Noncoding RNA-nucleated heterochromatin spreading is intrinsically labile and requires accessory elements for epigenetic stability

Abstract

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.

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

“…We hypothesized that for gene barrier activity to be ineffective in the 3′ orientation, heterochromatin must be able to at least partially invade the gene from the 3′ end, which is depleted for H3K4me3 [32]. This would be consistent with our previous results that showed that a 3′ oriented reporter can experience intermediate repression, which correlated with intermediate levels of RNA, H3K4me3, and H3K9me2 [33]. Partial invasion may then lead to transcriptional silencing, which would inherently down-regulate H3K4me.…”

“…To investigate ectopic invasion of heterochromatin, we employed our previously described heterochromatin spreading sensor (HSS) [33,34] in the euchromatic region proximal to the MAT Inverted Repeat Right (IR-R) boundary [35]. This HSS system contains two central components: (1) the spreading sensor, a monomeric Kusabira-Orange 2 fluorescent protein driven by the validated ade6 promoter, hereafter referred to as "orange", integrated 0.7kb outside IR-R, and (2) the control, a E2Crimson fluorescent protein driven by the same promoter, hereafter referred to as "red", integrated at a constitutive euchromatic locus [33] (Figure 1A). We use flow cytometry to capture information from tens of thousands of single cells.…”

“…We first examined the normalized (legend to Figure 1 and [33] ) orange fluorescence of a strain with a WT boundary (epe1+, B-box+) and detect no silencing in the population distribution ( Figure 1B). We define a threshold for silencing as the mean of the appropriate WT (epe1+) strain less two standard deviations.…”

“…To do this, we moved the HSS to the euchromatic ura4 locus (Figure 1E) downstream of a previously described RNAi-based heterochromatin nucleator [37]. We have demonstrated that this construct can generate spreading up to 8kb downstream [33] without the need to remove any boundary factors, thus avoiding confounding global effects on growth in the screen. At this locus, a reporter cassette encoding "green", a third fluorescent protein driven by ade6p, is integrated 1kb downstream of the RNAi element while the "orange" cassette is integrated 3kb downstream from "green" in the same orientation with respect to spreading as at IR-R.…”

“…At this locus, a reporter cassette encoding "green", a third fluorescent protein driven by ade6p, is integrated 1kb downstream of the RNAi element while the "orange" cassette is integrated 3kb downstream from "green" in the same orientation with respect to spreading as at IR-R. While nucleation at this locus is not as robust compared to endogenous heterochromatin domains, we can apply a computational gate to isolate successfully nucleated cells (greenOFF, described in [33]) and assess their spreading state. In the WT background, the nucleation gated "orange" signal in this strain resembles the behavior seen in the ∆boundary IR-R HSS strain (compare Figure 1F, black line and Figure 1D), exhibiting both gene silencing and fully expressed states.…”