Mécanismes moléculaires de la dynamique des chromosomes

Publications de l’équipe

Année de publication : 2020

Daniele Fachinetti, Hiroshi Masumoto, Natalay Kouprina (2020 Sep 27)

Artificial chromosomes.

Experimental cell research : 112302 : DOI : S0014-4827(20)30551-6 En savoir plus
Résumé

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Sebastiaan Jw van den Berg, Lars Et Jansen (2020 Sep 22)

Centromeres: genetic input to calibrate an epigenetic feedback loop.

The EMBO journal : e106638 : DOI : 10.15252/embj.2020106638 En savoir plus
Résumé

Centromeres are chromatin domains maintained by a self-templating feedback loop based on nucleosomes bearing the histone H3 variant CENP-A. The underlying centromeric DNA sequence is largely dispensable, yet paradoxically, it has highly conserved features. Hoffmann et al (2020) now uncover that when the epigenetic chromatin cycle falters, a genetically hardwired mechanism offers robustness to a dynamic epigenetic feedback loop ensuring long-term centromere inheritance.

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Marie Dumont, Daniele Fachinetti (2020 Sep 18)

Centromere strength: just a sense of proportion.

Molecular & cellular oncology : 1742063 : DOI : 10.1080/23723556.2020.1742063 En savoir plus
Résumé

The overall structure and composition of human centromeres have been well reported, but how these elements vary between individual chromosomes and influence the chromosome-specific behavior during mitosis remains untested. In our study, we discover the existence of heterogeneity of centromeric DNA features that dictates the chromosome segregation fidelity during mitosis.

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Sebastian Hoffmann, Helena M Izquierdo, Riccardo Gamba, Florian Chardon, Marie Dumont, Veer Keizer, Solène Hervé, Shannon M McNulty, Beth A Sullivan, Nicolas Manel, Daniele Fachinetti (2020 Sep 18)

A genetic memory initiates the epigenetic loop necessary to preserve centromere position.

The EMBO journal : e105505 : DOI : 10.15252/embj.2020105505 En savoir plus
Résumé

Centromeres are built on repetitive DNA sequences (CenDNA) and a specific chromatin enriched with the histone H3 variant CENP-A, the epigenetic mark that identifies centromere position. Here, we interrogate the importance of CenDNA in centromere specification by developing a system to rapidly remove and reactivate CENP-A (CENP-A ). Using this system, we define the temporal cascade of events necessary to maintain centromere position. We unveil that CENP-B bound to CenDNA provides memory for maintenance on human centromeres by promoting de novo CENP-A deposition. Indeed, lack of CENP-B favors neocentromere formation under selective pressure. Occasionally, CENP-B triggers centromere re-activation initiated by CENP-C, but not CENP-A, recruitment at both ectopic and native centromeres. This is then sufficient to initiate the CENP-A-based epigenetic loop. Finally, we identify a population of CENP-A-negative, CENP-B/C-positive resting CD4 T cells capable to re-express and reassembles CENP-A upon cell cycle entry, demonstrating the physiological importance of the genetic memory.

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Riccardo Gamba, Daniele Fachinetti (2020 Mar 17)

From evolution to function: Two sides of the same CENP-B coin?

Experimental cell research : 111959 : DOI : S0014-4827(20)30167-1 En savoir plus
Résumé

The centromere is the nucleoproteic chromosomal structure necessary for accurate chromosome segregation during cell division. One of the earliest centromeric proteins to be discovered was CENP-B, the only one capable of recognizing a specific centromeric DNA binding motif. The phylogenetic history of this protein and of its DNA binding site shows independent events of function acquisition across different species and raises questions on the evolutionary dynamics of CENP-B, including what may be the selective advantage provided by its role at the centromere. Recent results have provided insight into potential functions of CENP-B in chromosome dynamics, however, its function is still object of debate. The recurrent appearance of CENP-B centromeric activity along phylogenesis, together with its dispensability, represent strictly intertwined facets of this controversy. This chapter focuses on the evolution, function and homeostasis of CENP-B and its importance in centromere biology.

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Année de publication : 2019

Marie Dumont, Riccardo Gamba, Pierre Gestraud, Sjoerd Klaasen, Joseph T Worrall, Sippe G De Vries, Vincent Boudreau, Catalina Salinas-Luypaert, Paul S Maddox, Susanne Ma Lens, Geert Jpl Kops, Sarah E McClelland, Karen H Miga, Daniele Fachinetti (2019 Nov 22)

Human chromosome-specific aneuploidy is influenced by DNA-dependent centromeric features.

The EMBO journal : e102924 : DOI : 10.15252/embj.2019102924 En savoir plus
Résumé

Intrinsic genomic features of individual chromosomes can contribute to chromosome-specific aneuploidy. Centromeres are key elements for the maintenance of chromosome segregation fidelity via a specialized chromatin marked by CENP-A wrapped by repetitive DNA. These long stretches of repetitive DNA vary in length among human chromosomes. Using CENP-A genetic inactivation in human cells, we directly interrogate if differences in the centromere length reflect the heterogeneity of centromeric DNA-dependent features and whether this, in turn, affects the genesis of chromosome-specific aneuploidy. Using three distinct approaches, we show that mis-segregation rates vary among different chromosomes under conditions that compromise centromere function. Whole-genome sequencing and centromere mapping combined with cytogenetic analysis, small molecule inhibitors, and genetic manipulation revealed that inter-chromosomal heterogeneity of centromeric features, but not centromere length, influences chromosome segregation fidelity. We conclude that faithful chromosome segregation for most of human chromosomes is biased in favor of centromeres with high abundance of DNA-dependent centromeric components. These inter-chromosomal differences in centromere features can translate into non-random aneuploidy, a hallmark of cancer and genetic diseases.

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Reito Watanabe, Masatoshi Hara, Ei-Ichi Okumura, Solène Hervé, Daniele Fachinetti, Mariko Ariyoshi, Tatsuo Fukagawa (2019 Nov 3)

CDK1-mediated CENP-C phosphorylation modulates CENP-A binding and mitotic kinetochore localization.

The Journal of cell biology : DOI : jcb.201907006 En savoir plus
Résumé

The kinetochore is essential for faithful chromosome segregation during mitosis. To form a functional kinetochore, constitutive centromere-associated network (CCAN) proteins are assembled on the centromere chromatin that contains the centromere-specific histone CENP-A. CENP-C, a CCAN protein, directly interacts with the CENP-A nucleosome to nucleate the kinetochore structure. As CENP-C is a hub protein for kinetochore assembly, it is critical to address how the CENP-A-CENP-C interaction is regulated during cell cycle progression. To address this question, we investigated the CENP-C C-terminal region, including a conserved CENP-A-binding motif, in both chicken and human cells and found that CDK1-mediated phosphorylation of CENP-C facilitates its binding to CENP-A in vitro and in vivo. We observed that CENP-A binding is involved in CENP-C kinetochore localization during mitosis. We also demonstrate that the CENP-A-CENP-C interaction is critical for long-term viability in human RPE-1 cells. These results provide deeper insights into protein-interaction network plasticity in centromere proteins during cell cycle progression.

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Simon Gemble, Anthony Simon, Carole Pennetier, Marie Dumont, Solène Hervé, Franz Meitinger, Karen Oegema, Raphaël Rodriguez, Geneviève Almouzni, Daniele Fachinetti, Renata Basto (2019 Sep 10)

Centromere Dysfunction Compromises Mitotic Spindle Pole Integrity.

Current biology : CB : 3072-3080.e5 : DOI : S0960-9822(19)30932-7 En savoir plus
Résumé

Centromeres and centrosomes are crucial mitotic players. Centromeres are unique chromosomal sites characterized by the presence of the histone H3-variant centromere protein A (CENP-A) [1]. CENP-A recruits the majority of centromere components, collectively named the constitutive centromere associated network (CCAN) [2]. The CCAN is necessary for kinetochore assembly, a multiprotein complex that attaches spindle microtubules (MTs) and is required for chromosome segregation [3]. In most animal cells, the dominant site for MT nucleation in mitosis are the centrosomes, which are composed of two centrioles, surrounded by a protein-rich matrix of electron-dense pericentriolar material (PCM) [4]. The PCM is the site of MT nucleation during mitosis [5]. Even if centromeres and centrosomes are connected via MTs in mitosis, it is not known whether defects in either one of the two structures have an impact on the function of the other. Here, using high-resolution microscopy combined with rapid removal of CENP-A in human cells, we found that perturbation of centromere function impacts mitotic spindle pole integrity. This includes release of MT minus-ends from the centrosome, leading to PCM dispersion and centriole mis-positioning at the spindle poles. Mechanistically, we show that these defects result from abnormal spindle MT dynamics due to defective kinetochore-MT attachments. Importantly, restoring mitotic spindle pole integrity following centromere inactivation lead to a decrease in the frequency of chromosome mis-segregation. Overall, our work identifies an unexpected relationship between centromeres and maintenance of the mitotic pole integrity necessary to ensure mitotic accuracy and thus to maintain genetic stability.

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Robert F Lera, Roshan X Norman, Marie Dumont, Alexandra Dennee, Joanne Martin-Koob, Daniele Fachinetti, Mark E Burkard (2019 Aug 31)

Plk1 protects kinetochore-centromere architecture against microtubule pulling forces.

EMBO reports : e48711 : DOI : 10.15252/embr.201948711 En savoir plus
Résumé

During mitosis, sister chromatids attach to microtubules which generate ~ 700 pN pulling force focused on the centromere. We report that chromatin-localized signals generated by Polo-like kinase 1 (Plk1) maintain the integrity of the kinetochore and centromere against this force. Without sufficient Plk1 activity, chromosomes become misaligned after normal condensation and congression. These chromosomes are silent to the mitotic checkpoint, and many lag and mis-segregate in anaphase. Their centromeres and kinetochores lack CENP-A, CENP-C, CENP-T, Hec1, Nuf2, and Knl1; however, CENP-B is retained. CENP-A loss occurs coincident with secondary misalignment and anaphase onset. This disruption occurs asymmetrically prior to anaphase and requires tension generated by microtubules. Mechanistically, centromeres highly recruit PICH DNA helicase and PICH depletion restores kinetochore disruption in pre-anaphase cells. Furthermore, anaphase defects are significantly reduced by tethering Plk1 to chromatin, including H2B, and INCENP, but not to CENP-A. Taken as a whole, this demonstrates that Plk1 signals are crucial for stabilizing centromeric architecture against tension.

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Andrea Scelfo, Daniele Fachinetti (2019 Aug 21)

Keeping the Centromere under Control: A Promising Role for DNA Methylation.

Cells : DOI : E912 En savoir plus
Résumé

In order to maintain cell and organism homeostasis, the genetic material has to be faithfully and equally inherited through cell divisions while preserving its integrity. Centromeres play an essential task in this process; they are special sites on chromosomes where kinetochores form on repetitive DNA sequences to enable accurate chromosome segregation. Recent evidence suggests that centromeric DNA sequences, and epigenetic regulation of centromeres, have important roles in centromere physiology. In particular, DNA methylation is abundant at the centromere, and aberrant DNA methylation, observed in certain tumors, has been correlated to aneuploidy and genomic instability. In this review, we evaluate past and current insights on the relationship between centromere function and the DNA methylation pattern of its underlying sequences.

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Yael Nechemia-Arbely, Karen H Miga, Ofer Shoshani, Aaron Aslanian, Moira A McMahon, Ah Young Lee, Daniele Fachinetti, John R Yates, Bing Ren, Don W Cleveland (2019 Jun 5)

DNA replication acts as an error correction mechanism to maintain centromere identity by restricting CENP-A to centromeres.

Nature cell biology : 743-754 : DOI : 10.1038/s41556-019-0331-4 En savoir plus
Résumé

Chromatin assembled with the histone H3 variant CENP-A is the heritable epigenetic determinant of human centromere identity. Using genome-wide mapping and reference models for 23 human centromeres, CENP-A binding sites are identified within the megabase-long, repetitive α-satellite DNAs at each centromere. CENP-A is shown in early G1 to be assembled into nucleosomes within each centromere and onto 11,390 transcriptionally active sites on the chromosome arms. DNA replication is demonstrated to remove ectopically loaded, non-centromeric CENP-A. In contrast, tethering of centromeric CENP-A to the sites of DNA replication through the constitutive centromere associated network (CCAN) is shown to enable precise reloading of centromere-bound CENP-A onto the same DNA sequences as in its initial prereplication loading. Thus, DNA replication acts as an error correction mechanism for maintaining centromere identity through its removal of non-centromeric CENP-A coupled with CCAN-mediated retention and precise reloading of centromeric CENP-A.

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Matteo Gentili, Xavier Lahaye, Francesca Nadalin, Guilherme P F Nader, Emilia Puig Lombardi, Solène Herve, Nilushi S De Silva, Derek C Rookhuizen, Elina Zueva, Christel Goudot, Mathieu Maurin, Aurore Bochnakian, Sebastian Amigorena, Matthieu Piel, Daniele Fachinetti, Arturo Londoño-Vallejo, Nicolas Manel (2019 Mar 28)

The N-Terminal Domain of cGAS Determines Preferential Association with Centromeric DNA and Innate Immune Activation in the Nucleus.

Cell reports : 3798 : DOI : S2211-1247(19)30365-1 En savoir plus
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Viviana Barra, Glennis A Logsdon, Andrea Scelfo, Sebastian Hoffmann, Solène Hervé, Aaron Aslanian, Yael Nechemia-Arbely, Don W Cleveland, Ben E Black, Daniele Fachinetti (2019 Jan 13)

Phosphorylation of CENP-A on serine 7 does not control centromere function.

Nature communications : 175 : DOI : 10.1038/s41467-018-08073-1 En savoir plus
Résumé

CENP-A is the histone H3 variant necessary to specify the location of all eukaryotic centromeres via its CENP-A targeting domain and either one of its terminal regions. In humans, several post-translational modifications occur on CENP-A, but their role in centromere function remains controversial. One of these modifications of CENP-A, phosphorylation on serine 7, has been proposed to control centromere assembly and function. Here, using gene targeting at both endogenous CENP-A alleles and gene replacement in human cells, we demonstrate that a CENP-A variant that cannot be phosphorylated at serine 7 maintains correct CENP-C recruitment, faithful chromosome segregation and long-term cell viability. Thus, we conclude that phosphorylation of CENP-A on serine 7 is dispensable to maintain correct centromere dynamics and function.

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Année de publication : 2018

Nechemia-Arbely, Y., Miga, K., Shoshani, O., Aslanian, A., McMahon, M.A., Young Lee, A., Fachinetti, D., Yates, J.R., Ren, B. and Cleveland, D.W. (2018 Dec 31)

DNA replication-mediated error correction of ectopic CENP-A deposition maintains centromere identity.

BioRxiv En savoir plus
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V Barra, D Fachinetti (2018 Oct 20)

The dark side of centromeres: types, causes and consequences of structural abnormalities implicating centromeric DNA.

Nature communications : 4340 : DOI : 10.1038/s41467-018-06545-y En savoir plus
Résumé

Centromeres are the chromosomal domains required to ensure faithful transmission of the genome during cell division. They have a central role in preventing aneuploidy, by orchestrating the assembly of several components required for chromosome separation. However, centromeres also adopt a complex structure that makes them susceptible to being sites of chromosome rearrangements. Therefore, preservation of centromere integrity is a difficult, but important task for the cell. In this review, we discuss how centromeres could potentially be a source of genome instability and how centromere aberrations and rearrangements are linked with human diseases such as cancer.

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