Human Telomeres Are Tethered to the Nuclear Envelope during Postmitotic Nuclear Assembly

SummaryTelomeres are essential for nuclear organization in yeast and during meiosis in mice. Exploring telomere dynamics in living human cells by advanced time-lapse confocal microscopy allowed us to evaluate the spatial distribution of telomeres within the nuclear volume. We discovered an unambiguous enrichment of telomeres at the nuclear periphery during postmitotic nuclear assembly, whereas telomeres were localized more internally during the rest of the cell cycle. Telomere enrichment at the nuclear rim was mediated by physical tethering of telomeres to the nuclear envelope, most likely via specific interactions between the shelterin subunit RAP1 and the nuclear envelope protein Sun1. Genetic interference revealed a critical role in cell-cycle progression for Sun1 but no effect on telomere positioning for RAP1. Our results shed light on the dynamic relocalization of human telomeres during the cell cycle and suggest redundant pathways for tethering telomeres to the nuclear envelope

Analyse moléculaire de la structure et du raccourcissement des télomères (rôle de WRN et de MRE11)

Localisés à l'extrémité des chromosomes linéaires, les télomères jouent un rôle essentiel dans la préservation de la stabilité du génome, le vieillissement, et l'apparition de cancers. La mise en place d'un crible, ciblé sur l'identification de mutations influençant la dynamique de raccourcissement des télomères, a permis d'identifier deux protéines, Mre11 et WRN. L'étude du rôle de WRN aux télomères a démontré que l'activité hélicase de cette protéine est essentielle à la réplication des répétitions télomériques riches en G. Les télome res dysfonctionnels représentent un des facteurs déclenchant l'instabilité génomique, liée à la forte incidence de cancers, qui caractérisent le Syndrome de Werner. La dynamique d'érosion des télomères de cellules ne possédant pas l'activité télomérase est dépendante de l'activité de la nucléase Mre11. De nombreuses indications permettent de dresser un lien fonctionnel entre Mre11 et TRF2, qui coopèrent pour maintenir l'intégrité des télomères.MONTPELLIER-BU Pharmacie (341722105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Functional human telomeres are recognized as DNA damage in G2 of the cell cycle

Telomeres have to be distinguished from DNA breaks that initiate a DNA damage response. Proteins involved in the DNA damage response have previously been found at telomeres in transformed cells; however, the importance of these factors for telomere function has not been understood. Here, we show that telomeres of telomerase-negative primary cells recruit Mre11, phosphorylated NBS1, and ATM in every G2 phase of the cell cycle. This recruitment correlates with a partial release of telomeric POT1; moreover, telomeres were found to be accessible to modifying enzymes at this time in the cell cycle, suggesting that they are unprotected. Degradation of the MRN complex, as well as inhibition of ATM, led to telomere dysfunction. Consequentially, we propose that a localized DNA damage response at telomeres after replication is essential for recruiting the processing machinery that promotes formation of a chromosome end protection complex

Defective telomere lagging strand synthesis in cells lacking WRN helicase activity

Cells from Werner syndrome patients are characterized by slow growth rates, premature senescence, accelerated telomere shortening rates, and genome instability. The syndrome is caused by the loss of the RecQ helicase WRN, but the underlying molecular mechanism is unclear. Here we report that cells lacking WRN exhibit deletion of telomeres from single sister chromatids. Only telomeres replicated by lagging strand synthesis were affected, and prevention of loss of individual telomeres was dependent on the helicase activity of WRN. Telomere loss could be counteracted by telomerase activity. We propose that WRN is necessary for efficient replication of G-rich telomeric DNA, preventing telomere dysfunction and consequent genomic instability

MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells

Summary: Mapping the binding sites of DNA- or chromatin-interacting proteins is essential to understanding biological processes. DNA adenine methyltransferase identification (DamID) has emerged as a comprehensive method to map genome-wide occupancy of proteins of interest. A caveat of DamID is the specificity of Dam methyltransferase for GATC motifs that are not homogenously distributed in the genome. Here, we developed an optimized method named MadID, using proximity labeling of DNA by the methyltransferase M.EcoGII. M.EcoGII mediates N6-adenosine methylation in any DNA sequence context, resulting in deeper and unbiased coverage of the genome. We demonstrate, using m6A-specific immunoprecipitation and deep sequencing, that MadID is a robust method to identify protein-DNA interactions at the whole-genome level. Using MadID, we revealed contact sites between human telomeres, repetitive sequences devoid of GATC sites, and the nuclear envelope. Overall, MadID opens the way to identification of binding sites in genomic regions that were largely inaccessible. : Mapping the binding sites of DNA- or chromatin-interacting proteins is essential to understanding biological processes. Sobecki et al. developed an optimized method named MadID based on proximity labeling of DNA by the bacterial methyltransferase M.EcoGII. MadID results in deep and unbiased coverage for genome-wide mapping studies. Keywords: MadID, M.EcoGII, m6A, LADs, telomeres, nuclear envelope, proximity labeling, methylatio

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

Impact of exogenous stress on TGF-β inducible early gene 1 in human skin cells.

International audienc

Non-random spatial organization of telomeres varies during the cell cycle and requires LAP2 and BAF

International audienceSpatial genome organization within the nucleus influences major biological processes and is impacted by the configuration of linear chromosomes. Here, we applied 3D spatial statistics and modeling on high-resolution telomere and centromere 3D-structured illumination microscopy images in cancer cells. We found a multi-scale organization of telomeres that dynamically evolved from a mixed clustered-and-regular distribution in early G1 to a purely regular distribution as cells progressed through the cell cycle. In parallel, our analysis revealed two pools of peripheral and internal telomeres, the proportions of which were inverted during the cell cycle. We then conducted a targeted screen using MadID to identify the molecular pathways driving or maintaining telomere anchoring to the nuclear envelope observed in early G1. Lamina-associated polypeptide (LAP) proteins were found transiently localized to telomeres in anaphase, a stage where LAP2α initiates the reformation of the nuclear envelope, and impacted telomere redistribution in the next interphase together with their partner barrier-to-autointegration factor (BAF)

Spatial modeling of telomere intra-nuclear distribution reveals non-random organization that varies during cell cycle and depends on LAP2 and BAF

Abstract Genome organization within the 3D nuclear volume influences major biological processes but is completely lost during mitosis, which represents a major challenge to maintain cellular identity and cell fate. To restore a functional G1 nucleus for the next cell cycle, it is imperative to reestablish genome organization during post-mitotic nuclear assembly. Importantly, the configuration of linear chromosomes has been shown to directly impact spatial genome architecture. Both centromeres and telomeres are known to associate with nuclear structures, such as the nuclear envelope, and support chromatin distribution. Here, using high-resolution 3D imaging combined with 3D spatial statistics and modeling, we showed that telomeres generally followed a regular distribution compared to what is expected under a random organization. While the preferential localization of telomeres at nuclear periphery was restricted to early G1, we found a strong clustering of centromeres in addition to their predominant peripheral localization at all cell cycle stages. We then conducted a targeted screen using MadID to identify the molecular pathways driving or maintaining telomere anchoring to the nuclear envelope. Among these factors, we could show that LAP2α transiently localizes to telomeres in anaphase, at a stage where LAP2α initiates the reformation of the nuclear envelope. Moreover, co-depletion of LAP proteins and their partner BAF impacted telomere redistribution in the next interphase. There results suggest that in addition to their crucial role in genome protection, telomeres also participate in reshaping functional G1 nuclei after mitosis