Genome-wide approaches to determining origin distribution.

International audienceGenome integrity depends upon a highly co-ordinated process that ensures the exact duplication of the genome at each cell cycle. Genomic mapping of DNA replication starting points in mammals, known as origins of replication, is an important step towards our understanding of how this essential mechanism is regulated throughout complex genomes. Two recent studies carried out in both human and mouse cells have revealed a strong association between replication origins and transcriptional regulatory elements. This strong overlap raises the question of how gene deserts, also lacking replication origins, are properly replicated in conditions where replication is disrupted. It also provides valuable information forward the identification of key regulatory factors of DNA replication initiation. Here, we review what these large-scale mappings of replication origins have brought to our understanding of replication initiation and what are the future prospects

Impact of the DNA polymerase Theta on the DNA replication program

The physiological function of the human DNA polymerase θ (pol θ) is still unclear despite its in vitro translesion synthesis capacity during DNA damage repair process. However this DNA polymerase is always present along the cell cycle in the absence of replication stress and DNA damage. Is there a different molecular function? We present the genomic data of replication timing in depleted pol θ cells (GSE49693) and in cells overexpressing pol θ (GSE53070) indicating that Pol θ holds a novel role in the absence of external stress as a critical determinant of the replication timing program in human cells

Replication Stress, Genomic Instability, and Replication Timing: A Complex Relationship

The replication-timing program constitutes a key element of the organization and coordination of numerous nuclear processes in eukaryotes. This program is established at a crucial moment in the cell cycle and occurs simultaneously with the organization of the genome, thus indicating the vital significance of this process. With recent technological achievements of high-throughput approaches, a very strong link has been confirmed between replication timing, transcriptional activity, the epigenetic and mutational landscape, and the 3D organization of the genome. There is also a clear relationship between replication stress, replication timing, and genomic instability, but the extent to which they are mutually linked to each other is unclear. Recent evidence has shown that replication timing is affected in cancer cells, although the cause and consequence of this effect remain unknown. However, in-depth studies remain to be performed to characterize the molecular mechanisms of replication-timing regulation and clearly identify different cis- and trans-acting factors. The results of these studies will potentially facilitate the discovery of new therapeutic pathways, particularly for personalized medicine, or new biomarkers. This review focuses on the complex relationship between replication timing, replication stress, and genomic instability

The relationship between DNA replication and human genome organization.

International audienceAssessment of the impact of DNA replication on genome architecture in Eukaryotes has long been hampered by the scarcity of experimental data. Recent work, relying on computational predictions of origins of replication, suggested that replication might be a major determinant of gene organization in human (Huvet et al., 2007). Here, we address this question by analyzing the first large-scale dataset of experimentally determined origins of replication in human: 283 origins identified in HeLa cells, in 1% of the genome covered by ENCODE regions (Cadoret et al., 2008). We show that origins of replication are not randomly distributed, as they display significant overlap with promoter regions and CpG islands. The hypothesis of a selective pressure to avoid frontal collisions between replication and transcription polymerases is not supported by experimental data, as we find no evidence for gene orientation bias in the proximity of origins of replication. The lack of a significant orientation bias remains manifest even when considering only genes expressed at a high rate, or in a wide number of tissues, and is not affected by the regional replication timing. Gene expression breadth does not appear to be correlated with the distance from the origins of replication. We conclude that the impact of DNA replication on human genome organization is considerably weaker than previously proposed

Characterization of the replication timing program of 6 human model cell lines

During the S-phase, the DNA replication process is finely orchestrated and regulated by two programs: the spatial program that determines where replication will start in the genome (Cadoret et al. (2008 Oct 14), Cayrou et al. (2011 Sep), Picard et al. (2014 May 1) [1–3]), and the temporal program that determines when during the S phase different parts of the genome are replicated and when origins are activated. The temporal program is so well conserved for each cell type from independent individuals [4] that it is possible to identify a cell type from an unknown sample just by determining its replication timing program. Moreover, replicative domains are strongly correlated with the partition of the genome into topological domains (determined by the Hi-C method, Lieberman-Aiden et al. (2009 Oct 9), Pope et al. (2014 Nov 20) [5,6]). On the one hand, replicative areas are well defined and participate in shaping the spatial organization of the genome for a given cell type. On the other hand, studies on the timing program during cell differentiation showed a certain plasticity of this program according to the stage of cell differentiation Hiratani et al. (2008 Oct 7, 2010 Feb) [7,8]. Domains where a replication timing change was observed went through a nuclear re-localization. Thus the temporal program of replication can be considered as an epigenetic mark Hiratani and Gilbert (2009 Feb 16) [9]. We present the genomic data of replication timing in 6 human model cell lines: U2OS (GSM2111308), RKO (GSM2111309), HEK 293T (GSM2111310), HeLa (GSM2111311), MRC5-SV (GSM2111312) and K562 (GSM2111313). A short comparative analysis was performed that allowed us to define regions common to the 6 cell lines. These replication timing data can be taken into account when performing studies that use these model cell lines

The contribution of culturomics to the repertoire of isolated human bacterial and archaeal species

Abstract After a decade of research and metagenomic analyses, our knowledge of the human microbiota appears to have reached a plateau despite promising results. In many studies, culture has proven to be essential in describing new prokaryotic species and filling metagenomic gaps. In 2015, only 2172 different prokaryotic species were reported to have been isolated at least once from the human body as pathogens or commensals. In this review, we update the previous repertoire by reporting the different species isolated from the human body to date, increasing it by 28% to reach a total of 2776 species associated with human beings. They have been classified into 11 different phyla, mostly the Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. Finally, culturomics contributed up to 66.2% towards updating this repertoire by reporting 400 species, of which 288 were novel. This demonstrates the need to continue the culturing work, which seems essential in order to decipher the hidden human microbial content

Dissipation of excess energy triggered by blue light in cyanobacteria with CP43' (isiA)

International audienceThe chlorophyll-protein CP43V(isiA gene) induced by stress conditions in cyanobacteria is shown to serve as an antenna for Photosystem II (PSII), in addition to its known role as an antenna for Photosystem I (PSI). At high light intensity, this antenna is converted to an efficient trap for chlorophyll excitations that protects system II from photo-inhibition. In contrast to the denergy-dependent non-photochemical quenchingT (NPQ) in chloroplasts, this photoprotective energy dissipation in cyanobacteria is triggered by blue light. The induction is proportional to light intensity. Induction and decay of the quenching exhibit the same large temperature-dependence

Progerin impairs 3D genome organization and induces fragile telomeres by limiting the dNTP pools

International audienceChromatin organization within the nuclear volume is essential to regulate many aspects of its function and to safeguard its integrity. A key player in this spatial scattering of chromosomes is the nuclear envelope (NE). The NE tethers large chromatin domains through interaction with the nuclear lamina and other associated proteins. This organization is perturbed in cells from Hutchinson–Gilford progeria syndrome (HGPS), a genetic disorder characterized by premature aging features. Here, we show that HGPS-related lamina defects trigger an altered 3D telomere organization with increased contact sites between telomeres and the nuclear lamina, and an altered telomeric chromatin state. The genome-wide replication timing signature of these cells is perturbed, with a shift to earlier replication for regions that normally replicate late. As a consequence, we detected a higher density of replication forks traveling simultaneously on DNA fibers, which relies on limiting cellular dNTP pools to support processive DNA synthesis. Remarkably, increasing dNTP levels in HGPS cells rescued fragile telomeres, and improved the replicative capacity of the cells. Our work highlights a functional connection between NE dysfunction and telomere homeostasis in the context of premature aging

A role for DNA polymerase θ in the timing of DNA replication.

International audienceAlthough DNA polymerase θ (Pol θ) is known to carry out translesion synthesis and has been implicated in DNA repair, its physiological function under normal growth conditions remains unclear. Here we present evidence that Pol θ plays a role in determining the timing of replication in human cells. We find that Pol θ binds to chromatin during early G1, interacts with the Orc2 and Orc4 components of the Origin recognition complex and that the association of Mcm proteins with chromatin is enhanced in G1 when Pol θ is downregulated. Pol θ-depleted cells exhibit a normal density of activated origins in S phase, but early-to-late and late-to-early shifts are observed at a number of replication domains. Pol θ overexpression, on the other hand, causes delayed replication. Our results therefore suggest that Pol θ functions during the earliest steps of DNA replication and influences the timing of replication initiation

Low Replicative Stress Triggers Cell-Type Specific Inheritable Advanced Replication Timing

International audienceDNA replication timing (RT), reflecting the temporal order of origin activation, is known as a robust and conserved cell-type specific process. Upon low replication stress, the slowing of replication forks induces well-documented RT delays associated to genetic instability, but it can also generate RT advances that are still uncharacterized. In order to characterize these advanced initiation events, we monitored the whole genome RT from six independent human cell lines treated with low doses of aphidicolin. We report that RT advances are cell-type-specific and involve large heterochromatin domains. Importantly, we found that some major late to early RT advances can be inherited by the unstressed next-cellular generation, which is a unique process that correlates with enhanced chromatin accessibility, as well as modified replication origin landscape and gene expression in daughter cells. Collectively, this work highlights how low replication stress may impact cellular identity by RT advances events at a subset of chromosomal domains