Compartmentation and Dynamics of Nuclear Functions

Keywords: chromatin, nucleus, epigenetic, transcription, fragile sites

The eukaryotic genome is packaged into large chromatin structures that occupy distinct domains in the cell nucleus. This organisation is thought to be crucial for the normal functions of the genome and, hence, of the cell; defects in architectural elements of the nucleus, for example, are responsible for several human diseases, demonstrating the importance of nuclear organisation for correct cellular function. Not only is the genome organised in three-dimensions but also this spatial organisation changes throughout the cell cycle, at different stage of development and in different metabolic states.

Our aim is to understand what determines the spatial and temporal behaviour of chromatin and how this affects two essential functions of the genome: gene expression and the maintenance of genome integrity. To understand these fundamental processes, we are using yeast as a model system to which we can apply genetics, molecular biology and advanced microscopy of living cells.

Studies of the yeast, Saccharomyces cerevisiae, have demonstrated that a gene's location in the nucleus can play an active role in regulating its expression. Distinct compartments dedicated to either gene silencing or activation of specific genes are located around the periphery of the nucleus (Fig. 1).

Fig. 1 

To gain insights into the mechanisms underlying this compartmentalisation at the nuclear periphery, we address three related questions:

• What mechanisms establish and maintain silent compartments at the nuclear periphery?
• How and why are some activated genes recruited to the nuclear periphery?
• What are the relationships between repressive and activating nuclear compartments?

Recent studies of yeast have indicated that nuclear architecture is important also for genome integrity, i.e. the repair of damaged DNA. Exactly how nuclear organisation influences the formation and processing of DNA lesions, however, remains poorly understood. We are focusing on two aspects of this issue:

• How and where in the nucleus are DNA double-strand breaks recognised, processed, and repaired?
• Where in the nucleus do breaks or gaps in the DNA arise due to perturbations in DNA synthesis at so-called 'fragile sites'?

Fig. 2 

We hope that our research will lead us to a better understanding of the mechanisms underlying the formation of functional nuclear compartments. We aim to discover general rules that apply to the self-organisation of the compartments. These mechanisms are likely to be conserved across species and we therefore plan to test for them in higher eukaryotic cells in collaboration with other laboratories in our Unit UMR218 at the Institut Curie.

Last update: February 2010

Key publications

2009

• Angela Taddei, Griet Van Houwe, Shigeki Nagai, Ionas Erb,
  Erik van Nimwegen and Susan M. Gasser
  The functional importance of telomere clustering:
  Global changes in gene expression result from SIR
  factor dispersion
  Genome Research, 19:611-625 - Abstract
  Budding yeast telomeres and cryptic mating-type loci are enriched at the nuclear envelope, forming foci that sequester silent information regulators (SIR factors), much as heterochromatic chromocenters in higher eukaryotes sequester HP1. Here we examine the impact of such subcompartments for regulating transcription genome-wide. We show that the efficiency of subtelomeric reporter gene repression depends not only on the strength of SIR factor recruitment by cisacting elements, but also on the accumulation of SIRs in such perinuclear foci. To monitor the effects of disrupting this subnuclear compartment, we performed microarray analyses under conditions that eliminate telomere anchoring, while preserving SIR complex integrity. We found 60 genes reproducibly misregulated. Among those with increased expression, 22% were within 20 kb of a telomere, confirming that the nuclear envelope (NE) association of telomeres helps repress natural subtelomeric genes. In contrast, loci that were down-regulated were distributed over all chromosomes. Half of this ectopic repression was SIR complex dependent. We conclude that released SIR factors can promiscuously repress transcription at nontelomeric genes despite the presence of ‘‘anti-silencing'' mechanisms. Bioinformatic analysis revealed that promoters bearing the PAC (RNA Polymerase A and C promoters) or Abf1 binding consenses are consistently downregulated by mislocalization of SIR factors. Thus, the normal telomeric sequestration of SIRs both favors subtelomeric repression and prevents promiscuous effects at a distinct subset of promoters. This demonstrates that patterns of gene expression can be regulated by changing the spatial distribution of repetitive DNA sequences that bind repressive factors.

2008

• Myriam Ruault, Marion Dubarry et Angela Taddei
  Re-positioning genes to the nuclear
  envelope in mammalian cells: impact on transcription
  Trends In Genetics, 24(11): 574-581 - Abstract
  The spatial organization of the genome within the nucleus is thought to contribute to genome functions. A key component of the nuclear architecture is the
  nuclear envelope, which is often associated with inactive chromatin. Studies in budding yeast indicate that nuclear position can directly affect gene function. However, the causal relationship between gene position and gene activity in mammalian cells has been more elusive. Several groups recently addressed this issue by tethering
  genes to the inner nuclear membrane. Their studies show that the nuclear periphery is not refractory to gene transcription, but can modulate the activity of certain genes. The 3D organization of the genome might, thus, provide an additional level of regulation necessary for fine-tuning gene expression.
• Nagai S., Dubrana K., Rsai-Pflugfelder M., Davidson M.B., Roberts T.M., Brown G.W., Varela E., Hediger F., Gasser S.M. & Krogan N.J.
  Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase
  Science, 322, 597-602

2007

• Taddei, A.
  Active genes at the nuclear pore complex
  Curr. Opin. Cell Biol., vol 19(3), 306-10
• Meister P., Taddei A., Ponti A., Baldacci G.#, Gasser S.M.#
  (# These authors contributed equally to this work)
  Spatio-temporal organization of replication is modulated by S-phase checkpoint kinases in fission yeast
  Embo J., 26(5), 1315-1326 - Abstract
  Although the molecular enzymology of DNA replication is well characterized, how and why it occurs in discrete nuclear foci is unclear. Using fission yeast, we show that replication takes place in a limited number of replication foci, whose distribution changes with progression through S phase. These sites define replication factories which contain on average 14 replication forks. We show for the first time that entire foci are mobile, able both to fuse and re-segregate. These foci form distinguishable patterns during S-phase, whose succession is reproducible, defining early-, mid- and late- S phase. In wild-type cells, this same temporal sequence can be detected in the presence of hydroxyurea (HU), despite the reduced rate of replication. However, in cells lacking Cds1, an intra-S checkpoint kinase, replication factories dismantle on HU. Intriguingly, even in the absence of DNA damage the replication foci in cds1 cells assume a novel distribution that is not present in wild-type cells, arguing that Cds1 kinase activity contributes to the spatio-temporal organisation of replication during normal cell growth.

2006

• Meister P., Taddei A., Gasser S.M.
  In and out of the replication factory
  Cell, 125, 1233-1235
• Taddei A., Van Houwe M., Hediger F., Kalck V., Cubizolles F., Schober H., Gasser S.M.
  Nuclear pore association confers optimal expression levels for an inducible yeast gene
  Nature, 441, 774-778 - Abstract
  The organization of the nucleus into subcompartments creates micro-environments that are thought to facilitate distinct nuclear functions 1. In budding yeast, regions of silent chromatin, such as telomeres and mating-type loci, create zones that favor gene repression by clustering at the nuclear envelope1, 2. Other reports indicate that gene transcription occurs at the nuclear periphery, apparently due to an association with nuclear pore complexes 3-5. Here we report that transcriptional activation of a subtelomeric gene HXK1 by growth on a non-glucose carbon source led to its relocalization to nuclear pores. This relocation required the 3'UTR which is essential for efficient message processing and export (consistent with data of Cabal et al. 6). On the other hand, activation of HXK1 by an alternative pathway based on the transactivator VP16 moved the locus away from the nuclear periphery and abrogated the normal induction of HXK1 by galactose. Importantly, when we interfered with HXK1 localization by either antagonizing association with the pore, or promoting it, transcript levels were respectively reduced or enhanced. From this we conclude that nuclear position has an active role in determining optimal gene expression levels.

2005

• Taddei A.#, Meister P.#, Vernis L., Poidevin M., Gasser S.M., Baldacci G.
  (# These authors contributed equally to this work)
  Intra-S checkpoint represses recombination at stalled replication forks
  J. Cell Biol., 168: 537-544

2004

• Taddei A., Hediger F., Neumann F.R., Gasser S.M.
  The function of nuclear architecture: a genetic approach. Annual Review of Genetics
  Annual Rev. Genet., 38, 305-345
• Taddei A., Hediger F., Bauer, C., Neumann F.R., Gasser S.M.
  Separation of silencing from perinuclear anchoring functions in yeast Ku80, Sir4 and Esc1 proteins
  EMBO J., 23, 1301-1312