Heterochromatin is regulated by numerous factors acting through interconnected pathways, yet identifying individual components does not explain how these pathways work together. We therefore use systematic genetics combined with sensitive, quantitative reporter assays to identify regulators of gene silencing and determine how their functions depend on chromosomal context.
Over several studies, genome-wide mutant screens have allowed us to uncover factors controlling distinct aspects of heterochromatin regulation. These approaches contributed, for example, to identifying mechanisms involving Epe1 degradation, PAF complex-dependent restriction of heterochromatin spreading, the nuclear membrane protein Lem2, and euchromatin protection by Mst2 (Braun et al., Cell, 2011; Verrier et al., Open Biol, 2015; Barrales et al., Genes Dev, 2016; Flury et al., Mol Cell, 2017).
More recently, we expanded this strategy into a systematic quantitative analysis of heterochromatin regulation across the genome. By screening mutant libraries with reporters positioned at pericentromeres, the silent mating-type locus, subtelomeres and telomeres, we identified 369 mutants with reduced or enhanced silencing. The sensitivity and breadth of this approach substantially expanded the spectrum of factors implicated in heterochromatin silencing compared with previous genetic screens, including partial and context-dependent phenotypes that would otherwise be difficult to detect (Muhammad, Sarkadi et al., Nucleic Acids Res, 2024).
Rather than revealing a single universal silencing pathway, these screens uncovered striking quantitative and qualitative differences between heterochromatin domains. Core heterochromatin factors act broadly, whereas many other pathways affect only selected chromosomal regions; for example, several metabolic pathways preferentially influence subtelomeric silencing.
The resulting multidimensional phenotypic profiles provide additional information beyond individual mutant phenotypes. Factors with related functions tend to display similar profiles, allowing us to identify functional relationships, distinguish regulatory pathways, and even resolve submodules within larger chromatin complexes. This illustrates how quantitative genetics can move from identifying individual regulators toward understanding the organization of heterochromatin control at the systems level.
Systematic quantitative screening identifies heterochromatin regulators and reveals domain-specific phenotypic profiles. Reporter screens across distinct heterochromatin domains uncover factors with reduced or enhanced silencing and substantially expand the spectrum of regulators identified by previous genetic screens (Muhammad, Sarkadi et al., Nucleic Acids Res, 2024).
From screening hit to mechanism: Dhm2
One factor emerging from our systematic screens is Dhm2, a previously poorly characterized protein required for robust heterochromatin maintenance and the inheritance of repressive chromatin states. Loss of Dhm2 also causes defects in DNA replication and increased replication stress, pointing to an unexpected connection between heterochromatin inheritance and DNA replication. We are currently investigating the molecular function of Dhm2 and how it contributes to the faithful propagation of heterochromatin through cell division (Muhammad, Sarkadi et al., Nucleic Acids Res, 2024).
Our long-term goal is to reconstruct the functional architecture of silencing networks. We are therefore extending the reporter-based approach by systematically combining mutations and examining their effects across different heterochromatin domains, reporter systems and environmental conditions.
Using principles of epistasis mini-array profiling (E-MAP), these multidimensional genetic interaction profiles can reveal whether factors act in the same pathway, in parallel pathways, or in distinct chromatin contexts. Ultimately, this should allow us to organize silencing factors into functional pathways and regulatory networks and determine how these networks are reshaped across genomic locations and cellular conditions.