The euchromatic histone mark H3K36me3 preserves heterochromatin through sequestration of an acetyltransferase complex in fission yeast

Abstract

Maintaining the identity of chromatin states requires mechanisms that ensure their structural integrity through the concerted actions of histone modifiers, readers, and erasers. Histone H3K9me and H3K27me are hallmarks of repressed heterochromatin, whereas H3K4me and H3K36me are associated with actively transcribed euchromatin. Paradoxically, several studies have reported that loss of Set2, the methyltransferase responsible for H3K36me, causes de-repression of heterochromatin. Here we show that unconstrained activity of the acetyltransferase complex Mst2C, which antagonizes heterochromatin, is the main cause of the silencing defects observed in Set2-deficient cells. As previously shown, Mst2C is sequestered to actively transcribed chromatin via binding to H3K36me3 that is recognized by the PWWP domain protein Pdp3. We demonstrate that combining deletions of set2 ⁺ and pdp3 ⁺ results in an epistatic silencing phenotype. In contrast, deleting mst2 ⁺ , or other members of Mst2C, fully restores silencing in Set2-deficient cells. Suppression of the silencing defect in set2Δ cells is specific for pericentromeres and subtelomeres, which are marked by H3K9me, but is not seen for loci that lack genuine heterochromatin. Mst2 is known to acetylate histone H3K14 redundantly with the HAT Gnc5. Further, it is involved in the acetylation of the non-histone substrate and E3 ubiquitin ligase Brl1, resulting in increased H2B-K119 ubiquitylation at euchromatin. However, we reveal that none of these mechanisms are responsible for the Set2-dependent silencing pathway, implying that Mst2 targets another, unknown substrate critical for heterochromatin silencing. Our findings demonstrate that maintenance of chromatin states requires spatial constraint of opposing chromatin activities.

Keywords: acetyltransferase; chromatin; heterochromatin; histone modification; silencing.

Figure 1. FIGURE 1: Loss of Mst2 recues the silencing defect caused by set2⁺ deletion.

(A) Scheme depicting genetic interactions of set2⁺, pdp3⁺ and mst2⁺ contributing to heterochromatic silencing and potential parallel pathways in which H3K36me3 may be also involved. Black lines indicate positive regulations, red lines indicate negative regulations. (B) Silencing reporter assay with the imr::ura4⁺ reporter. Fivefold serial dilutions of wild-type (WT) cells and single and double deletion mutants of mst2⁺ and set2⁺; (N/S) nonselective medium. (C) RT-qPCR analysis. Shown are heterochromatic transcript levels of the strains used in (B). The schemes display the positions of the ura4⁺ reporter insertion and endogenous heterochromatic transcripts from pericentromeric (left) and subtelomeric heterochromatin (right); transcript levels have been normalized to act1⁺ and are shown relative to WT for each transcript. Circles and horizontal lines represent individual data and median from 6-12 independent experiments. (D) ChIP-qPCR analysis for H3K9me2 (top), H3K36me3 (middle) and H3 (bottom) at pericentromeric and subtelomeric heterochromatin; ade2⁺ (right panels) was used as control for euchromatin. Circles and horizontal lines represent individual data and median from 3 independent experiments. Input-normalized ChIP data were corrected for variation in IP efficiency by normalizing to the mean of cendg and cendh for H3K9me2, or the mean of three euchromatic loci (tef3⁺, ade2⁺, act1⁺) for H3K36me3 and H3. Note that H3K9me2 is largely unaltered at the dh repeats in set2Δ [34].

Figure 2. FIGURE 2: Loss of heterochromatin silencing in set2Δ is dependent on functional Mst2C.

(A) Scheme displaying genetic interactions and genes mutated for experiments shown in (B) and (C). (B, C) RT-qPCR analysis of transcript levels at pericentromeric (B) and subtelomeric HC (C) RT-qPCR data analysis and primer positions as in Figure 1C. Circles and horizontal lines represent individual data and median from 6 independent experiments unless specified (i.e. WT: n = 12; ptf1Δ, ptf2Δ, eaf6Δ, ptf1Δ set2Δ, ptf2Δ set2Δ, and eaf6Δ set2Δ: n = 3).

Figure 3. FIGURE 3: The target of Mst2 in heterochromatin is not Brl1.

(A) Scheme depicting the described Mst2C pathway (Flury et al., 2017) involving Brl1-K242 acetylation and H2B ubiquitylation and a potential alternative pathway on HC silencing; black arrows represent positive regulation and red lines represent negative regulation. (B) ChIP-qPCR analysis for H2B-K119ub in wild-type cells. Circles and horizontal lines represent individual data and median from 3 (left panel) and 4 independent experiments (right panel). Input-normalized ChIP data are shown relative to the median of the ChIP signals for act1⁺ (mid). (C, D) ChIP-qPCR analysis for H2B-K119ub in mutants affecting Brl1 acetylation. Circles and horizontal lines represent individual data and median from 3 independent experiments (except: WT, set2Δ: n = 4). ChIP analysis as in (B), except that ChIP data were corrected for variation in IP efficiency by normalizing to act1⁺ (mid). Note that H2B-K119ub at act1⁺ is not largely affected by Set2. (E) RT-qPCR analysis of transcript levels at pericentromeric and subtelomeric HC. Data analysis as in Figure 1C (n = 3 individual experiments).

Figure 4. FIGURE 4: The silencing defect at ‘knobs' in set2Δ is not associated with the Mst2 pathway.

(A) Scheme depicting expression sites of the non-coding RNA TERRA and the positions of several loci within the ‘knob' regions on chromosomes I and II. Chromosomal positions refer to annotations in www.pombase.org but differ from the absolute positions due to missing sequences at the chromosomal termini. (B) RT-qPCR analysis in WT and set2Δ strains comparing transcript levels at pericentromeric and subtelomeric HC (shaded in grey) to loci in the ‘knob' region. Transcript levels from 12 independent experiments are shown relative to act1⁺. (C, D) ChIP-qPCR analysis of H3K9me2 and H3K36me enrichments in WT cells at loci analyzed in (B). Shown are ChIP data from 2-3 individual experiments analyzed as described in Figure 1D. (E) RT-qPCR analysis in mst2Δ and set2Δ single and double mutants; data from independent experiments analyzed as described in Figure 1C (WT n = 12; mst2Δ, mst2Δ set2Δ: n = 6; set2Δ: n = 9). (F) ChIP-qPCR analysis of TERRA and different loci located within the ‘knob' region; data from independent experiments analyzed as described in Figure 1D (n = 3).

Figure 5. FIGURE 5: Model for Mst2C-dependent functional pathways in the presence of H3K36me3 (sequestered to euchromatin) and in the absence of H3K36me3-mediated anchoring (promiscuous access to heterochromatin).