Chromatin acts as a key regulator of DNA-related processes such as DNA damage repair. Although ChIP-chip is a powerful technique to provide high-resolution maps of protein-genome interactions, its use to study DNA double strand break (DSB) repair has been hindered by the limitations of the available damage induction methods. We have developed a human cell line that permits induction of multiple DSBs randomly distributed and unambiguously positioned within the genome. Using this system, we have generated the first genome-wide mapping of gammaH2AX around DSBs. We found that all DSBs trigger large gammaH2AX domains, which spread out from the DSB in a bidirectional, discontinuous and not necessarily symmetrical manner. The distribution of gammaH2AX within domains is influenced by gene transcription, as parallel mappings of RNA Polymerase II and strand-specific expression showed that gammaH2AX does not propagate on active genes. In addition, we showed that transcription is accurately maintained within gammaH2AX domains, indicating that mechanisms may exist to protect gene transcription from gammaH2AX spreading and from the chromatin rearrangements induced by DSBs.

Chromatin undergoes major remodeling around DNA double-strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high-resolution map of γH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit of the cohesin complex, previously characterized as required for repair by homologous recombination. We found that recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs in human cells. In addition, we show that cohesin controls γH2AX distribution within domains. Indeed, as we reported previously for transcription, cohesin binding antagonizes γH2AX spreading. Remarkably, depletion of cohesin leads to an increase of γH2AX at cohesin-bound genes, associated with a decrease in their expression level after DSB induction. We propose that, in agreement with their function in chromosome architecture, cohesin could also help to isolate active genes from some chromatin remodelling and modifications such as the ones that occur when a DSB is detected on the genome.

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN homologue exhibit many phenotypic features of WS, including a prooxidant status and a shorter mean life span compared to wild-type animals. Here, we show that Wrn mutant mice also develop premature liver sinusoidal endothelial defenestration along with inflammation and metabolic syndrome. Vitamin C supplementation rescued the shorter mean life span of Wrn mutant mice and reversed several age-related abnormalities in adipose tissues and liver endothelial defenestration, genomic integrity, and inflammatory status. At the molecular level, phosphorylation of age-related stress markers like Akt kinase-specific substrates and the transcription factor NF-κB, as well as protein kinase Cδ and Hif-1α transcription factor levels, which are increased in the liver of Wrn mutants, were normalized by vitamin C. Vitamin C also increased the transcriptional regulator of lipid metabolism PPARα. Finally, microarray and gene set enrichment analyses on liver tissues revealed that vitamin C decreased genes normally up-regulated in human WS fibroblasts and cancers, and it increased genes involved in tissue injury response and adipocyte dedifferentiation in obese mice. Vitamin C did not have such effect on wild-type mice. These results indicate that vitamin C supplementation could be beneficial for patients with WS.

No abstract

No abstract

Werner syndrome (WS) is a rare disorder characterized by the premature onset of a number of age-related diseases. The gene responsible for WS is believed to be involved in different aspects of transcription, replication and/or DNA repair. The poly(ADP-ribose) polymerase-1 (PARP-1) enzyme is also involved in DNA repair and is known to affect transcription of several genes. In this study, we examined the expression profile of cells lacking the normal function of either or both enzymes. All mutant cells exhibited altered expression of genes normally responding to oxidative stress. Interestingly, more than 58% of misregulated genes identified in double mutant cells were not altered in cells with either the Wrn or PARP-1 mutation alone. So, the impact on gene expression profile when both Wrn and PARP-1 are mutated was greater than a simple addition of individual mutant genotype. In addition, double mutant cultured cells showed major misregulation of genes involved in apoptosis, cell cycle control, embryonic development, metabolism and signal transduction. More importantly, in vivo analyses of double mutant mice have confirmed the increased apoptosis and the developmental defects in embryos as well as the major increase in intracellular phosphorylation and oxidative DNA damage in adult tissues. They also exhibited a progressive increase in oxidative stress with age. Thus, a major result of this study is that changes in expression of several genes and physiological functions identified in vitro were confirmed in mouse embryonic and adult tissues.

Vertebrate Hox genes act as developmental architects by patterning embryonic structures like axial skeletal elements, limbs, brainstem territories, or neural crest derivatives. While active during the patterning steps of development, these genes turn out to be down-regulated in specific differentiation programs like that leading to chondrogenesis. To investigate why chondrocyte differentiation is correlated to the silencing of a Hox gene, we generated transgenic mice allowing Cre-mediated conditional misexpression of Hoxa2 and induced this gene in Collagen 2 alpha 1-expressing cells committed to enter chondrogenesis. Persistent Hoxa2 expression in chondrogenic cells resulted in overall chondrodysplasia with delayed cartilage hypertrophy, mineralization, and ossification but without proliferation defects. The absence of skeletal patterning anomaly and the regular migration of precursor cells indicated that the condensation step of chondrogenesis was normal. In contrast, closer examination at the differentiation step showed severely impaired chondrocyte differentiation. In addition, this inhibition affected structures independently of their embryonic origin. In conclusion, for the first time here, by a cell-type specific misexpression, we precisely uncoupled the patterning function of Hoxa2 from its involvement in regulating differentiation programs per se and demonstrate that Hoxa2 displays an anti-chondrogenic activity that is distinct from its patterning function.

SummaryWerner syndrome is a rare disorder characterized by genome instability and the premature onset of several pathologies associated with aging. The gene responsible for Werner syndrome codes for a RecQ-type DNA helicase and is believed to be involved in different aspects of DNA repair, replication, and transcription. The human Werner protein (WRN) translocates from nucleoli to the nucleoplasm upon DNA damage. Here, for the first time we show WRN translocation following treatment with chloroquine (CHL) or trichostatin A (TSA), agents that alter chromatin structure without producing DNA breaks. In contrast to normal cells, WRN deficient human and murine cells incurred extensive DNA breaks upon CHL or TSA treatment, indicating a functional role for WRN in the proper response to these agents. Cells deficient for another RecQ-type helicase, Bloom syndrome, were not sensitive to these agents. WRN is known from in vitro studies to bind and stimulate the activity of topoisomerase I (TopoI). CHL enhanced the association between WRN and TopoI, suggesting that topological stress elicits a requirement for the stimulation of TopoI by WRN. Supporting this idea, overexpression of TopoI reduced CHL and TSA-induced DNA breaks in WRN null cells. We thus describe a novel function for WRN in ensuring genome stability to act in concert with TopoI to prevent DNA breaks, following alterations in chromatin topology.