Set1/COMPASS repels heterochromatin invasion at euchromatic sites by disrupting Suv39/Clr4 activity and nucleosome stability

Abstract

Protection of euchromatin from invasion by gene-repressive heterochromatin is critical for cellular health and viability. In addition to constitutive loci such as pericentromeres and subtelomeres, heterochromatin can be found interspersed in gene-rich euchromatin, where it regulates gene expression pertinent to cell fate. While hetero-and euchromatin are globally poised for mutual antagonism, the mechanisms underlying precise spatial encoding of heterochromatin containment within euchromatic sites remain opaque. We investigated ectopic heterochromatin invasion by manipulating the fission yeast mating type locus boundary, using a single-cell spreading reporter system. We found that heterochromatin repulsion is locally encoded by Set1/COMPASS on certain actively transcribed genes and that this protective role is most prominent at heterochromatin islands, small domains interspersed in euchromatin that regulate cell fate specifiers. Interestingly, this effect can be gene orientation dependent. Sensitivity to invasion by heterochromatin, surprisingly, is not dependent on Set1 altering overall gene expression levels. At least two independent pathways direct this Set1 activity-inhibition of catalysis by Suv39/Clr4 and disruption of nucleosome stability. Taken together, these results describe a mechanism for spatial encoding of euchromatic signals that repel heterochromatin invasion.

Local chromatin context dictates the genetic determinants of the heterochromatin spreading reaction

“…Several Bioneer collection mutants were independently validated and produced de novo. For the ectopic locus HSS reporter strain, the screen was performed essentially as described (Greenstein et al 2019). Briefly, the parent HSS reporter strain was crossed to the library.…”

“…Crosses were performed as described (Verrier et al 2015; Barrales et al 2016; Greenstein et al 2018; Greenstein et al 2019) using a RoToR HDA colony pinning robot (Singer). For the MAT HSS reporter strains, the screen was performed essentially as described (Greenstein et al 2019) with the exception that crosses were generated using a 96 well manual pinner. Note that the MAT ΔcenH, we used an OFF isolate as described (Greenstein et al 2018).…”

“…Silencing is instructed by assembly factors, such as HP1, that recognize heterochromatic chemical modifications (Jacobs et al 2001; Lachner et al 2001). The spreading of silencing structures occurs in at least two very different chromatin contexts: (1) Constitutive heterochromatin, which is generally gene-poor and therefore depleted of activities associated with active genes known to antagonize heterochromatin (Scott et al 2006; Greenstein et al 2019); or (2) heterochromatin involved in regulating cellular differentiation, which is either seeded at new nucleation sites or invades gene-rich euchromatin de-novo from existing nucleation sites (Wen et al 2009; Zhu et al 2013; Zylicz et al 2015; Zylicz et al 2018; Nicetto and Zaret 2019). In either scenario, specific factors may intrinsically promote or antagonize this process.…”

“…This inheritance promotes modification of nearby nucleosomes due to the “read-write” positive feedback intrinsic to heterochromatin histone modifiers (Zhang et al 2008; Al-Sady et al 2013; Ragunathan et al 2015). When the spreading process invades active chromatin de novo, as occurs in differentiation, it will encounter chromatin modifications that can specifically antagonize heterochromatin (Greenstein et al 2019). Thus, during this initial invasion process, heterochromatin spreading does not benefit from the inheritance through replication of pre-existing marked nucleosomes.…”

“…However, less is understood about factors specifically required for the spreading process. With our previously established fluorescent reporter-based heterochromatin spreading sensor, we can segregate the central output of heterochromatin (gene silencing) from the spatial control of the reaction (spreading) (Greenstein et al 2018; Greenstein et al 2019). This allows us to address the following questions: (1) Are there known or novel regulators of heterochromatin that primarily regulate spreading, versus nucleation?…”