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 epigenetic states are stably inherited independently of the underlying DNA sequence. The stability of chromatin domains is reinforced by positive feedback loops that maintain the settings required for establishment. These feedback loops further promote the expansion of heterochromatin along the chromatin fiber (spreading). However, while many of the components and enzymes contributing to the establishment and spreading of heterochromatin have been identified, we still have a poor knowledge of the spatiotemporal control of this process: Where and when do heterochromatic domains form? How is their spreading restricted? Which factors are involved? What are the underlying mechanisms? How are these pathways coordinated? What are the intrinsic and extrinsic signals of this regulation?
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
Through reporter-based genetic screens, we have identified a large number of factors that modulate the silent state of heterochromatin domains (our unpublished data). To understand the roles of these novel factors in heterochromatin regulation, we analyze their genetic interactions quantitatively and 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 on assessing systematically the pair-wise genetic interactions of all identified factors by an advanced version of the E-MAP (Epistasis Mini-Array Profile) approach and examining 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 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 the methyl-reader Pdp3 recruits the acetyltransferase Mst2 to euchromatin via recognition of methylated H3K36. Whereas Mst2 protects euchromatin from ectopic heterochromatin assembly by acetylating Brl1, a histone H2B ubiquitin ligase involved in transcription, lack of Pdp3 results in the mistargeting of Mst2 to heterochromatin triggering a silencing defect. This illustrates how opposing feedback loops maintain the integrity of chromatin domains (Flury et al. Mol Cell 2017). [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 comain. 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]