this active chromatin dependent on the presence of exogeneous Mg2+ ATP or on the addition of a mixture of histones H2A and H2B are shown to lead to the selective repression of Xenopus 5S RNA genes. Preexisting transcription complexes on 5S DNA are disrupted following the reorganization of a "disordered" histone-DNA complex into a structure consisting of physiologically spaced nucleosomes. Thus, we demonstrate that chromatin structural transitions can have dominant and specific effects on transcription.

Regulated formation of extrachromosomal circular DNA molecules during development in Xenopus laevis.

Extrachromosomal circular DNA molecules of chromosomal origin have been detected in many organisms and are thought to reflect genomic plasticity in eukaryotic cells. Here we report a developmentally regulated formation of extrachromosomal circular DNA that occurs de novo in preblastula Xenopus embryos. This specific DNA population is not detected in the male or female germ cells and is dramatically reduced in later developmental stages and in adult tissues. The activity responsible for the de novo production of extrachromosomal circles is maternally inherited, is stored in the unfertilized egg, and requires genomic DNA as a template. The formation of circular molecules does not require genomic DNA replication but both processes can occur simultaneously in the early development. The production of extrachromosomal circular DNA does not proceed at random since multimers of the tandemly repeated sequence satellite 1 were over-represented in the circle population, while other sequences (such as ribosomal DNA and JCC31 repeated sequence) were not detected. This phenomenon reveals an unexpected plasticity of the embryonic genome which is restricted to the early developmental stage.

Selective and rapid nuclear translocation of a c-Myc-containing complex after fertilization of Xenopus laevis eggs.

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.

Translocation of a store of maternal cytoplasmic c-myc protein into nuclei during early development.

The c-myc proto-oncogene is expressed as a maternal protein during oogenesis in Xenopus laevis, namely, in nondividing cells. A delayed translation of c-myc mRNA accumulated in early oocytes results in the accumulation of the protein during late oogenesis. The oocyte c-myc protein is unusually stable and is located in the cytoplasm, contrasting with its features in somatic cells. A mature oocyte contains a maternal c-myc protein stockpile of 4 x 10(5) to 6 x 10(5) times the level in a somatic growing cell. This level of c-myc protein is preserved only during the cleavage stage of the embryo. Fertilization triggers its rapid migration into the nuclei of the cleaving embryo and a change in the phosphorylation state of the protein. The c-myc protein content per nucleus decreases exponentially during the cleavage stage until a stoichiometric titration by the embryonic nuclei is reached during a 0.5-h period at the midblastula stage. Most of the maternal c-myc store is degraded by the gastrula stage. These observations implicate the participation of c-myc in the events linked to early embryonic development and the midblastula transition.

Comparative analysis of the intracellular localization of c-Myc, c-Fos, and replicative proteins during cell cycle progression.

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.

A novel cell-free system reveals a mechanism of circular DNA formation from tandem repeats.

One characteristic of genomic plasticity is the presence of extrachromosomal circular DNA (eccDNA). High levels of eccDNA are associated with genomic instability, exposure to carcinogens and aging. We have recently reported developmentally regulated formation of eccDNA that occurs preferentially in pre-blastula Xenopus laevis embryos. Multimers of tandemly repeated sequences were over-represented in the circle population while dispersed sequences were not detected, indicating that circles were not formed at random from any chromosomal sequence. Here we present detailed mechanistic studies of eccDNA formation in a cell-free system derived from Xenopus egg extracts. We show that naked chromosomal DNA from sperm or somatic tissues serves as a substrate for direct tandem repeat circle formation. Moreover, a recombinant bacterial tandem repeat can generate eccDNA in the extract through a de novo mechanism which is independent of DNA replication. These data suggest that the presence of a high level of any direct tandem repeat can confer on DNA the ability to be converted into circular multimers in the early embryo irrespective of its sequence and that homologous recombination is involved in this process.

Chromatin remodelling and DNA replication: from nucleosomes to loop domains.

Organization of DNA into chromatin is likely to participate in the control of the timing and selection of DNA replication origins. Reorganization of the chromatin is carried out by chromatin remodelling machines, which may affect the choice of replication origins and efficiency of replication. Replication itself causes a profound rearrangement in the chromatin structure, from nucleosomes to DNA loop domains, allowing to retain or switch an epigenetic state. The present review considers the effects of chromatin remodelling on replication and vice versa.

Analysis of c-Myc and Max binding to the c-myc promoter: evidence that autosuppression occurs via an indirect mechanism.

c-myc negatively autoregulates its expression at the level of transcriptional initiation, although the precise mechanism remains debated. While conclusive evidence for c-Myc binding in its own promoter has not been found, it has been proposed that c-Myc binds to a site upstream of the human c-myc gene which may also be a component of a replication origin (Ariga et al., 1989). In an attempt to clarify this issue, sequences flanking the c-myc gene were screened for c-Myc or Max binding sites using a novel procedure to facilitate the detection of DNA binding dependent upon long distance interactions or DNA secondary structure. Since the sequence specificity of DNA binding proteins may also be mediated by interaction with other proteins or by protein modification, this affinity capture assay was used in conjunction with nuclear extracts, potentially allowing the selection of novel in vivo DNA binding specificities. Using conditions that gave strong binding to an internal control sequence, c-Myc or Max binding elements were not detected in genomic sequences extending 5.4 kb upstream of the Xenopus c-myc gene. Identical results were obtained using both purified proteins and a variety of nuclear extracts, suggesting c-myc autosuppression most likely involves an indirect pathway.

Cloning and characterization of a cDNA from Xenopus laevis coding for a protein homologous to human and murine p53.

A Xenopus laevis oocyte cDNA library was screened with a murine p53 cDNA probe for the presence of p53-related clones. Several such clones were isolated and analysed. The nucleotide sequence of the largest cDNA clone (2.2 kb) showed a high degree of homology with the human (68%) and murine (70%) p53 coding sequences. This clone contains a single large open-reading frame, coding for a protein of 363 amino acids, which is 51% homologous to human p53 and 57% homologous to murine p53. Furthermore, five highly conserved internal regions were found in all three proteins. The three proteins have a highly similar amino acid composition (including, notably, the presence of a high proportion of proline residues), and they display a comparable distribution of charged amino acids and hydropathic index profile. The in vitro transcription-translation products of the X. laevis clone were successfully immunoprecipitated by human anti-p53 sera, demonstrating that there is at least one epitope in common between the X. laevis protein and human, and possibly murine, p53.

Detection of proto-oncogenes in the genome of the amphibian Xenopus laevis.

The Xenopus laevis genome was probed by Southern Blot analysis for the presence of sequences homologous to mammalian or avian proto-oncogenes. Hybridization conditions were strictly defined with a known proto-oncogene to detect a positive signal with DNA sequences having at least 60 to 64% homology. In such conditions thirteen genes representing different oncogene families exhibited positive hybridizations with specific DNA restriction fragments. Members of the protein kinase oncogene family were detected including abl, erbB, fes, fms, ros, raf and mos. Ets, rel, and the steroid hormone related receptor erbA also gave positive signals with specific Xenopus DNA fragments. Proto-oncogenes raf and the ras family, N-ras, H-ras and c-ral, gave the strongest hybridizations and the signals remained positive in high stringency wash conditions. This study confirms the relative conservation of these genes during evolution and opens the possibility of studying their role in one of the best characterized systems of embryonic development.

A functional analysis of p53 during early development of Xenopus laevis.

p53 is a nuclear protein that acts like a tumor suppressor and is involved in regulation of cellular growth. In Xenopus, the p53 protein is highly expressed during oogenesis and is strictly cytoplasmic in the oocyte. We have analysed its participation in DNA replication and transcription during early development, using the egg and oocyte as model-systems. The injection of sperm nuclei into Xenopus eggs is followed by DNA replication and mitotic events. We show that the endogenous p53 enters the nuclei and moves through a series of discrete sub-nuclear loci whose distribution is S-phase specific. A specific peripheral nuclear localization of p53 is observed before entry into S-phase, followed by an internal localization which is strictly dependent on ongoing DNA synthesis. At no stage in the cell cycle, however, did we observe any co-localization with RPA or PCNA, which were used as initiation or elongation markers for DNA replication. We also show that injection into the nucleus of the oocyte of small amounts of either Xenopus or human p53 - less than 10% of the cytoplasmic storage - is sufficient to block RNA polymerase II-dependent transcription from a coinjected TATA-box-containing reporter plasmid. Transcription is rescued by microinjection of the TATA-box binding protein (TBP), suggesting that nuclear exclusion of p53 during oogenesis may be necessary for transcription of maternal genes. These characteristics are discussed in relation to the regulation of nuclear activities during early embryogenesis.

Conservation of structural domains and biochemical activities of the MDM2 protein from Xenopus laevis.

The Mdm2 gene is the best known cellular regulator of p53 tumor suppressor activity. We report here the cloning and characterization of Xdm2, its homolog in Xenopus laevis. Human, mouse and Xenopus MDM2 proteins are more than 65% identical in several regions which are likely to be important for the biological activities of MDM2. Region I is sufficient for binding p53 and inhibiting its G1 arrest and apoptosis functions. Region II contains most of a central acidic region required for interaction with the L5 ribosomal protein and a putative C4 zinc finger. Region III is nearly identical from Xenopus to human and comprises the RING finger domain. We show that this structural conservation is associated with the conservation of three biochemical activities of MDM2; binding to the p53 and L5 proteins and specifically to RNA. Lastly, Xdm2 expression during early development is mainly restricted from the oocyte stage I/II to the blastula stage and is possibly independent of transcriptional activation by p53. These data as well as the utilization of Xenopus laevis to investigate the roles of MDM2 and p53 during early embryogenesis are discussed.

Xenopus egg extracts: a model system for chromatin replication.

A cell-free system derived from Xenopus eggs enables in vitro reproduction of the steps occurring during eukaryotic DNA replication. With a circular single-stranded DNA template, extracts obtained from high-speed centrifugation perform complementary DNA strand synthesis coupled to chromatin assembly. Nucleosomes are formed on the newly replicated DNA and the overall reaction mimics the events occurring during chromosomal replication on the lagging strand at the replication fork. ATP is necessary at all steps examined individually, including RNA priming, elongation of DNA strands and chromatin assembly. Although not required for nucleosome formation, ATP is involved in the correct spacing of nucleosomes and the stability of the assembled chromatin. Replication of double-stranded DNA was observed only with extracts obtained from low-speed centrifugation using demembraned sperm nuclei as substrate. Nuclei are reconstituted around the DNA and then undergo a series of events characteristic of a cell cycle. In contrast, neither DNA elongation or chromatin assembly require formation of the nucleus, and both are independent of the cell cycle.

DNA polymerase-alpha from regenerating rat liver. Catalytic properties of the highly purified enzyme.

DNA polymerase-alpha from the cytosol of regenerating rat liver has been highly purified by a procedure which includes affinity chromatography. The purified enzyme sediments at 7.4 S in high ionic strength and at 9--10 S in low ionic strength, i.e. under in vitro polymerization conditions. This enzyme has all the properties of the other mammalian DNA polymerases-alpha: sensitivity to sulfhydryl-blocking agents, to heparin, and to the level of salt in the assay, neutral pH optimum, use of ribonucleotide-initiated DNA templates, and inability to copy the ribostrand of hybrids. After chromatography on denatured DNA-cellulose, the alpha-polymerase is completely devoid of exo- and endonuclease activities. Template competition experiments indicate that the binding of the enzyme to the template can be distinguished from the polymerization itself and that the in vitro synthesis catalyzed by this alpha-polymerase is not distributive in a classical sense. These facts are discussed.

Nerve-dependent expression of c-myc protein during forelimb regeneration of Xenopus laevis froglets.

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.

Genes and mechanisms involved in early embryonic development in Xenopus laevis.

Our laboratory is studying genes involved in the regulation of the balance between cell growth and differentiation during embryonic development in Xenopus. We have analyzed the developmental expression of the proto-oncogenes c-myc, and KiRas 2B, the proliferating cell nuclear antigen (PCNA), and the tumor suppressor gene p53. These genes, usually expressed during cell proliferation, are expressed in the oocyte in large quantities, but the majority of their maternal RNAs are degraded by the gastrula stage. The expression of c-myc and the localization of the protein indicate that c-myc has the characteristics expected for a gene involved in the regulation of the mid-blastula transition, when zygotic expression is turned on in the embryo. Its expression during late development or during regeneration indicates that it enables the cells to remain competent for cycling during organogenesis. In vitro systems that reproduce the principal cellular functions during early development are used as model systems to understand the mechanisms involved in early embryogenesis.