Chromatin epigenetics and nuclear lamina keep the nucleus in shape: Examples from natural and accelerated aging

As the repository of genetic information, the cell nucleus must protect DNA integrity from mechanical stresses. The nuclear lamina, which resides within the nuclear envelope (NE), is made up of lamins, intermediate filaments bound to DNA. The nuclear lamina provides the nucleus with the ability to deal with inward as well as outward mechanical stimuli. Chromatin, in turn, through its degrees of compaction, shares this role with the nuclear lamina, thus, ensuring the plasticity of the nucleus. Perturbation of chromatin condensation or the nuclear lamina has been linked to a plethora of biological conditions, that range from cancer and genetic diseases (laminopathies) to aging, both natural and accelerated, such as the case of Hutchinson-Gilford Progeria Syndrome (HGPS). From the experimental results accumulated so far on the topic, a direct link between variations of the epigenetic pattern and nuclear lamina structure would be suggested, however, it has never been clarified thoroughly. This relationship, instead, has a downstream important implication on nucleus shape, genome preservation, force sensing, and, ultimately, aging-related disease onset. With this review, we aim to collect recent studies on the importance of both nuclear lamina components and chromatin status in nuclear mechanics. We also aim to bring to light evidence of the link between DNA methylation and nuclear lamina in natural and accelerated aging

A Glimpse into Chromatin Organization and Nuclear Lamina Contribution in Neuronal Differentiation

During embryonic development, stem cells undergo the differentiation process so that they can specialize for different functions within the organism. Complex programs of gene transcription are crucial for this process to happen. Epigenetic modifications and the architecture of chromatin in the nucleus, through the formation of specific regions of active as well as inactive chromatin, allow the coordinated regulation of the genes for each cell fate. In this mini-review, we discuss the current knowledge regarding the regulation of three-dimensional chromatin structure during neuronal differentiation. We also focus on the role the nuclear lamina plays in neurogenesis to ensure the tethering of the chromatin to the nuclear envelop

Nesprins are mechanotransducers that discriminate epithelial-mesenchymal transition programs

© 2020 Déjardin et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).LINC complexes are transmembrane protein assemblies that physically connect the nucleoskeleton and cytoskeleton through the nuclear envelope. Dysfunctions of LINC complexes are associated with pathologies such as cancer and muscular disorders. The mechanical roles of LINC complexes are poorly understood. To address this, we used genetically encoded FRET biosensors of molecular tension in a nesprin protein of the LINC complex of fibroblastic and epithelial cells in culture. We exposed cells to mechanical, genetic, and pharmacological perturbations, mimicking a range of physiological and pathological situations. We show that nesprin experiences tension generated by the cytoskeleton and acts as a mechanical sensor of cell packing. Moreover, nesprin discriminates between inductions of partial and complete epithelial-mesenchymal transitions. We identify the implicated mechanisms, which involve α-catenin capture at the nuclear envelope by nesprin upon its relaxation, thereby regulating β-catenin transcription. Our data thus implicate LINC complex proteins as mechanotransducers that fine-tune β-catenin signaling in a manner dependent on the epithelial-mesenchymal transition program.This material is based on work supported by the Centre national de la recherche scientifique (CNRS), Agence nationale de la recherche (ANR; grants ANR-13-JSV5-0007 and ANR-14-CE09-0006), France BioImaging (ANR-10-INBS-04), la Ligue contre le Cancer (REMX17751 to P.M. Davidson), and the Fondation ARC pour la Recherche sur le Cancer (PDF20161205227 to P.M. Davidson). P.S. Carollo has received funding from the European Union’s Horizon 2020 Framework Programme for Research and Innovation (Marie Skłodowska-Curie grant agreement 665850-INSPIRE) and acknowledges the Ecole Doctorale Frontières de l'Innovation en Recherche et Éducation (FIRE) Programme Bettencourt. E.R. Gomes was supported by a European Research Council consolidator grant (617676).info:eu-repo/semantics/publishedVersio

Investigating the inhibition of FTSJ1 a tryptophan tRNA-specific 2’-O-methyltransferase by NV TRIDs, as a mechanism of readthrough in nonsense mutated CFTR

Abstract: Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR gene, coding for the CFTR chloride channel. About 10% of the CFTR gene mutations are "stop" mutations, which generate a Premature Termination Codon (PTC), thus synthesizing a truncated CFTR protein. A way to bypass PTC relies on ribosome readthrough, which is the ri-bosome’s capacity to skip a PTC, thus generating a full-length protein. “TRIDs” are molecules exerting ribosome readthrough; for some, the mechanism of action is still under debate. We in-vestigate a possible mechanism of action (MOA) by which our recently synthesized TRIDs, namely NV848, NV914, and NV930, could exert their readthrough activity by in silico analysis and in vitro studies. Our results suggest a likely inhibition of FTSJ1, a tryptophan tRNA-specific 2’-O-methyltransferase

Readthrough Approach Using NV Translational Readthrough-Inducing Drugs (TRIDs): A Study of the Possible Off-Target Effects on Natural Termination Codons (NTCs) on TP53 and Housekeeping Gene Expression

Nonsense mutations cause several genetic diseases such as cystic fibrosis, Duchenne muscular dystrophy, β-thalassemia, and Shwachman–Diamond syndrome. These mutations induce the formation of a premature termination codon (PTC) inside the mRNA sequence, resulting in the synthesis of truncated polypeptides. Nonsense suppression therapy mediated by translational readthrough-inducing drugs (TRIDs) is a promising approach to correct these genetic defects. TRIDs generate a ribosome miscoding of the PTC named “translational readthrough” and restore the synthesis of full-length and potentially functional proteins. The new oxadiazole-core TRIDs NV848, NV914, and NV930 (NV) showed translational readthrough activity in nonsense-related in vitro systems. In this work, the possible off-target effect of NV molecules on natural termination codons (NTCs) was investigated. Two different in vitro approaches were used to assess if the NV molecule treatment induces NTC readthrough: (1) a study of the translational-induced p53 molecular weight and functionality; (2) the evaluation of two housekeeping proteins’ (Cys-C and β2M) molecular weights. Our results showed that the treatment with NV848, NV914, or NV930 did not induce any translation alterations in both experimental systems. The data suggested that NV molecules have a specific action for the PTCs and an undetectable effect on the NTCs

Le complexe LINC est un mécanotransducteur qui régule la signalisation de la caténine au cours des transitions épithélo-mésenchymateuses

In multicellular organisms, cells generate and experience mechanical forces. As a consequence, these forces can regulate cellular behaviour as well as tissue organization through a process known as “mechanotransduction”, by which cells convert mechanical stimuli into biochemical signals. In animal cells, the nucleus is mechanically coupled by the cytoskeleton to cell adhesion complexes, such that extracellular mechanical cues can affect the position and shape of the nucleus. Such mechanical coupling is provided by outer nuclear transmembrane proteins, nesprins, whose KASH domain interacts with inner nuclear transmembrane SUN proteins in the perinuclear space. The cytoplasmic domain of nesprins can bind to the cytoskeleton and the nucleoplasmic domain of SUNs to the nucleoskeleton to form the so-called LINC complex: Linker of Nucleoskeleton and Cytoskeleton. Mutations in, or loss of LINC complex proteins impair nuclear envelope integrity, nucleus anchoring, chromosome positioning, DNA repair, genome transcription and replication and, in addition, LINC complex disruption negatively impacts nuclear translocation of transcription co-factors. However, it is still unclear whether the consequences of a dysfunctional LINC complex result from an impairment of mechanotransduction. In this thesis, we focused on nesprin-2 giant (nesprin2G), which forms a complex with and regulates the nuclear localization of -catenin, a major transcription co-factor in several morphogenetic processes. Upon induction of epithelial-mesenchymal transition (EMT) -a process through which epithelial cells can gradually acquire increased motility and decreased intercellular adhesion-, epithelial cell packing regulates β-catenin signalling. We thus hypothesized that the LINC complex could participate in this mechanical regulation. To this aim, we combined molecular tension microscopy, involving a genetically encoded FRET biosensor, with mechanical, genetic and pharmacological perturbations of fibroblastic and epithelial cells in culture.We found that the LINC complex is mechanosensitive to cell packing. Moreover, nesprin2G tension increases upon induction of partial, but not complete EMT, thereby defining two mechanisms of β-catenin nuclear translocation. Upon induction of complete EMT, relaxed nesprin2G recruits α-catenin at the nuclear envelope, which results in nuclear translocation of both catenins. Upon partial EMT however, tensed nesprin2G does not recruit α-catenin and only β-catenin translocates to the nucleus. Finally, we found that α-catenin sequesters β -catenin in the nucleus in a transcriptionally less active form. We thus propose that, in a manner dependent on the EMT program, mechanosensitive nesprins may capture, at the nuclear envelope, the catenins and fine-tune their nuclear translocation and activities.Dans les organismes multicellulaires, les cellules génèrent et subissent des forces mécaniques. En conséquence, ces forces peuvent réguler le comportement cellulaire ainsi que l'organisation des tissus grâce à un processus appelé «mécanotransduction», par lequel les cellules convertissent les stimuli mécaniques en signaux biochimiques. Dans les cellules animales, le noyau est couplé mécaniquement par le cytosquelette aux complexes d’adhésion cellulaire, de sorte que des signaux mécaniques extracellulaires puissent affecter la position et la forme du noyau. Un tel couplage mécanique est assuré par les protéines transmembranaires nucléaires externes, les nesprines, dont le domaine KASH interagit avec les protéines SUN transmembranaires nucléaires internes dans l'espace périnucléaire. Le domaine cytoplasmique des nesprines peut se lier au cytosquelette et le domaine nucléoplasmique des SUN au nucléosquelette pour former le complexe dit LINC: Linker of Nucleoskeleton and Cytoskeleton. Les mutations ou la perte de protéines du complexe LINC altèrent l'intégrité de l'enveloppe nucléaire, l'ancrage du noyau, le positionnement des chromosomes, la réparation de l'ADN, la transcription et la réplication du génome et, de plus, la rupture du complexe LINC a un impact négatif sur la translocation nucléaire des co-facteurs de transcription. Cependant, il n’est pas clair si les conséquences d'un complexe LINC dysfonctionnel résultent d'une déficience de la mécanotransduction.Dans cette thèse, nous nous sommes concentrés sur la nesprine-2 géante (nesprine2G), qui forme un complexe avec et régule la localisation nucléaire de la β-caténine, un co-facteur de transcription majeur dans plusieurs processus morphogénétiques. Lors de l'induction de la transition épithélium-mésenchyme (TEM) -processus par lequel les cellules épithéliales peuvent acquérir progressivement une motilité accrue et une adhésion intercellulaire réduite- la compaction des cellules épithéliales régule la signalisation de la β-caténine. Nous avons donc émis l’hypothèse que le complexe LINC pourrait participer à cette régulation mécanique. À cette fin, nous avons combiné la microscopie de tension moléculaire, impliquant un biosenseur FRET codé génétiquement, à des perturbations mécaniques, génétiques et pharmacologiques de cellules fibroblastiques et épithéliales en culture.Nous avons constaté que le complexe LINC est mécanosensible à la compaction des cellules. De plus, la tension de nesprin2G augmente lors de l'induction d'une TEM partielle, mais pas d’une TEM complète, définissant ainsi deux mécanismes de translocation nucléaire de la β-caténine. Lors de l'induction de la TEM complète, la nesprine2G détendue recrute l'α-caténine au niveau de l'enveloppe nucléaire, ce qui entraîne une translocation nucléaire des deux caténines. Cependant, en cas de TEM partielle, la nesprine2G sous tension ne recrute pas d'α-caténine et seule la β-caténine effectue une translocation dans le noyau. Enfin, nous avons constaté que l'α-caténine séquestrait la β-caténine dans le noyau sous une forme transcriptionnellement moins active. Nous proposons donc que, d'une manière dépendant du programme de TEM, les nesprines mécanosensibles peuvent capturer, au niveau de l'enveloppe nucléaire, les caténines et réguler finement leur translocations et activités nucléaires

Be careful on DNA methylation alterations

DNA methylation is an epigenetic modification involved in DNA compaction and in the regulation of gene expression. Also, DNA methylation is found in repetitive DNA including centromere and telomere. Recently, the research has been focused on repetitive sequences at the centromere due to its role in chromosome segregation, an event essential to cell fitness. So far, studies revealed that tumour cells are usually hypomethylated at repetitive sequences and that DNA hypomethylation leads to the loss of chromatid cohesion and to aneuploidy as well[1-2]. However, in the centromeric sequences there are no coding genes so it is still unclear what role DNA methylation plays in this context. In order to answer this question we used and compared immortalized (RPE-1) and tumour (DLD-1) cell lines, both engineered to allow an inducible DNA hypomethylation (in collaboration with Institut Curis, Paris). We took advantage of the Auxin Inducible Degron (AID) system[3] to degrade the endogenous AID-tagged DNMT1 (DNA-methyl- Transferase1). The induced DNMT1-AID degradation for several cell cycles leads to passive DNA hypomethylation. Our results showed that, once hypomethylated, immortalized non-tumour cells suffered a reduced growth rate. Both cell lines acquired aneuploidy and chromatids cohesion defects. We also observed an increase of mitotic errors, especially misaligned and lagging chromosomes, which strongly suggest a centromere/kinetochore malfunctioning. To this regard, by microscopy we noticed a reduced amount of centromeric proteins (CENPs) at centromere. Lastly, cells underwent nuclear and cytoskeleton defects that could also contribute to the genetic instability typical of tumour cells. In summary, our study reveals that methylation of repetitive DNA is indeed important to cells survival and fitness, probably by maintaining centromere stability and function and by impacting on nuclear/cytoskeleton mechanics. This also suggests cautiousness in the use of hypomethylating drug as anti-cancer therapy that may have a risky effect on normal cells

Specific Irreversible Cell-Cycle Arrest and Depletion of Cancer Cells Obtained by Combining Curcumin and the Flavonoids Quercetin and Fisetin

Background: Induced senescence could be exploited to selectively counteract the proliferation of cancer cells and target them for senolysis. We examined the cellular senescence induced by curcumin and whether it could be targeted by fisetin and quercetin, flavonoids with senolytic activity. Methods: Cell-cycle profiles, chromosome number and structure, and heterochromatin markers were evaluated via flow cytometry, metaphase spreads, and immunofluorescence, respectively. The activation of p21(waf1/cip1) was assessed via RT-qPCR and immunoblotting. Senescent cells were detected via SA-beta-Galactosidase staining. Results: We report that curcumin treatment specifically triggers senescence in cancer cells by inducing mitotic slippage and DNA damage. We show that curcumin-induced senescence is p21(waf1/cip1)-dependent and characterized by heterochromatin loss. Finally, we found that flavonoids clear curcumin-induced senescent cancer cells. Conclusions: Our findings expand the characterization of curcumin-induced cellular senescence in cancer cells and lay the foundation for the combination of curcumin and flavonoids as a possible anti-cancer therapy

Inhibition of FTSJ1, a tryptophan tRNA-specific 2’-O-methyltransferase as possible mechanism to readthrough premature termination codons (UGAs) of the CFTR mRNA

Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR gene, coding for the CFTR chloride channel. About 10 % of the mutations affecting the CFTR gene are "stop" mutations, which generate a Premature Termination Codon (PTC), thus resulting in the synthesis of a truncated CFTR protein. A way to bypass PTC relies on ribosome readthrough, that is the capacity of the ribosome to skip a PTC, thus generating a full-length protein. “TRIDs” are molecules exerting ribosome readthrough and for some of them the mechanism of action is still under debate. By in silico analysis as well as in vitro studies, we investigate a possible mechanism of action (MOA) by which our recently synthesized TRIDs, namely NV848, NV914 and NV930, could exert their readthrough activity. Our results suggest a likely inhibition of FTSJ1, a tryptophan tRNA-specific 2’-O-methyltransferase. In addition, we report that our TRIDs do not exert readthrough on natural termination codons