Jean-Charles Cadoret

Bio: Jean-Charles Cadoret is an academic researcher from University of Paris. The author has contributed to research in topics: Replication timing & DNA replication. The author has an hindex of 5, co-authored 13 publications receiving 198 citations. Previous affiliations of Jean-Charles Cadoret include Paris Diderot University.

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

Abstract: Although 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.

Chromatin conformation regulates the coordination between DNA replication and transcription

Abstract: Chromatin is the template for the basic processes of replication and transcription, making the maintenance of chromosomal integrity critical for cell viability. To elucidate how dividing cells respond to alterations in chromatin structure, here we analyse the replication programme of primary cells with altered chromatin configuration caused by the genetic ablation of the HMGB1 gene, or three histone H1 genes. We find that loss of chromatin compaction in H1-depleted cells triggers the accumulation of stalled forks and DNA damage as a consequence of transcription-replication conflicts. In contrast, reductions in nucleosome occupancy due to the lack of HMGB1 cause faster fork progression without impacting the initiation landscape or fork stability. Thus, perturbations in chromatin integrity elicit a range of responses in the dynamics of DNA replication and transcription, with different consequences on replicative stress. These findings have broad implications for our understanding of how defects in chromatin structure contribute to genomic instability.

Histone H4K20 tri‐methylation at late‐firing origins ensures timely heterochromatin replication

Abstract: Among other targets, the protein lysine methyltransferase PR‐Set7 induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4‐20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K20 methylation to DNA replication remains unclear. Here, we show that H4K20 mutation in mammalian cells, unlike in Drosophila , partially impairs S‐phase progression and protects from DNA re‐replication induced by stabilization of PR‐Set7. Using Epstein–Barr virus‐derived episomes, we further demonstrate that conversion of H4K20me1 to higher H4K20me2/3 states by Suv4‐20h is not sufficient to define an efficient origin per se , but rather serves as an enhancer for MCM2‐7 helicase loading and replication activation at defined origins. Consistent with this, we find that Suv4‐20h‐mediated H4K20 tri‐methylation (H4K20me3) is required to sustain the licensing and activity of a subset of ORCA/LRWD1‐associated origins, which ensure proper replication timing of late‐replicating heterochromatin domains. Altogether, these results reveal Suv4‐20h‐mediated H4K20 tri‐methylation as a critical determinant in the selection of active replication initiation sites in heterochromatin regions of mammalian genomes.

A hypothesis-driven approach identifies CDK4 and CDK6 inhibitors as candidate drugs for treatments of adrenocortical carcinomas.

Abstract: High proliferation rate and high mutation density are both indicators of poor prognosis in adrenocortical carcinomas. We performed a hypothesis-driven association study between clinical features in adrenocortical carcinomas and the expression levels of 136 genes involved in DNA metabolism and G1/S phase transition. In 79 samples downloaded from The Cancer Genome Atlas portal, high Cyclin Dependent Kinase 6 (CDK6) mRNA levels gave the most significant association with shorter time to relapse and poorer survival of patients. A hierarchical clustering approach assembled most tumors with high levels of CDK6 mRNA into one group. These tumors tend to cumulate mutations activating the Wnt/β-catenin pathway and show reduced MIR506 expression. Actually, the level of MIR506 RNA is inversely correlated with the levels of both CDK6 and CTNNB1 (encoding β-catenin). Together these results indicate that high CDK6 expression is found in aggressive tumors with activated Wnt/β-catenin pathway. Thus we tested the impact of Food and Drug Administration-approved CDK4 and CDK6 inhibitors, namely palbociclib and ribociclib, on SW-13 and NCI-H295R cells. While both drugs reduced viability and induced senescence in SW-13 cells, only palbociclib was effective on the retinoblastoma protein (pRB)-negative NCI-H295R cells, by inducing apoptosis. In NCI-H295R cells, palbociclib induced an increase of the active form of Glycogen Synthase Kinase 3β (GSK3β) responsible for the reduced amount of active β-catenin, and altered the amount of AXIN2 mRNA. Taken together, these data underline the impact of CDK4 and CDK6 inhibitors in treating adrenocortical carcinomas.

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

Abstract: 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], [2], [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.

Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination

Abstract: The alternative non-homologous end-joining (NHEJ) machinery facilitates several genomic rearrangements, some of which can lead to cellular transformation. This error-prone repair pathway is triggered upon telomere de-protection to promote the formation of deleterious chromosome end-to-end fusions. Using next-generation sequencing technology, here we show that repair by alternative NHEJ yields non-TTAGGG nucleotide insertions at fusion breakpoints of dysfunctional telomeres. Investigating the enzymatic activity responsible for the random insertions enabled us to identify polymerase theta (Polθ; encoded by Polq in mice) as a crucial alternative NHEJ factor in mammalian cells. Polq inhibition suppresses alternative NHEJ at dysfunctional telomeres, and hinders chromosomal translocations at non-telomeric loci. In addition, we found that loss of Polq in mice results in increased rates of homology-directed repair, evident by recombination of dysfunctional telomeres and accumulation of RAD51 at double-stranded breaks. Lastly, we show that depletion of Polθ has a synergistic effect on cell survival in the absence of BRCA genes, suggesting that the inhibition of this mutagenic polymerase represents a valid therapeutic avenue for tumours carrying mutations in homology-directed repair genes.

Gene expression profiling reveals a new classification of adrenocortical tumors and identifies molecular predictors of malignancy and survival

Abstract: PURPOSE Adrenocortical tumors, especially cancers, remain challenging both for their diagnosis and prognosis assessment. The aim of this article is to identify molecular predictors of malignancy and of survival. PATIENTS AND METHODS One hundred fifty-three unilateral adrenocortical tumors were studied by microarray (n = 92) or reverse transcription quantitative polymerase chain reaction (n = 148). A two-gene predictor of malignancy was built using the disease-free survival as the end point in a training cohort (n = 47), then validated in an independent validation cohort (n = 104). The best candidate genes were selected using Cox models, and the best gene combination was validated using the log-rank test. Similarly, for malignant tumors, a two-gene predictor of survival was built using the overall survival as the end point in a training cohort (n = 23), then tested in an independent validation cohort (n = 35). RESULTS Unsupervised clustering analysis discriminated robustly the malignant and benign tumors, and identified two groups of malignant tumors with very different outcome. The combined expression of DLG7 and PINK1 was the best predictor of disease-free survival (log-rank P approximately 10(-12)), could overcome the uncertainties of intermediate pathological Weiss scores, and remained significant after adjustment to the Weiss score (P < 1.3 x 10(-2)). Among the malignant tumors, the combined expression of BUB1B and PINK1 was the best predictor of overall survival (P < 2 x 10(-6)), and remained significant after adjusting for MacFarlane staging (P < .005). CONCLUSION Gene expression analysis unravels two distinct groups of adrenocortical carcinomas. The molecular predictors of malignancy and of survival are reliable and provide valuable independent information in addition to pathology and tumor staging. These original tools should provide important improvements for adrenal tumors management.

Mechanism of suppression of chromosomal instability by DNA polymerase POLQ

Abstract: Although a defect in the DNA polymerase POLQ leads to ionizing radiation sensitivity in mammalian cells, the relevant enzymatic pathway has not been identified. Here we define the specific mechanism by which POLQ restricts harmful DNA instability. Our experiments show that Polq-null murine cells are selectively hypersensitive to DNA strand breaking agents, and that damage resistance requires the DNA polymerase activity of POLQ. Using a DNA break end joining assay in cells, we monitored repair of DNA ends with long 3' single-stranded overhangs. End joining events retaining much of the overhang were dependent on POLQ, and independent of Ku70. To analyze the repair function in more detail, we examined immunoglobulin class switch joining between DNA segments in antibody genes. POLQ participates in end joining of a DNA break during immunoglobulin class-switching, producing insertions of base pairs at the joins with homology to IgH switch-region sequences. Biochemical experiments with purified human POLQ protein revealed the mechanism generating the insertions during DNA end joining, relying on the unique ability of POLQ to extend DNA from minimally paired primers. DNA breaks at the IgH locus can sometimes join with breaks in Myc, creating a chromosome translocation. We found a marked increase in Myc/IgH translocations in Polq-defective mice, showing that POLQ suppresses genomic instability and genome rearrangements originating at DNA double-strand breaks. This work clearly defines a role and mechanism for mammalian POLQ in an alternative end joining pathway that suppresses the formation of chromosomal translocations. Our findings depart from the prevailing view that alternative end joining processes are generically translocation-prone.

The impact of replication stress on replication dynamics and DNA damage in vertebrate cells.

Abstract: The interplay between replication stress and the S phase checkpoint is a key determinant of genome maintenance, and has a major impact on human diseases, notably, tumour initiation and progression. Recent studies have yielded insights into sequence-dependent and sequence-independent sources of endogenous replication stress. These stresses result in nuclease-induced DNA damage, checkpoint activation and genome-wide replication fork slowing. Several hypotheses have been proposed to account for the mechanisms involved in this complex response. Recent results have shown that the slowing of the replication forks most commonly results from DNA precursor starvation. By concomitantly increasing the density of replication initiation, the cell elicits an efficient compensatory strategy to avoid mitotic anomalies and the inheritance of damage over cell generations.

Control of DNA replication timing in the 3D genome.

Abstract: The 3D organization of mammalian chromatin was described more than 30 years ago by visualizing sites of DNA synthesis at different times during the S phase of the cell cycle. These early cytogenetic studies revealed structurally stable chromosome domains organized into subnuclear compartments. Active-gene-rich domains in the nuclear interior replicate early, whereas more condensed chromatin domains that are largely at the nuclear and nucleolar periphery replicate later. During the past decade, this spatiotemporal DNA replication programme has been mapped along the genome and found to correlate with epigenetic marks, transcriptional activity and features of 3D genome architecture such as chromosome compartments and topologically associated domains. But the causal relationship between these features and DNA replication timing and the regulatory mechanisms involved have remained an enigma. The recent identification of cis-acting elements regulating the replication time and 3D architecture of individual replication domains and of long non-coding RNAs that coordinate whole chromosome replication provide insights into such mechanisms. Different genomic regions are replicated at different times during the S phase of the cell cycle, forming early- and late-replicating domains that occupy different locations in the nucleus. The recent identification of specific DNA sequences and long non-coding RNAs that regulate DNA replication timing is providing key insights into the roles of replication timing and into timing and 3D organization.