The research of our lab focuses on the identification of regulatory factors and elucidating the molecular mechanisms by which they control heterochromatin.

Eukaryotic genomes are organized into distinct functional domains that are transcriptionally active (euchromatin) or repressed (heterochromatin). Once established, these chromatin domains are stably maintained by positive feedback loops and often independently of the underlying DNA sequence contributing to epigenetic inheritance. These feedback loops also promote the expansion of heterochromatin along with the chromatin fiber (spreading). However, while many components and enzymes contributing to heterochromatin establishment and spreading have been identified, we still have poor knowledge of the spatiotemporal control of this process. In particular, we seek to understand:

  • Which factors control when and where heterochromatin forms?
  • What are the underlying molecular mechanisms?
  • How are these pathways coordinated and dynamically regulated?
  • What limits its spreading beyond natural boundaries?
  • How does the nuclear organization contribute to gene expression?
  • How is the genome integrity maintained, and how is the stability of repetitive elements controlled?
  • How are these processes altered in response to the environment?

To address these questions, our lab applies several strategies. Using functional genome-wide screens, we seek to identify novel factors that modulate the heterochromatic state. Using functional genomics, we examine how these factors cooperate by dissecting regulatory pathways and networks. Using live-cell imaging, molecular biology, and biochemistry methods, we investigate the mechanism of heterochromatin regulation by these factors.


[1] Identifying novel factors and dissecting regulatory networks

We have identified a large number of factors that modulate the silent state of heterochromatin domains through reporter-based genetic screens (our unpublished data). To understand the roles of these novel factors in heterochromatin regulation, we analyze the behavior of the mutants systematically under different conditions and in the context of other mutations (genetic interactions) at the genome-wide level using SGA (Synthetic Gene Arrays).

This allows us to dissect mutations within the same pathway displaying non-additive phenotypes from mutants of parallel pathways exhibiting synthetic phenotypes (Verrier et al. Open Biol 2015; Barrales et al. Genes Dev 2016; Flury et al. Mol Cell 2017; Salas-Pino et al. JCB 2017). Future work aims to systematically assess the pair-wise genetic interactions of all identified factors by an advanced version of the E-MAP (Epistasis Mini-Array Profile) approach and examine the role of stress in heterochromatin regulation. [Read more]

[2] Demarcation and protection of heterochromatin domains

Partitioning into active and silent chromatin requires mechanisms that specify boundaries and maintain the identity of chromatin domains. We previously discovered a novel mechanism by which the chromatin distribution of an anti-silencing factor is shaped through ubiquitin-dependent degradation. Removal takes place within the body of heterochromatin but not at the boundaries, where this anti-silencing factor prevents heterochromatin from spreading into neighboring euchromatin. This demonstrates that chromatin domains can be formed through the selective removal of associated factors, adding a novel concept that we coined ‘chromatin sculpting‘ (Braun et al. Cell 2011). The integrity of chromatin domains can also be protected through anchoring to specific chromatin sites, thereby preventing promiscuous binding. In collaboration with Marc Bühler’s lab, we showed that sequestration of the acetyltransferase Mst2 to actively transcribed chromatin protects euchromatin from ectopic heterochromatin assembly. The same mechanism prevents Mst2 mistargeting to heterochromatin. This illustrates how opposing feedback loops maintain the integrity of chromatin domains (Flury et al. Mol Cell 2017; Georgescu et al., Microbial Cell 2020). [Read more]

[3] Regulation at the nuclear periphery

The nuclear periphery provides a specialized subcompartment that promotes the establishment and maintenance of silent chromatin. We showed that the inner nuclear membrane protein Lem2 is involved in silencing and localization of heterochromatin and part of a network of multiple redundant silencing pathways (Barrales et al. Genes Dev 2016).

Lem2 belongs to the family of LEM domain-containing proteins conserved from yeast to humans. While Lem2 associates with centromeric chromatin via its LEM domain, it mediates silencing through another conserved domain called the MSC domain. Thus, centromere localization and silencing can be mechanistically separated. These findings reveal an unanticipated complexity and shift the focus from the well-studied LEM domain to the poorly understood MSC domain. [Read more]

[4] Genome maintenance of repetitive sequences

DNA repeats are often embedded into heterochromatin to prevent unwarranted recombination and genome instability. However, during DNA damage or loss, recombination of these repeats may become necessary. We showed recently that repeat-like sequences present at subtelomeres contain metastable nucleosomes and are hyper-recombinogenic, promoting telomere plasticity and recombination (van Emden, Forn, et al., 2019).

This is an intrinsic property of the underlying DNA sequences and counteracted by Ccq1, a member of the conserved shelterin complex. Ccq1 recruits heterochromatin factors to stabilize nucleosomes at these refractory sequences. How recombination. However, how Ccq1 prevents subtelomeric rearrangements and what is the physiological relevance is yet poorly understood. We favor the hypothesis that these fragile DNA sequences play an active role in telomere maintenance independent of telomerase, similar to the ALT (alternative lengthening of telomeres) in human cancer cells.

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