Nuclear Dynamics and Genome Plasticity

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