The heterochromatin spreading reaction is a central contributor to the formation of gene-repressive structures, which are re-established with high positional precision, or fidelity, following replication. How the spreading reaction contributes to this fidelity is not clear. To resolve the origins of stable inheritance of repression, we probed the intrinsic character of spreading events in fission yeast using a system that quantitatively describes the spreading reaction in live single cells. We show that spreading triggered by noncoding RNA-nucleated elements is stochastic, multimodal, and fluctuates dynamically across time. This lack of stability correlates with high histone turnover. At the mating type locus, this unstable behavior is restrained by an accessory cis-acting element REIII, which represses histone turnover. Further, REIII safeguards epigenetic memory against environmental perturbations. Our results suggest that the most prevalent type of spreading, driven by noncoding RNA-nucleators, is epigenetically unstable and requires collaboration with accessory elements to achieve high fidelity.

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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 heterochromatin 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. Sensitivity to invasion by heterochromatin, surprisingly, is not dependent on Set1 altering overall gene expression levels. Rather, the gene-protective effect is strictly dependent on Set1's catalytic activity. H3K4 methylation, the Set1 product, antagonizes spreading in two ways: directly inhibiting catalysis by Suv39/Clr4 and locally disrupting nucleosome stability. Taken together, these results describe a mechanism for spatial encoding of euchromatic signals that repel heterochromatin invasion.

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.

Heterochromatin spreading, the expansion of repressive chromatin structure from sequence-specific nucleation sites, is critical for stable gene silencing. Spreading re-establishes gene-poor constitutive heterochromatin across cell cycles but can also invade gene-rich euchromatin de novo to steer cell fate decisions. How chromatin context (i.e. euchromatic, heterochromatic) or different nucleation pathways influence heterochromatin spreading remains poorly understood. Previously, we developed a single-cell sensor in fission yeast that can separately record heterochromatic gene silencing at nucleation sequences and distal sites. Here we couple our quantitative assay to a genetic screen to identify genes encoding nuclear factors linked to the regulation of heterochromatin nucleation and the distal spreading of gene silencing. We find that mechanisms underlying gene silencing distal to a nucleation site differ by chromatin context. For example, Clr6 histone deacetylase complexes containing the Fkh2 transcription factor are specifically required for heterochromatin spreading at constitutive sites. Fkh2 recruits Clr6 to nucleation-distal chromatin sites in such contexts. In addition, we find that a number of chromatin remodeling complexes antagonize nucleation-distal gene silencing. Our results separate the regulation of heterochromatic gene silencing at nucleation versus distal sites and show that it is controlled by context-dependent mechanisms. The results of our genetic analysis constitute a broad community resource that will support further analysis of the mechanisms underlying the spread of epigenetic silencing along chromatin.

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Heterochromatin spreading, the propagation of repressive chromatin along the chromosome, is a reaction critical to genome stability and defense, as well as maintenance of unique cell fates. Here, we discuss the intrinsic properties of the spreading reaction and circumstances under which its products, formed distal to DNA-encoded nucleation sites, can be epigenetically maintained. Finally, we speculate that the epigenetic properties of heterochromatin evolved together with the need to stabilize cellular identity.

Heterochromatin spreading, the expansion of gene-silencing structures from DNA-encoded nucleation sites, occurs in distinct chromatin contexts. Spreading re-establishes gene-poor constitutive heterochromatin every cell cycle, but also invades gene-rich euchromatin de novo to steer fate decisions.Unlike heterochromatin nucleation and assembly, the determinants of the spreading process remain poorly understood. Our heterochromatin spreading sensor separately records nucleation site-proximal, and distal, heterochromatin gene silencing. By screening a nuclear function gene deletion library in fission yeast, we identified regulators that alter the propensity, both positively and negatively, of a nucleation site to spread heterochromatin. Critically, the involvement of many regulators is conditioned by the chromatin context within which spreading occurs. We find spreading, but not nucleation, within constitutive heterochromatin, requires distinct Clr6 histone deacetylase complexes. However, spreading is universally antagonized by a suite of chromatin remodelers. Our results disentangle the machineries that control lateral heterochromatin spreading from those that instruct DNA-directed assembly. 109 ΔcenH. Within each neighborhood, the distribution of "orange" expression, especially for MAT ΔREIII 110 and ECT, is graded from above to below the expression level of the parent(s), revealing a continuum of 111 mutants with enhanced or abrogated spreading. We could not find mutants that display more repression 112 than the parental strains of MAT ΔcenH, which are highly repressed in the OFF state, as previously 113 described (Grewal and Klar 1996;Greenstein et al. 2018). However, we did observe mutants located 114 out of the area of their chromatin context-driven "neighborhood". First, the major known 115 heterochromatin mutants, Δclr4, Δswi6, Δclr3 among others, from each chromatin context formed a 116 cluster with high expression of "green" and "orange" (Figure 1I enlarged region, exemplified by Δclr3), 117segregating from the rest of the population. Second, we observed mutants, such as Δcdt2, Δepe1 and 118 Δchp1, that segregate out of neighborhood only for selected chromatin contexts, indicating specificity 119 (highlighted in Figure 1I). The t-SNE analysis visualized the relationship of all four chromatin contexts, 120 and mutants therein, with respect to their nucleation and spreading behavior, directly revealing the 121 graded nature of mutant phenotypes. This is particularly the case with respect to spreading in ECT and 122 MAT ΔREIII neighborhoods (Figure 1I) 123 However, in the t-SNE analysis the phenotype patterns are weighted by the intrinsic behavior of 124 each parent's chromatin context. To quantify how much each mutation impacted the heterochromatin 125 state in each chromatin context, we performed Earth Mover's Distance analysis (EMD, Figure 1J see 126 also materials and methods and (Orlova et al. 2016)). We express the contribution of each mutant 127relative to the parental isolates by a quotient of their...