Dynamique de la Chromatine

Publications de l’équipe

Année de publication : 2016

Salomé Adam, Juliette Dabin, Odile Chevallier, Olivier Leroy, Céline Baldeyron, Armelle Corpet, Patrick Lomonte, Olivier Renaud, Geneviève Almouzni, Sophie E Polo (2016 Sep 20)

Real-Time Tracking of Parental Histones Reveals Their Contribution to Chromatin Integrity Following DNA Damage.

Molecular cell : DOI : S1097-2765(16)30461-0 En savoir plus
Résumé

Chromatin integrity is critical for cell function and identity but is challenged by DNA damage. To understand how chromatin architecture and the information that it conveys are preserved or altered following genotoxic stress, we established a system for real-time tracking of parental histones, which characterize the pre-damage chromatin state. Focusing on histone H3 dynamics after local UVC irradiation in human cells, we demonstrate that parental histones rapidly redistribute around damaged regions by a dual mechanism combining chromatin opening and histone mobilization on chromatin. Importantly, parental histones almost entirely recover and mix with new histones in repairing chromatin. Our data further define a close coordination of parental histone dynamics with DNA repair progression through the damage sensor DDB2 (DNA damage-binding protein 2). We speculate that this mechanism may contribute to maintaining a memory of the original chromatin landscape and may help preserve epigenome stability in response to DNA damage.

Replier
Christèle Maison, Delphine Bailly, Jean-Pierre Quivy, Geneviève Almouzni (2016 Jul 19)

The methyltransferase Suv39h1 links the SUMO pathway to HP1α marking at pericentric heterochromatin.

Nature communications : 12224 : DOI : 10.1038/ncomms12224 En savoir plus
Résumé

The trimethylation of histone H3 on lysine 9 (H3K9me3) – a mark recognized by HP1 that depends on the Suv39h lysine methyltransferases (KMTs) – has provided a basis for the reader/writer model to explain HP1 accumulation at pericentric heterochromatin in mammals. Here, we identify the Suv39h1 paralog, as a unique enhancer of HP1α sumoylation both in vitro and in vivo. The region responsible for promoting HP1α sumoylation (aa1-167) is distinct from the KMT catalytic domain and mediates binding to Ubc9. Tethering the 1-167 domain of Suv39h1 to pericentric heterochromatin, but not mutants unable to bind Ubc9, accelerates the de novo targeting of HP1α to these domains. Our results establish an unexpected feature of Suv39h1, distinct from the KMT activity, with a major role for heterochromatin formation. We discuss how linking Suv39h1 to the SUMO pathway provides conceptual implications for our general view on nuclear domain organization and physiological functions.

Replier
C Clément, I Vassias, D Ray-Gallet, G Almouzni (2016 Jul 4)

Functional Characterization of Histone Chaperones Using SNAP-Tag-Based Imaging to Assess De Novo Histone Deposition.

Methods in enzymology : 97-117 : DOI : 10.1016/bs.mie.2016.04.004 En savoir plus
Résumé

Histone chaperones-key actors in the dynamic organization of chromatin-interact with the various histone variants to ensure their transfer in and out of chromatin. In vitro chromatin assembly assays and isolation of protein complexes using tagged histone variants provided first clues concerning their binding specificities and mode of action. Here, we describe an in vivo method using SNAP-tag-based imaging to assess the de novo deposition of histones and the role of histone chaperones. This method exploits cells expressing SNAP-tagged histones combined with individual cell imaging to visualize directly de novo histone deposition in vivo. We show how, by combining this method with siRNA-based depletion, we could assess the function of two distinct histone chaperones. For this, we provide the details of the method as applied in two examples to characterize the function of the histone chaperones CAF-1 and HIRA. In both cases, we document the impact of their depletion on the de novo deposition of the histone variants H3.1 and H3.3, first in a normal context and second in response to DNA damage. We discuss how this cellular assay offers means to define in a systematic manner the function of any chosen chaperone with respect to the deposition of a given histone variant.

Replier
Charles Auffray, Rudi Balling, Inês Barroso, László Bencze, Mikael Benson, Jay Bergeron, Enrique Bernal-Delgado, Niklas Blomberg, Christoph Bock, Ana Conesa, Susanna Del Signore, Christophe Delogne, Peter Devilee, Alberto Di Meglio, Marinus Eijkemans, Paul Flicek, Norbert Graf, Vera Grimm, Henk-Jan Guchelaar, Yi-Ke Guo, Ivo Glynne Gut, Allan Hanbury, Shahid Hanif, Ralf-Dieter Hilgers, Ángel Honrado, D Rod Hose, Jeanine Houwing-Duistermaat, Tim Hubbard, Sophie Helen Janacek, Haralampos Karanikas, Tim Kievits, Manfred Kohler, Andreas Kremer, Jerry Lanfear, Thomas Lengauer, Edith Maes, Theo Meert, Werner Müller, Dörthe Nickel, Peter Oledzki, Bertrand Pedersen, Milan Petkovic, Konstantinos Pliakos, Magnus Rattray, Josep Redón I Màs, Reinhard Schneider, Thierry Sengstag, Xavier Serra-Picamal, Wouter Spek, Lea A I Vaas, Okker van Batenburg, Marc Vandelaer, Peter Varnai, Pablo Villoslada, Juan Antonio Vizcaíno, John Peter Mary Wubbe, Gianluigi Zanetti (2016 Jun 25)

Making sense of big data in health research: Towards an EU action plan.

Genome medicine : 71 : DOI : 10.1186/s13073-016-0323-y En savoir plus
Résumé

Medicine and healthcare are undergoing profound changes. Whole-genome sequencing and high-resolution imaging technologies are key drivers of this rapid and crucial transformation. Technological innovation combined with automation and miniaturization has triggered an explosion in data production that will soon reach exabyte proportions. How are we going to deal with this exponential increase in data production? The potential of « big data » for improving health is enormous but, at the same time, we face a wide range of challenges to overcome urgently. Europe is very proud of its cultural diversity; however, exploitation of the data made available through advances in genomic medicine, imaging, and a wide range of mobile health applications or connected devices is hampered by numerous historical, technical, legal, and political barriers. European health systems and databases are diverse and fragmented. There is a lack of harmonization of data formats, processing, analysis, and data transfer, which leads to incompatibilities and lost opportunities. Legal frameworks for data sharing are evolving. Clinicians, researchers, and citizens need improved methods, tools, and training to generate, analyze, and query data effectively. Addressing these barriers will contribute to creating the European Single Market for health, which will improve health and healthcare for all Europeans.

Replier
Lisa Prendergast, Sebastian Müller, Yiwei Liu, Hongda Huang, Florent Dingli, Damarys Loew, Isabelle Vassias, Dinshaw J Patel, Kevin F Sullivan, Geneviève Almouzni (2016 Jun 11)

The CENP-T/-W complex is a binding partner of the histone chaperone FACT.

Genes & development : 1313-26 : DOI : 10.1101/gad.275073.115 En savoir plus
Résumé

The CENP-T/-W histone fold complex, as an integral part of the inner kinetochore, is essential for building a proper kinetochore at the centromere in order to direct chromosome segregation during mitosis. Notably, CENP-T/-W is not inherited at centromeres, and new deposition is absolutely required at each cell cycle for kinetochore function. However, the mechanisms underlying this new deposition of CENP-T/-W at centromeres are unclear. Here, we found that CENP-T deposition at centromeres is uncoupled from DNA synthesis. We identified Spt16 and SSRP1, subunits of the H2A-H2B histone chaperone facilitates chromatin transcription (FACT), as CENP-W binding partners through a proteomic screen. We found that the C-terminal region of Spt16 binds specifically to the histone fold region of CENP-T/-W. Furthermore, depletion of Spt16 impairs CENP-T and CENP-W deposition at endogenous centromeres, and site-directed targeting of Spt16 alone is sufficient to ensure local de novo CENP-T accumulation. We propose a model in which the FACT chaperone stabilizes the soluble CENP-T/-W complex in the cell and promotes dynamics of exchange, enabling CENP-T/-W deposition at centromeres.

Replier
Zachary A Gurard-Levin, Laurence O W Wilson, Vera Pancaldi, Sophie Postel-Vinay, Fabricio G Sousa, Cécile Reyes, Elisabetta Marangoni, David Gentien, Alfonso Valencia, Yves Pommier, Paul Cottu, Genevieve Almouzni (2016 May 20)

Chromatin regulators as a guide for cancer treatment choice.

Molecular cancer therapeutics : DOI : molcanther.1008.2015 En savoir plus
Résumé

The limited capacity to predict a patient’s response to distinct chemotherapeutic agents is a major hurdle in cancer management. The efficiency of a large fraction of current cancer therapeutics (radio- and chemotherapies) is influenced by chromatin structure. Reciprocally, alterations in chromatin organization may impact resistance mechanisms. Here, we explore how the mis-expression of chromatin regulators-factors involved in the establishment and maintenance of functional chromatin domains-can inform about the extent of docetaxel response. We exploit gene Affymetrix and NanoString gene expression data for a set of chromatin regulators generated from breast cancer patient-derived xenograft (PDX) models and patient samples treated with docetaxel. Random Forest classification reveals specific panels of chromatin regulators, including key components of the SWI/SNF chromatin remodeler, which readily distinguish docetaxel high-responders and poor-responders. Further exploration of SWI/SNF components in the comprehensive NCI-60 dataset reveals that the expression inversely correlates with docetaxel sensitivity. Finally, we show that loss of the SWI/SNF subunit BRG1 (SMARCA4) in a model cell line leads to enhanced docetaxel sensitivity. Altogether, our findings identify chromatin regulators as biomarkers for drug response as well as therapeutic targets to sensitize patients towards docetaxel and combat drug resistance.

Replier
Geneviève Almouzni, Howard Cedar (2016 May 4)

Maintenance of Epigenetic Information.

Cold Spring Harbor perspectives in biology : DOI : 10.1101/cshperspect.a019372 En savoir plus
Résumé

SUMMARYThe genome is subject to a diverse array of epigenetic modifications from DNA methylation to histone posttranslational changes. Many of these marks are somatically stable through cell division. This article focuses on our knowledge of the mechanisms governing the inheritance of epigenetic marks, particularly, repressive ones, when the DNA and chromatin template are duplicated in S phase. This involves the action of histone chaperones, nucleosome-remodeling enzymes, histone and DNA methylation binding proteins, and chromatin-modifying enzymes. Last, the timing of DNA replication is discussed, including the question of whether this constitutes an epigenetic mark that facilitates the propagation of epigenetic marks.

Replier

Année de publication : 2015

Anne Lafon, Surayya Taranum, Federico Pietrocola, Florent Dingli, Damarys Loew, Sandipan Brahma, Blaine Bartholomew, Manolis Papamichos-Chronakis (2015 Dec 15)

INO80 Chromatin Remodeler Facilitates Release of RNA Polymerase II from Chromatin for Ubiquitin-Mediated Proteasomal Degradation.

Molecular cell : 784-96 : DOI : 10.1016/j.molcel.2015.10.028 En savoir plus
Résumé

Stalling of RNA Polymerase II (RNAPII) on chromatin during transcriptional stress results in polyubiquitination and degradation of the largest subunit of RNAPII, Rpb1, by the ubiquitin proteasome system (UPS). Here, we report that the ATP-dependent chromatin remodeling complex INO80 is required for turnover of chromatin-bound RNAPII in yeast. INO80 interacts physically and functionally with Cdc48/p97/VCP, a component of UPS required for degradation of RNAPII. Cells lacking INO80 are defective in Rpb1 degradation and accumulate tightly bound ubiquitinated Rpb1 on chromatin. INO80 forms a ternary complex with RNAPII and Cdc48 and targets Rpb1 primed for degradation. The function of INO80 in RNAPII turnover is required for cell growth and survival during genotoxic stress. Our results identify INO80 as a bona fide component of the proteolytic pathway for RNAPII degradation and suggest that INO80 nucleosome remodeling activity promotes the dissociation of ubiquitinated Rpb1 from chromatin to protect the integrity of the genome.

Replier
Claire Bonneau, Zachary A Gurard-Levin, Fabrice Andre, Lajos Pusztai, Roman Rouzier (2015 Sep 27)

Predictive and Prognostic Value of the TauProtein in Breast Cancer.

Anticancer research : 5179-84 En savoir plus
Résumé

Predictive markers for response to chemotherapy are required in breast cancer. The Tau protein is a microtubule-associated protein variably expressed in breast cancer. The objective of our study was to describe drug resistance induced by the tau protein, and its predictive and prognostic value in breast cancer.

Replier
Sophie E Polo, Geneviève Almouzni (2015 Sep 15)

Chromatin dynamics after DNA damage: The legacy of the access-repair-restore model.

DNA repair : 114-21 : DOI : 10.1016/j.dnarep.2015.09.014 En savoir plus
Résumé

Eukaryotic genomes are packaged into chromatin, which is the physiological substrate for all DNA transactions, including DNA damage and repair. Chromatin organization imposes major constraints on DNA damage repair and thus undergoes critical rearrangements during the repair process. These rearrangements have been integrated into the « access-repair-restore » (ARR) model, which provides a molecular framework for chromatin dynamics in response to DNA damage. Here, we take a historical perspective on the elaboration of this model and describe the molecular players involved in damaged chromatin reorganization in human cells. In particular, we present our current knowledge of chromatin assembly coupled to DNA damage repair, focusing on the role of histone variants and their dedicated chaperones. Finally, we discuss the impact of chromatin rearrangements after DNA damage on chromatin function and epigenome maintenance.

Replier
Carlos Rivera, Francisco Saavedra, Francisca Alvarez, César Díaz-Celis, Valentina Ugalde, Jianhua Li, Ignasi Forné, Zachary A Gurard-Levin, Geneviève Almouzni, Axel Imhof, Alejandra Loyola (2015 Sep 7)

Methylation of histone H3 lysine 9 occurs during translation.

Nucleic acids research : 9097-106 : DOI : 10.1093/nar/gkv929 En savoir plus
Résumé

Histone post-translational modifications are key contributors to chromatin structure and function, and participate in the maintenance of genome stability. Understanding the establishment and maintenance of these marks, along with their misregulation in pathologies is thus a major focus in the field. While we have learned a great deal about the enzymes regulating histone modifications on nucleosomal histones, much less is known about the mechanisms establishing modifications on soluble newly synthesized histones. This includes methylation of lysine 9 on histone H3 (H3K9), a mark that primes the formation of heterochromatin, a critical chromatin landmark for genome stability. Here, we report that H3K9 mono- and dimethylation is imposed during translation by the methyltransferase SetDB1. We discuss the importance of these results in the context of heterochromatin establishment and maintenance and new therapeutic opportunities in pathologies where heterochromatin is perturbed.

Replier
Camille Clément, Geneviève Almouzni (2015 Aug 6)

MCM2 binding to histones H3-H4 and ASF1 supports a tetramer-to-dimer model for histone inheritance at the replication fork.

Nature structural & molecular biology : 587-9 : DOI : 10.1038/nsmb.3067 En savoir plus
Résumé

Replier
Guillermo A Orsi, Sivakanthan Kasinathan, Gabriel E Zentner, Steven Henikoff, Kami Ahmad (2015 Apr 2)

Mapping regulatory factors by immunoprecipitation from native chromatin.

Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] : 21.31.1-25 : DOI : 10.1002/0471142727.mb2131s110 En savoir plus
Résumé

Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin (ORGANIC) is a high-resolution method that can be used to quantitatively map protein-DNA interactions with high specificity and sensitivity. This method uses micrococcal nuclease (MNase) digestion of chromatin and low-salt solubilization to preserve protein-DNA complexes, followed by immunoprecipitation and paired-end sequencing for genome-wide mapping of binding sites. In this unit, we describe methods for isolation of nuclei and MNase digestion of unfixed chromatin, immunoprecipitation of protein-DNA complexes, and high-throughput sequencing to map sites of bound factors.

Replier
Hiroaki Tachiwana, Sebastian Müller, Julia Blümer, Kerstin Klare, Andrea Musacchio, Geneviève Almouzni (2015 Apr 1)

HJURP involvement in de novo CenH3(CENP-A) and CENP-C recruitment.

Cell reports : 22-32 : DOI : 10.1016/j.celrep.2015.03.013 En savoir plus
Résumé

Although our understanding of centromere maintenance, marked by the histone H3 variant CenH3(CENP-A) in most eukaryotes, has progressed, the mechanism underlying the de novo formation of centromeres remains unclear. We used a synthetic system to dissect how CenH3(CENP-A) contributes to the accumulation of CENP-C and CENP-T, two key components that are necessary for the formation of functional kinetochores. We find that de novo CENP-T accumulation depends on CENP-C and that recruitment of these factors requires two domains in CenH3(CENP-A): the HJURP-binding region (CATD) and the CENP-C-binding region (CAC). Notably, HJURP interacts directly with CENP-C and is critical for de novo accumulation of CENP-C at synthetic centromeres. On the basis of our findings, we propose that HJURP serves a dual chaperone function in coordinating CenH3(CENP-A) and CENP-C recruitment.

Replier
Sihem Cheloufi, Ulrich Elling, Barbara Hopfgartner, Youngsook L Jung, Jernej Murn, Maria Ninova, Maria Hubmann, Aimee I Badeaux, Cheen Euong Ang, Danielle Tenen, Daniel J Wesche, Nadezhda Abazova, Max Hogue, Nilgun Tasdemir, Justin Brumbaugh, Philipp Rathert, Julian Jude, Francesco Ferrari, Andres Blanco, Michaela Fellner, Daniel Wenzel, Marietta Zinner, Simon E Vidal, Oliver Bell, Matthias Stadtfeld, Howard Y Chang, Genevieve Almouzni, Scott W Lowe, John Rinn, Marius Wernig, Alexei Aravin, Yang Shi, Peter J Park, Josef M Penninger, Johannes Zuber, Konrad Hochedlinger (2015 Feb 23)

The histone chaperone CAF-1 safeguards somatic cell identity.

Nature : 218-24 : DOI : 10.1038/nature15749 En savoir plus
Résumé

Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.

Replier