Dynamique de la chromatine silencieuse sur différents états métaboliques

The tri-dimensional organization of the genome emerges as an important, still poorly understood, control mechanism in genomic function. Studies in S. cerevisiae have broadly contributed to demonstrate the functional importance of nuclear organization. Upon logaritmic growth, the 16 chromosomes of a S. cerevisiae haploid nucleus are organized into the Rabl conformation, with centromeres bound at the SPB and telomeres grouped in 3-4 foci localized at the nuclear periphery. Telomere clusters allow the concentration of silencing proteins (SIRs) and appear important for genome functions. The aim of my doctorate work was to study telomeric silent chromatin upon major metabolic transitions. We found that the genome of long-lived quiescent cells undergoes a major spatial re-organization following carbon source exhaustion. This change in nuclear architecture is driven by the grouping of telomeres into a unique focus (hypercluster) localized in the center of the nucleus. We also show that this reorganization is a programmed event triggered by reactive oxigen species (ROS) produced upon early respiration and involves the DNA damage checkpoint pathway. Finally, we report that excess of Sir2 activity counteracts telomere clustering upon quiescence and has a negative role on chronological life span. Our work suggests that the drastic genome reorganization due to telomere grouping favors survival upon quiescence, and unravels a novel connection between metabolism, nuclear organization and aging.L'organisation tridimensionnelle du génome émerge comme un mécanisme de contrôle important dans la fonction génomique. Les études chez S. cerevisiae ont largement contribué à démontrer l'importance fonctionnelle de l'organisation nucléaire. Pendant la fermentation, les 16 chromosomes d'un noyau haploïde de S. cerevisiae sont organisées avec centromères liés à la SPB et télomères regroupés en 3-4 foyers localisés à la périphérie nucléaire. Cet organisation permet la concentration de protéines silencieuse (SIRS) et semble importants pour les fonctions du génome. Le but de mon travail de doctorat était d'étudier la chromatine télomérique silencieuse dans different transitions métaboliques.Nous avons constaté que le génome de cellules quiescente subit une réorganisation spatiale majeure suite à la source de carbone épuisement. Cette modification de l'architecture nucléaire est entraîné par le regroupement des télomères en un foyer unique (hypercluster) localisée au centre du noyau. Nous montrons également que cette réorganisation est un événement programmé déclenché par les espèces réactives de oxigen (ROS) produits lors de la respiration. Enfin, nous déclarons que l'excès d'activité Sir2 contrecarre le regroupement des télomères lors de la quiescence et a un rôle négatif sur la durée de vie en quiescence. Notre travail suggère que la réorganisation drastique du génome en ‘hypercluster’ de télomères favorise la survie lors de quiescence, et dénoue un lien entre le métabolisme, l'organisation nucléaire et le vieillissement

Spatial reorganization of telomeres in long-lived quiescent cells

International audienceBackground : The spatiotemporal behavior of chromatin is an important control mechanism of genomic function. Studies in Saccharomyces cerevisiae have broadly contributed to demonstrate the functional importance of nuclear organization. Although in the wild yeast survival depends on their ability to withstand adverse conditions, most of these studies were conducted on cells undergoing exponential growth. In these conditions, as in most eukaryotic cells, silent chromatin that is mainly found at the 32 telomeres accumulates at the nuclear envelope, forming three to five foci. Results : Here, combining live microscopy, DNA FISH and chromosome conformation capture (HiC) techniques, we report that chromosomes adopt distinct organizations according to the metabolic status of the cell. In particular, following carbon source exhaustion the genome of long-lived quiescent cells undergoes a major spatial re-organization driven by the grouping of telomeres into a unique focus or hypercluster localized in the center of the nucleus. This change in genome conformation is specific to quiescent cells able to sustain long-term viability. We further show that reactive oxygen species produced by mitochondrial activity during respiration commit the cell to form a hypercluster upon starvation. Importantly, deleting the gene encoding telomere associated silencing factor SIR3 abolishes telomere grouping and decreases longevity, a defect that is rescued by expressing a silencing defective SIR3 allele competent for hypercluster formation. Conclusions : Our data show that mitochondrial activity primes cells to group their telomeres into a hypercluster upon starvation, reshaping the genome architecture into a conformation that may contribute to maintain longevity of quiescent cells

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Related to Fig. 3. Animated 3D reconstruction of the entire contact map of long-lived SP cells (isolated from a SP culture by density gradient). Same annotations as in Additional file 2. (GIF 12057 kb

Additional file 3: Figure S2. of Spatial reorganization of telomeres in long-lived quiescent cells

SIR-mediated telomere clustering drives chromosome conformation in the dense fraction of SP cells. a Mean contacts frequencies between 100-kb centromeres windows in G1 (blue) and G0 quiescent cells (red). Black and green curves: contacts between 100-kb segments randomly sampled in both conditions, to illustrate the absence of coverage biases after normalization. b Chromosome organization of WT and sir3∆ quiescent cells (the cryptic mating type locus HML was deleted to prevent pseudo-diploid effect). ii) Normalized contact matrix obtained for hml∆* (left) and hml∆ sir3∆ (right) cells. Color scale: contact frequencies from rare (white) to frequent (dark blue). Red arrowheads: centromeres contacts; green and yellow arrowheads: telomere–telomere contacts in hml∆ and hml∆ sir3∆ G0 cells, respectively. The 3D representations of the hml∆ and hml∆ sir3∆ matrices are represented next to the contact maps. Each chromosome is represented as a chain of beads (1 bead = 20 kb), with color code reflecting the chromosome arm lengths, from short (blue) to long (red) arms. Yellow beads: subtelomeric regions; black beads: centromeres; purple beads: boundaries of the rDNA cluster. c Contact maps of W303 strain during exponentially growth (EXPO, left) and quiescence (G0, right). Red arrowheads: centromere clustering; green and yellow arrowheads: telomere–telomere contacts of two chromosomes (XIII and XV) in expo and G0 cells, respectively. Because of the low sequencing coverage and quality, the signal is not as strong as for data in Fig. 3 and the bins are larger (1 vector: 80 DpnII RFs). d Quantification of colocalization of 30-kb telomeric regions (red dots) compared with the distribution of the colocalization scores (box plot, two standard deviations) computed for 1000 random sets of 32 windows of 30 kb in the genome (excluding centromeric regions). The colocalization score is normalized by the sequencing depth for each dataset

Additional file 1: Figure S1. of Spatial reorganization of telomeres in long-lived quiescent cells

Characterization of the SP silent chromatin hypercluster. a Western blot against Rap1 on crude extracts from exponential, respiratory, or stationary cultures of a WT strain (yAT1684). H2A antibody was used for the loading control. b Representative fluorescent images of wild-type (WT) strains tagged with Rap1-GFP “yAT 1684”, GFP-Sir2 “yAT405”, Sir3-GFP “yAT779” and GFP-Sir4 “yAT431” strains. Overnight liquid cultures were diluted to 0.2 OD600nm/ml and images were acquired after 5 h (1 OD600nm/ml, fermentation phase) and 7 days (40 OD600nm/ml, stationary phase). c Representative fluorescent image of a Rap1-GFP Sir3-mCherry-tagged strain “yAT194” from stationary phase cultures. We note that Sir3 associates with both telomeres and the rDNA in stationary phase cells. d Representative fluorescent images of Rap1-GFP in stationary cultures of WT “yAT1684” and sir4∆ “yAT2092” strains. e Representative fluorescent images of the nucleolar protein Sik1 tagged with mCherry during fermentation, respiration, and stationary phase (“yAT340”). f Representative fluorescent image of Rap1-GFP Dad2-mRFP (Duo1 And Dam1 interacting, an essential component of the microtubule–kinetochore interface) tagged stationary phase cells (“yAT2279”). g Representative fluorescent image of Sir3-mCherry Cse4-GFP-tagged strain “yAT2280” from stationary phase. Scale bar is 1 μm. (PDF 1343 kb

Guidi et al. 2015

<p>Raw images corresponding to Guidi et al. 2015.</p> <p>For each figure of the manuscript Guidi et al. containing microscopy data we show Z projection (max intensity) of cropped images normalized to be comparable within each experiment. Here we provide for each crop the corresponding Z stack and the Z projection of the whole field image.</p