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.

[Comparison of leakage at the implant to abutment connection in several implants types using a gas flow method]. / Fuites de la connexion implantaire : comparaison de plusieurs types d'implants par la méthode de diffusion gazeuse.

INTRODUCTION: The aim of this study was to compare the leakage at the implant to abutment connection in several implants, using a new gas diffusion method. MATERIAL AND METHODS: Sixty-eight implants of 13 different types were used. Nitrogen leaking was measured after screwing the connections to the torque levels recommended by the manufacturers. RESULTS: A significant tightness difference was observed between the different implant types. This difference cannot be explained by the various connection designs (flat, conical) or by the various torques recommended by the manufacturers. CONCLUSION: The authors suggest that the tightness difference between the various implant systems could be mainly associated with quality and precision of machining.

A 'DNA replication' signature of progression and negative outcome in colorectal cancer.

Colorectal cancer is one of the most frequent cancers worldwide. As the tumor-node-metastasis (TNM) staging classification does not allow to predict the survival of patients in many cases, additional prognostic factors are needed to better forecast their outcome. Genes involved in DNA replication may represent an underexplored source of such prognostic markers. Indeed, accidents during DNA replication can trigger 'replicative stress', one of the main features of cancer from earlier stages onward. In this study, we assessed the expression of 47 'DNA replication' genes in primary tumors and adjacent normal tissues from a homogeneous series of 74 patients. We found that genes coding for translesional (TLS) DNA polymerases, initiation of DNA replication, S-phase signaling and protection of replication forks were significantly deregulated in tumors. We also observed that the overexpression of either the MCM7 helicase or the TLS DNA polymerase POLQ (if also associated with a concomitant overexpression of firing genes) was significantly related to poor patient survival. Our data suggest the existence of a 'DNA replication signature' that might represent a source of new prognostic markers. Such a signature could help in understanding the molecular mechanisms underlying tumor progression in colorectal cancer patients.

DNA replication origins: from sequence specificity to epigenetics.

Site-specific initiation of DNA replication is a conserved function in all organisms. In Escherichia coli and Saccharomyces cerevisiae, DNA replication origins are sequence specific, but in multicellular organisms, origins are not so clearly defined. In this article, I present a model of origin specification by epigenetic mechanisms that allows the establishment of stable chromatin domains, which are characterized by autonomous replication. According to this model, origins of DNA replication help to establish domains of gene expression for the generation of cell diversity.

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.

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.

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.

Rearrangement of chromatin domains during development in Xenopus.

A dynamic change in the organization of different gene domains transcribed by RNA polymerase I, II, or III occurs during the progression from quiescent [pre-midblastula transition (pre-MBT)] to active (post-MBT) embryos during Xenopus development. In the rDNA, c-myc, and somatic 5S gene domains, a transition from random to specific anchorage to the nuclear matrix occurs when chromatin domains become active. The keratin gene domain was also randomly associated to the nuclear matrix before MBT, whereas a defined attachment site was found in keratinocytes. In agreement with this specification, ligation-mediated (LM)-PCR genomic footprinting carried out on the subpopulation of 5S domains specifically attached to the matrix reveals the hallmarks of determined chromatin after the midblastula transition. In contrast, the same analysis performed on the total 5S gene population does not reveal specific chromatin organization, validating the use of nuclear matrix fractionation to unveil active chromatin domains. These data provide a means for the determination of active chromosomal territories in the embryo and emphasize the role of nuclear architecture in regulated gene expression during development.

Specification of chromatin domains and regulation of replication and transcription during development.

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.

XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis.

In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins. Formation and maintenance of pre-replication complexes is dependent upon CDC6 (ref. 1), a protein which allows assembly of MCM2-7 proteins, which are putative replicative helicases. The functional assembly of MCM proteins into chromatin corresponds to replication licensing. Removal of these proteins from chromatin in S phase is crucial in origins firing regulation. We have identified a protein that is required for the assembly of pre-replication complexes, in a screen for maternally expressed genes in Xenopus. This factor (XCDT1) is a relative of fission yeast cdt1, a protein proposed to function in DNA replication, and is the first to be identified in vertebrates. Here we show, using Xenopus in vitro systems, that XCDT1 is required for chromosomal DNA replication. XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis. Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2-7 proteins onto pre-replicative chromatin. These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase.

Stepwise regulated chromatin assembly of MCM2-7 proteins.

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.

T-antigen interactions with chromatin and p53 during the cell cycle in extracts from xenopus eggs.

The role of SV40 large tumor T-antigen in replication of viral DNA is well established, but it is still unclear how T-antigen triggers cellular replication and cell transformation in non-permissive cells. Here, we used Xenopus egg extracts which reproduce most nuclear events linked to the cell cycle in vitro to analyze its interaction with genomic chromatin during the cell cycle. We show that T-antigen associates with chromatin before the nuclear membrane formation, and further demonstrate that the nuclear membrane is not necessary for its import into the nucleus. We show that the interaction of T-antigen with the endogenous chromatin does not occur at replication foci nor at RPA pre-replication centers. Immunoprecipitations as well as sucrose gradient experiments, indicate that the endogenous pool of p53 interacts with T-antigen. In addition, a transient association of both proteins with the nuclear matrix is observed during the ongoing DNA synthesis. These data are discussed in view of the T-antigen and p53 activity during the cell cycle.

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.

Nuclear import of p53 during Xenopus laevis early development in relation to DNA replication and DNA repair.

The role of p53 in transcriptional activation of genes involved in cell cycle progression is well established. However, the wide range of functions attributed to this gene suggests that some of them might be unrelated to transcription. Here we investigated p53 localization and recruitment to chromatin during Xenopus early development when 12 rapid cell cycles occur without transcription of the genome. We show that after fertilization, part of the large store of p53 previously stored in the cytoplasm of the oocyte is imported into the nucleus. This import was further analyzed in relation with DNA replication and DNA repair using cell-free systems from Xenopus eggs. Formation of a nuclear lamina envelope is necessary for the import of p53 into the nucleus. p53 associates both with decondensed DNA and the nuclear lamina envelope, but no colocalization with prereplication or replication complexes is observed. We show that UV- or gamma-damaged nuclei recruit p53 as well as replication protein A (RPA) in large common foci. Together, these data suggest that p53 plays a role in the regulation of the accelerated S phases that occur during Xenopus early development, in a manner that does not rely on its transcription-mediated activity.

Initiation of DNA replication in eukaryotes: questioning the origin.

Although proteins involved in DNA replication in yeast have counterparts in multicellular organisms, the definition of an origin of DNA replication and its control in higher eukaryotes might obey to different rules. Origins of DNA replication that are site-specific have been found, supporting the notion that specific DNA regions are used to initiate DNA synthesis along metazoan chromosomes. However, the notion that specific sequences will define origins is still being debated. The variety and complexity of transcriptional programs that have to be regulated in multicellular organisms may impose a plasticity that would not be compatible with a fixed origin simply defined at the sequence level. Such a plasticity would be essential to developmental programs where the control of DNA replication could be more integrated to the control of gene expression than in unicellular eukaryotes.

Characterization of Xenopus RalB and its involvement in F-actin control during early development.

We describe the characterization and a functional analysis in Xenopus development of RalB, a small G protein. RalB RNA and protein are detectable during oogenesis and early development, but the gene is expressed only weakly in adult tissues. The RalB transcripts are processed by poly(A) extension during oocyte maturation and up to the gastrulation stage. Microinjection of wild-type or mutant RalB RNAs was performed in fertilized eggs in order to gain insight into the function of RalB during development. We show that during cleavage stages the activated GTP form of RalB specifically induces a cortical reaction that affects the localization of pigment granules. The use of different drugs suggests that this reaction is dependent on the outer cortical actin array. The relation between F-actin and RalB was shown by confocal analysis. Injection of mRNAs encoding the mutated activated form of RalB leads, at dependent doses, to a blocking of gastrulation or defects in closing of neural folding structures. In contrast, the inactivated form blocks only the closing of neural tube. Altogether, these observations suggest that RalB is part of a regulatory pathway that may affect the blastomere cytoskeleton and take part in early development.

Evidence for different MCM subcomplexes with differential binding to chromatin in Xenopus.

MCM proteins are molecular components of the DNA replication licensing system in Xenopus. These proteins comprise a conserved family made up of six distinct members which have been found to associate in large protein complexes. We have used a combination of biochemical and cytological methods to study the association of soluble and chromatin-bound Xenopus MCM proteins during the cell cycle. In interphase, soluble MCM proteins are found organized in a core salt-resistant subcomplex that includes MCM subunits which are known to have high affinity for histones. The interphasic complex is modified at mitosis and the subunit composition of the resulting mitotic subcomplexes is distinct, indicating that the stability of the MCM complex is under cell cycle control. Moreover, we provide evidence that the binding of MCM proteins to chromatin may occur in sequential steps involving the loading of distinct MCM subunits. Comparative analysis of the chromatin distribution of MCM2, 3, and 4 shows that the binding of MCM4 is distinct from that of MCM2 and 3. Altogether, these data suggest that licensing of chromatin by MCMs occurs in an ordered fashion involving discrete subcomplexes.

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.

Control of gene expression in Xenopus early development.

We examine the control of gene expression before and through the MBT in Xenopus laevis. The generalized repression of transcription that occurs before the midblastula transition (MBT) is regulated by a dynamic competition between chromatin and transcription complex assembly. Conditions favoring the access of basal factors (TBP) or transactivators can overcome this transcriptional repression. Changes in DNase I hypersensitivity patterns of the chromatin during early development show that it is more accessible to DNase I before the MBT (and by extension to other DNA interacting proteins) than after the MBT. We also show that at the level of genomic domains, organization of the chromatin matrix attachment sites is random before MBT. We propose that these three components, chromatin domain structure, DNA accessibility, and the transcription complex-chromatin dynamic competition, combine to regulate transcription in the embryo before and through the MBT.

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.