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Chromatin dynamics

Keywords : genome stability, chromatin dynamics, regulation of nuclear functions

Group Leader: Geneviève Almouzni

IN BRIEF

Beyond DNA that carries genomic information, we have come to the realization that its packaging into the nucleus in the form of chromatin provides a major source of information. How this information is imposed, changed and/or inherited is a central issue in the field of epigenetics.

Our team is interested by the dynamics of this organization during development and cellular division. How this is responding to various environmental conditions and possibly compromised in various pathologies will be important to decipher.

 

 

 

 


 

RESEARCH THEMATIC

In all eukaryotic cells, the genome is organized within the nucleus in a nucleoprotein structure called chromatin (Fig. 1).

Figure 1 : The various levels of chromatin organization. Left panel, Artistic representation; Right panel : Schematic representation (Probst et al., 2009)

Chromatin organisation in the nucleus is important both for the compaction of DNA into the nucleus and for the control of many genome functions including DNA transcription, replication, repair and recombination. This ‘packaging' of the genome provides a large repertoire of information in addition to that encoded genetically. This layer of so-called ‘epigenetic' information is stable and can be inherited through cell division, even though it is not encoded genetically (depicted artistically in Fig. 2).

 

 

Figure 2 : The establishment and maintenance of epigenetic profiles defines cellular identity, as illustrated on the cover of a special issue of Cell (2007) dedicated to epigenetics.

 

One way in which epigenetic information can be conveyed within chromatin is by modifying DNA or by using distinct variants of histones, the major protein components of chromatin and by apposing particular post-translational modification onto them. According to the ‘histone code hypothesis' these post-translational marks can be read by the cell and thus used to define active, repressed or inert chromatin states. Each cell type is thought to display a specific 'epigenome‘. Furthermore, other chromatin proteins, and non coding RNA can also contribute in potentiating chromatin states and nuclear organization.

Our team is interested in understanding how both genetic and epigenetic information is established, propagated, and maintained, as well as how it may change during development and in response to environmental cues. Potential errors can lead to misregulation of genome functions, which may have implications for various diseases, including cancer. We hope, therefore, that our research will contribute not only to a better understanding of nuclear organisation during the normal life of the cell but also to understanding pathological conditions such as cancer and, ultimately, to treating cancer.

Our general objective has been to dissect the mechanisms of chromatin assembly, from the basic structural unit, the nucleosome, up to higher-order structures in the nucleus (Fig. 3).

 

Figure 3 : Chromatin assembly, from the level of the nucleosome to entire nuclear domains, is important in different physiological contexts and during various DNA transactions (Polo and Almouzni, 2006). 

 

Over the past few years (2005-2010) we have focused on the roles of histone chaperones. Because histones are very basic proteins, they tend to interact non-specifically with more acidic proteins and with nucleic acids. These chaperone proteins help to transfer histones from one site to another, for example, to load histones onto DNA during chromatin assembly. Also, a constantly controlled flow of histones enables the cell to adapt to physiological demands during the cell cycle and development as well as in response to DNA damage.

We have characterized several key chaperones involved in nucleosome assembly: CAF-1, HIRA, ASF1a and ASF1b, and HJURP. We initially identified CAF-1 as a marker of cell proliferation in breast cancer and recently found that Asf1b proves an attractive prognostic marker correlating with high metastasis incidence. HJURP, the chaperone of the centromeric histone variant CENP-A is also promising. We also found these chaperones to be part of multiprotein complexes in vivo, with different specificities for individual histone H3 variants (Fig. 4).

 

Figure 4 : Asf-1, CAF-1, HIRA and HJURP, H3 chaperones which promote deposition of dimers of histones (Polo and Almouzni, 2006). H3-H4 dimers associate with histone chaperones in pre-deposition complexes (upper panel), suggesting that H3 and H4 are deposited as dimers like H2A and H2B in the course of de novo nucleosome formation (lower panel). Whether the reverse reaction can involve similar intermediates and complexes is not known. Red arrows highlight the binding interface involved both in H3 dimerization and H3-ASF1 interaction. H3 variants are indicated.

 

We were able to define the dynamics of new histone incorporation during repair of UV damage in chromatin. Interestingly, specific modifications can be found on histones even before their incorporation into chromatin. Taken together, our findings have thrown light on the fundamental issues of the dynamics, fate and inheritance of histone H3 variants together with their specific marks typical of particular chromatin domains. A current challenge is to understand how the maintenance and duplication of both genetic and epigenetic information is ensured and coordinated. Our working hypothesis is that histone chaperones function in an ‘assembly line', and that their specificity for individual histone variants contributes to the specific marking of defined regions of the genome.

Our long term plan is to analyse the regulatory pathways that target histone chaperones to control the assembly line and its connecting network. Our specific approach is based on tools and model systems that combine biochemistry, to study complexes at a molecular level, and cell biology, to integrate them in vivo and examine specific nuclear domains e.g. centromeric heterochromatin (Fig. 5).

Figure 5 : Centromere organization. A) Regions of heterochromatin within centromeric regions are composed of numerous DNA repeats that are known as major and minor satellite repeats. These repeats are heavily methylated. Hypoacetylated histones and methylated histone H3-K9 (histone methylation is shown by a blue star) are present as well as the H3-K9 histone methyltransferase,which is known as Suv39h. H3-K9 modification provides binding sites for HP1 proteins in mouse cells and Swi6 in S. pombe. The centric region of the inner domain is represented by a red eight-pointed star. The RNA component is illustrated as a red line between HP1 and H3. (Maison and Almouzni, 2004. B) Major (green) and minor (red) satellite DNA visualized by FISH define 3-dimensional domains in mouse interphase nuclei (Guenatri, et al., 2004). 

 

The centromere, an essential chromosomal component for proper genome maintenance represents a paradigm of a region which identity is epigenetically specified. Understanding the maintenance of this organization throughout multiple cellular division will be a central question in our research. How its function is influenced by the organization of the pericentric heterochromatin is also an important aspect that have to be considered. In particular, we have searched for parameters important for HP1 targeting to this loci. We find that modification of HP1 by sumoylation is key for its association with RNAs encoded in pericentric regions. This enables to promote de novo targeting of HP1 to pericentric heterochromatin.

Our developmental studies exploits mice as a mammalian system with a strong genetic and Xenopus, an amphibian, as a model organism in which we can validate the relevance of our findings from experiments in vitro. Together, these studies should ultimately help to develop medical applications. Our most recent results illustrate the critical role of chromatin assembly factors as well as non-coding RNAs in the control of higher-order chromatin organization in pre-implantation embryos in which a major genome reprogramming is observed.

Our team was a former co-ordinating member of the Epigenome Network of Excellence: the focal point for the European epigenetics research community, and member of the Research Training Network working on checkpoints, the DNA damage response and cancer. Now, we are coordinating the Epigenesys Network of Excellence building on the previous NoE to take the new challenges that move epigenetics towards systems biology. In addition, we are involved in two International Training Networks dealing respectively with Nucleosome variation in function, and with imaging DNA Damage Response.

For Epigenesys see : http://www.epigenesys.org

Key publications

  • Year of publication : 2011

  • HP1 enrichment at pericentric heterochromatin is considered important for centromere function. Although HP1 binding to H3K9me3 can explain its accumulation at pericentric heterochromatin, how it is initially targeted there remains unclear. Here, in mouse cells, we reveal the presence of long nuclear noncoding transcripts corresponding to major satellite repeats at the periphery of pericentric heterochromatin. Furthermore, we find that major transcripts in the forward orientation specifically associate with SUMO-modified HP1 proteins. We identified this modification as SUMO-1 and mapped it in the hinge domain of HP1alpha. Notably, the hinge domain and its SUMOylation proved critical to promote the initial targeting of HP1alpha to pericentric domains using de novo localization assays, whereas they are dispensable for maintenance of HP1 domains. We propose that SUMO-HP1, through a specific association with major forward transcript, is guided at the pericentric heterochromatin domain to seed further HP1 localization.

  • Proper genome packaging requires coordination of both DNA and histone metabolism. While histone gene transcription and RNA processing adequately provide for scheduled needs, how histone supply adjusts to unexpected changes in demand remains unknown. Here, we reveal that the histone chaperone nuclear autoantigenic sperm protein (NASP) protects a reservoir of soluble histones H3-H4. The importance of NASP is revealed upon histone overload, engagement of the reservoir during acute replication stress, and perturbation of Asf1 activity. The reservoir can be fine-tuned, increasing or decreasing depending on the level of NASP. Our data suggest that NASP does so by balancing the activity of the heat shock proteins Hsc70 and Hsp90 to direct H3-H4 for degradation by chaperone-mediated autophagy. These insights into NASP function and the existence of a tunable reservoir in mammalian cells demonstrate that contingency is integrated into the histone supply chain to respond to unexpected changes in demand.

  • Establishment of a proper chromatin landscape is central to genome function. Here, we explain H3 variant distribution by specific targeting and dynamics of deposition involving the CAF-1 and HIRA histone chaperones. Impairing replicative H3.1 incorporation via CAF-1 enables an alternative H3.3 deposition at replication sites via HIRA. Conversely, the H3.3 incorporation throughout the cell cycle via HIRA cannot be replaced by H3.1. ChIP-seq analyses reveal correlation between HIRA-dependent H3.3 accumulation and RNA pol II at transcription sites and specific regulatory elements, further supported by their biochemical association. The HIRA complex shows unique DNA binding properties, and depletion of HIRA increases DNA sensitivity to nucleases. We propose that protective nucleosome gap filling of naked DNA by HIRA leads to a broad distribution of H3.3, and HIRA association with Pol II ensures local H3.3 enrichment at specific sites. We discuss the importance of this H3.3 deposition as a salvage pathway to maintain chromatin integrity.

  • Year of publication : 2010

  • At the time of fertilization, the paternal genome lacks the typical configuration and marks characteristic of pericentric heterochromatin. It is thus essential to understand the dynamics of this region during early development, its importance during that time period and how a somatic configuration is attained. Here, we show that pericentric satellites undergo a transient peak in expression precisely at the time of chromocenter formation. This transcription is regulated in a strand-specific manner in time and space and is strongly biased by the parental asymmetry. The transcriptional upregulation follows a developmental clock, yet when replication is blocked chromocenter formation is impeded. Furthermore, interference with major satellite transcripts using locked nucleic acid (LNA)-DNA gapmers results in developmental arrest before completion of chromocenter formation. We conclude that the exquisite strand-specific expression dynamics at major satellites during the 2-cell stage, with both up and downregulation, are necessary events for proper chromocenter organization and developmental progression.

  • Year of publication : 2009

  • The histone H3 variant CenH3, called CENP-A in humans, is central in centromeric chromatin to ensure proper chromosome segregation. In the absence of an underlying DNA sequence, it is still unclear how CENP-A deposition at centromeres is determined. Here, we purified non-nucleosomal CENP-A complexes to identify direct CENP-A partners involved in such a mechanism and identified HJURP. HJURP was not detected in H3.1- or H3.3-containing complexes, indicating its specificity for CENP-A. HJURP centromeric localization is cell cycle regulated, and its transient appearance at the centromere coincides precisely with the proposed time window for new CENP-A deposition. Furthermore, HJURP downregulation leads to a major reduction in CENP-A at centromeres and impairs deposition of newly synthesized CENP-A, causing mitotic defects. We conclude that HJURP is a key factor for CENP-A deposition and maintenance at centromeres.

  • Studies that concern the mechanism of DNA replication have provided a major framework for understanding genetic transmission through multiple cell cycles. Recent work has begun to gain insight into possible means to ensure the stable transmission of information beyond just DNA, and has led to the concept of epigenetic inheritance. Considering chromatin-based information, key candidates have arisen as epigenetic marks, including DNA and histone modifications, histone variants, non-histone chromatin proteins, nuclear RNA as well as higher-order chromatin organization. Understanding the dynamics and stability of these marks through the cell cycle is crucial in maintaining a given chromatin state.

  • Year of publication : 2007

  • In eukaryotes, DNA is organized into chromatin in a dynamic manner that enables it to be accessed for processes such as transcription and repair. Histones, the chief protein component of chromatin, must be assembled, replaced or exchanged to preserve or change this organization according to cellular needs. Histone chaperones are key actors during histone metabolism. Here we classify known histone chaperones and discuss how they build a network to escort histone proteins. Molecular interactions with histones and their potential specificity or redundancy are also discussed in light of chaperone structural properties. The multiplicity of histone chaperone partners, including histone modifiers, nucleosome remodelers and cell-cycle regulators, is relevant to their coordination with key cellular processes. Given the current interest in chromatin as a source of epigenetic marks, we address the potential contributions of histone chaperones to epigenetic memory and genome stability.

  • Inheritance and maintenance of the DNA sequence and its organization into chromatin are central for eukaryotic life. To orchestrate DNA-replication and -repair processes in the context of chromatin is a challenge, both in terms of accessibility and maintenance of chromatin organization. To meet the challenge of maintenance, cells have evolved efficient nucleosome-assembly pathways and chromatin-maturation mechanisms that reproduce chromatin organization in the wake of DNA replication and repair. The aim of this Review is to describe how these pathways operate and to highlight how the epigenetic landscape may be stably maintained even in the face of dramatic changes in chromatin structure.

  • DNA replication in eukaryotes requires nucleosome disruption ahead of the replication fork and reassembly behind. An unresolved issue concerns how histone dynamics are coordinated with fork progression to maintain chromosomal stability. Here, we characterize a complex in which the human histone chaperone Asf1 and MCM2-7, the putative replicative helicase, are connected through a histone H3-H4 bridge. Depletion of Asf1 by RNA interference impedes DNA unwinding at replication sites, and similar defects arise from overproduction of new histone H3-H4 that compromises Asf1 function. These data link Asf1 chaperone function, histone supply, and replicative unwinding of DNA in chromatin. We propose that Asf1, as a histone acceptor and donor, handles parental and new histones at the replication fork via an Asf1-(H3-H4)-MCM2-7 intermediate and thus provides a means to fine-tune replication fork progression and histone supply and demand.

  • Year of publication : 2006

  • Histone posttranslational modifications (PTMs) and sequence variants regulate genome function. Although accumulating evidence links particular PTM patterns with specific genomic loci, our knowledge concerning where and when these PTMs are imposed remains limited. Here, we find that lysine methylation is absent prior to histone incorporation into chromatin, except at H3K9. Nonnucleosomal H3.1 and H3.3 show distinct enrichments in H3K9me, such that H3.1 contains more K9me1 than H3.3. In addition, H3.3 presents other modifications, including K9/K14 diacetylated and K9me2. Importantly, H3K9me3 was undetectable in both nonnucleosomal variants. Notably, initial modifications on H3 variants can potentiate the action of enzymes as exemplified with Suv39HMTase to produce H3K9me3 as found in pericentric heterochromatin. Although the set of initial modifications present on H3.1 is permissive for further modifications, in H3.3 a subset cannot be K9me3. Thus, initial modifications impact final PTMs within chromatin.

  • Chromatin organization is compromised during the repair of DNA damage. It remains unknown how and to what extent epigenetic information is preserved in vivo. A central question is whether chromatin reorganization involves recycling of parental histones or new histone incorporation. Here, we devise an approach to follow new histone deposition upon UV irradiation in human cells. We show that new H3.1 histones get incorporated in vivo at repair sites. Remarkably we find that H3.1, which is deposited during S phase, is also incorporated outside of S phase. Histone deposition is dependent on nucleotide excision repair (NER), indicating that it occurs at a postrepair stage. The histone chaperone chromatin assembly factor 1 (CAF-1) is directly involved in the histone deposition process in vivo. We conclude that chromatin restoration after damage cannot rely simply on histone recycling. New histone incorporation at repair sites both challenges epigenetic stability and possibly contributes to damage memory.

  • Year of publication : 2005

  • Maintenance of chromosomal integrity requires tight coordination of histone biosynthesis with DNA replication. Here, we show that extracts from human cells exposed to replication stress display an increased capacity to support replication-coupled chromatin assembly. While in unperturbed S phase, hAsf1 existed in equilibrium between an active form and an inactive histone-free pool, replication stress mobilized the majority of hAsf1 into an active multichaperone complex together with histones. This active multichaperone complex was limiting for chromatin assembly in S phase extracts, and hAsf1 was required for the enhanced assembly activity in cells exposed to replication stress. Consistently, siRNA-mediated knockdown of hAsf1 impaired the kinetics of S phase progression. Together, these data suggest that hAsf1 provides the cells with a buffering system for histone excess generated in response to stalled replication and explains how mammalian cells maintain a critical "active" histone pool available for deposition during recovery from replication stresses.

  • Year of publication : 2004

  • To investigate how the complex organization of heterochromatin is reproduced at each replication cycle, we examined the fate of HP1-rich pericentric domains in mouse cells. We find that replication occurs mainly at the surface of these domains where both PCNA and chromatin assembly factor 1 (CAF-1) are located. Pulse-chase experiments combined with high-resolution analysis and 3D modeling show that within 90 min newly replicated DNA become internalized inside the domain. Remarkably, during this time period, a specific subset of HP1 molecules (alpha and gamma) coinciding with CAF-1 and replicative sites is resistant to RNase treatment. Furthermore, these replication-associated HP1 molecules are detected in Suv39 knockout cells, which otherwise lack stable HP1 staining at pericentric heterochromatin. This replicative pool of HP1 molecules disappears completely following p150CAF-1 siRNA treatment. We conclude that during replication, the interaction of HP1 with p150CAF-1 is essential to promote delivery of HP1 molecules to heterochromatic sites, where they are subsequently retained by further interactions with methylated H3-K9 and RNA.

  • Deposition of the major histone H3 (H3.1) is coupled to DNA synthesis during DNA replication and possibly DNA repair, whereas histone variant H3.3 serves as the replacement variant for the DNA-synthesis-independent deposition pathway. To address how histones H3.1 and H3.3 are deposited into chromatin through distinct pathways, we have purified deposition machineries for these histones. The H3.1 and H3.3 complexes contain distinct histone chaperones, CAF-1 and HIRA, that we show are necessary to mediate DNA-synthesis-dependent and -independent nucleosome assembly, respectively. Notably, these complexes possess one molecule each of H3.1/H3.3 and H4, suggesting that histones H3 and H4 exist as dimeric units that are important intermediates in nucleosome formation. This finding provides new insights into possible mechanisms for maintenance of epigenetic information after chromatin duplication.

  • Year of publication : 2002

  • Post-translational modification of histone tails is thought to modulate higher-order chromatin structure. Combinations of modifications including acetylation, phosphorylation and methylation have been proposed to provide marks recognized by specific proteins. This is exemplified, in both mammalian cells and fission yeast, by transcriptionally silent constitutive pericentric heterochromatin. Such heterochromatin contains histones that are generally hypoacetylated and methylated by Suv39h methyltransferases at lysine 9 of histone H3 (H3-K9). Each of these modification states has been implicated in the maintenance of HP1 protein-binding at pericentric heterochromatin, in transcriptional silencing and in centromere function. In particular, H3-K9 methylation is thought to provide a marking system for the establishment and maintenance of stably repressed regions and heterochromatin subdomains. To address the question of how these two types of modifications, as well as other unidentified parameters, function to maintain pericentric heterochromatin, we used a combination of histone deacetylase inhibitors, RNAse treatments and an antibody raised against methylated branched H3-K9 peptides. Our results show that both H3-K9 acetylation and methylation can occur on independent sets of H3 molecules in pericentric heterochromatin. In addition, we identify an RNA- and histone modification-dependent structure that brings methylated H3-K9 tails together in a specific configuration required for the accumulation of HP1 proteins in these domains.