Elimination of shelterin components bypasses RNAi for pericentric heterochromatin assembly

Abstract

The RNAi pathway is required for heterochromatin assembly at repetitive DNA elements in diverse organisms. In fission yeast, loss of RNAi causes pericentric heterochromatin defects, compromising gene silencing and chromosome segregation. Here we show that deletion of telomere shelterin components restores pericentric heterochromatin and its functions in RNAi mutants. We further isolated a separation-of-function mutant of Poz1 and revealed that defective telomere silencing, but not telomere length control, is critical for bypassing RNAi. Further analyses demonstrated that compromising shelterin-mediated heterochromatin assembly in RNAi mutants releases heterochromatin protein Swi6, which is redistributed to pericentric regions through RNAiindependent heterochromatin assembly pathways. Given the high mobility of Swi6 protein and that increased levels of Swi6 facilitates heterochromatin spreading as well as ectopic heterochromatin assembly, our results suggest that constitutive heterochromatin domains use multiple pathways to form high-affinity platforms to restrain Swi6, thus limiting its availability and avoiding promiscuous heterochromatin formation.

Control of heterochromatin localization and silencing by the nuclear membrane protein Lem2

“…Remarkably, inhibiting the telomere-specific (Taz1) or mating type-specific (Atf1) silencing pathway restores silencing in a reciprocal manner at pericentromeres. This observation is in agreement with the previous notion that different heterochromatin domains compete for a limited pool of soluble silencing factors (HP1 in S. pombe and SIR proteins in S. cerevisiae) (Taddei et al 2009;Tadeo et al 2013). These results unveil a network of partially redundant pathways that cooperate in heterochromatin silencing.…”

The proper connection between shelterin components is required for telomeric heterochromatin assembly

“…Moreover, H3K9me2 depends on Poz1 and Tpz1, which are required to link Ccq1 with Rap1, but independent of Taz1 or Pot1, which are not expected to affect Ccq1-Rap1 interaction (Supplemental Fig. S2; Tadeo et al 2013). These results further support the role of Ccq1 in mediating CLRC-shelterin interaction.…”

“…For example, mutations of Taz1, Rap1, Poz1, Tpz1, and Ccq1 all resulted in silencing defects of reporter genes inserted near telomeric repeats (Cooper et al 1997;Nimmo et al 1998;Kanoh and Ishikawa 2001;Kanoh et al 2005;Sugiyama et al 2007;Fujita et al 2012;Tadeo et al 2013;Kallgren et al 2014). In addition, insertion of telomeric repeats at an internal chromosome location or artificial recruitment of shelterin component Rap1 to DNA induces ectopic heterochromatin in a shelterin-and CLRC-dependent manner, suggesting that shelterin recruits CLRC to telomeric DNA (Kanoh et al 2005;Tadeo et al 2013). However, mechanistic dissection of subtelomeric heterochromatin assembly has been difficult due to the multiple functions that shelterin performs.…”

A systematic genetic screen identifies new factors influencing centromeric heterochromatin integrity in fission yeast

“…However, here, RNAi is required only for establishment but not maintenance of heterochromatin, since alternative pathways act redundantly with RNAi to facilitate ongoing recruitment of chromatin modifiers to these regions [11][12][13]. Recent evidence suggests that alternative, RNAiindependent pathways can also promote heterochromatin assembly at centromeres, although the mechanisms and significance of these are as yet unclear [14][15][16]. Moreover, a proportion of H3K9 methylation is maintained in the absence of RNAi, and this has been shown to be dependent on the histone deacetylases (HDACs) Sir2 and Clr3/ SHREC [17,18].…”