Spatial Regulation of gene silencing

In most organisms, and most cell types, silent chromatin or heterochromatin is enriched at the nuclear periphery forming subnuclear compartments where general repressors of transcription concentrate, thus favoring silencing establishment at the nuclear periphery (Meister and Taddei, 2013)(Bizhanova and Kaufman, 2021).

In budding yeast, heterochromatin is mainly found at the 32 subtelomeres and the 2 cryptic mating type loci (HM loci) that cluster into 3_5 perinuclear foci. These so-called silencing foci concentrate the yeast silencing complex (SIR: made of Sir2, Sir3 and Sir4), in a manner similar to the mammalian chromocenters in which centromeres cluster together concentrating locally the HP1 protein.

We study silencing foci as models of repressive subnuclear compartments, focusing on the different aspects of their formation.

1. Initiating heterochromatin formation.  

Although the mechanism of initiation is well understood at telomeres and HM loci, we identified new sites of initiations (Dubarry et al, 2011; Loïodice and Taddei 2014; Hocher et al, 2018) along chromosome arms pointing to a yet uncharacterized mechanism of SIR recruitment that seems linked to genome instability (Nikolov and Taddei, 2015). We are currently investigating the mechanisms and function of the recruitment of silencing factors at those non-canonical sites.

1. Spreading (and stopping) heterochromatin.

Once recruited, silent chromatin spreads from defined loci, and diverse mechanisms prevent the ectopic spread of heterochromatin into euchromatin. We showed that the main barrier to heterochromatin is provided by specific histone marks H3K79me3 deposited by the conserved methyltransferase Dot1 preventing heterochromatin to spread into euchromatin (Hocher et al, 2018). Indeed, discrete subtelomeres domains are characterized by specific histone marks that are permissive for SIR spreading leading us to propose a new definition of subtelomeres (Hocher et al, 2018; Hocher and Taddei 2020).

Other limiting factors for SIR spreading are the cellular amounts of Sirs protein. This is why silencing establishment at subtelomeres requires a high local concentration that is achieved through telomere clustering (Meister and Taddei, 2013).

1. Heterochromatin clustering

Previous work from our team have shed light on the mechanisms that are involved in the formation of such subnuclear silent domains. Combining genetics, live microscopy and genetic approaches we first showed that Sir3 promotes telomere clustering independently of heterochromatin formation  (Ruault et al., 2011).

Comparing the dynamics of telomeres clusters with physical models (collaboration with the team of D. Holcman: ENS, Paris), we could show that telomere clusters are dynamic subnuclear compartments resulting from a process of dissociation – aggregation (Hozé et al., 2013).

More recently, combining quantitative microscopy with Hi-C (collaboration with the team of R. Kozsul, I. Pasteur, Paris) we demonstrated that Sir3 is the molecular bridge that mediates trans-interactions between Sir3 bound regions including subtelomeres, HM loci and rDNA, but also some euchromatic sites (Ruault et al., 2021).

Our current projects aim to better characterize the molecular and physical principles underlying the formation, maintenance and dynamics of these subnuclear compartments, focusing on 3 main questions:

• What are the mechanism and function of Sir3 recruitment at specific euchromatic loci?
• What is the physical nature of silencing foci? We address this question by using single molecule microscopy approaches to track individual molecules of Sir proteins at 30 nm and 20Mz resolution in different genetic and physiological contexts (Heltberg et al., 2021; Miné-Hattab and Taddei, 2019; Miné-Hattab et al., 2021)
• What regulate the dynamics of silencing foci in normal conditions and in response to changes in the environmental conditions such as genotoxic events (see project 2) or metabolic transitions (project 3: quiescence)?

The clustering of silent loci, and the recruitment of heterochromatin factors at unstable genomic loci are conserved features of eukaryotic genomes, therefore we anticipate that basic principles will emerge from this study that could apply in other species.

 

Bibliography

Bizhanova, A., and Kaufman, P.D. (2021). Close to the edge: Heterochromatin at the nucleolar and nuclear peripheries. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1864, 194666.

Heltberg, M.L., Miné-Hattab, J., Taddei, A., Walczak, A.M., and Mora, T. (2021). Physical observables to determine the nature of membrane-less cellular sub-compartments (Biophysics).

Hozé, N., Ruault, M., Amoruso, C., Taddei, A., and Holcman, D. (2013). Spatial telomere organization and clustering in yeast Saccharomyces cerevisiae nucleus is generated by a random dynamics of aggregation–dissociation. MBoC 24, 1791–1800.

Meister, P., and Taddei, A. (2013). Building silent compartments at the nuclear periphery: a recurrent theme. Current Opinion in Genetics & Development 23, 96–103.

Miné-Hattab, J., and Taddei, A. (2019). Physical principles and functional consequences of nuclear compartmentalization in budding yeast. Current Opinion in Cell Biology 58, 105–113.

Miné-Hattab, J., Heltberg, M., Villemeur, M., Guedj, C., Mora, T., Walczak, A.M., Dahan, M., and Taddei, A. (2021). Single molecule microscopy reveals key physical features of repair foci in living cells. ELife 10, e60577.

Ruault, M., De Meyer, A., Loïodice, I., and Taddei, A. (2011). Clustering heterochromatin: Sir3 promotes telomere clustering independently of silencing in yeast. The Journal of Cell Biology 192, 417–431.

Ruault, M., Scolari, V.F., Lazar-Stefanita, L., Hocher, A., Loïodice, I., Koszul, R., and Taddei, A. (2021). Sir3 mediates long-range chromosome interactions in budding yeast. Genome Res. 31, 411–425.