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

Abstract

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...

Repression of a large number of genes requires interplay between homologous recombination and HIRA

“…In wild-type cells, Bre1 stimulates histone eviction at DSBs by H2B ubiquitination ( 123 ). Furthermore, a recent study analysing regulators of heterochromatin spreading in different chromatin contexts demonstrated that the HIRA histone chaperone and genes involved in HR pathway, such as rad50 and sfr1 , act in opposite ways ( 124 ). The authors detected these actions mainly at the ectopic heterochromatin domain composed of dh element embedded in gene-rich euchromatin.…”

“…The authors detected these actions mainly at the ectopic heterochromatin domain composed of dh element embedded in gene-rich euchromatin. The HIRA complex has been implicated in heterochromatic domains expansion, while rad50 and sfr1 have been found to function in heterochromatin destabilisation ( 124 ). Consistently, we show that H3K9me2 nucleosomes are elevated in the dbl2Δ mutant at constitutive heterochromatic regions and LTRs (Figure 7D , E ).…”