Epigenetics. Restricted epigenetic inheritance of H3K9 methylation

Abstract

Posttranslational histone modifications are believed to allow the epigenetic transmission of distinct chromatin states, independently of associated DNA sequences. Histone H3 lysine 9 (H3K9) methylation is essential for heterochromatin formation; however, a demonstration of its epigenetic heritability is lacking. Fission yeast has a single H3K9 methyltransferase, Clr4, that directs all H3K9 methylation and heterochromatin. Using releasable tethered Clr4 reveals that an active process rapidly erases H3K9 methylation from tethering sites in wild-type cells. However, inactivation of the putative histone demethylase Epe1 allows H3K9 methylation and silent chromatin maintenance at the tethering site through many mitotic divisions, and transgenerationally through meiosis, after release of tethered Clr4. Thus, H3K9 methylation is a heritable epigenetic mark whose transmission is usually countered by its active removal, which prevents the unauthorized inheritance of heterochromatin.

Fig. 1. H3K9 methylation is rapidly lost upon release of tethered TetR-Clr4*

(A) Positions of 4xtetO, tethered TetR-Clr4* beside ade6⁺ at ura4, and surrounding S. pombe chromosome III genes. Dumbbells indicate primer pairs. (B and C) qChIP time course of FLAG-TetR-Clr4* (B) and H3K9me2 (C) levels on 4xtetO-ade6⁺ following AHT addition using indicated primers. Data are mean ±SD (n=3), P<0.05 (t-test).

Fig. 2. Tethering TetR-Clr4* at loci with low expression and histone turnover does not stablize H3K9 methylation

(A) Read distribution (log₂RPKM) from S. pombe polyA RNA-seq relative to gene length. ade6⁺, sib1⁺ and vps1302⁺ indicated. (B) qRT-PCR of ade6⁺, sib1⁺ and vps1302⁺ RNA levels. Data are mean ±SD (n=3) P<0.005 (t-test) (C) Recombination-induced tag exchange monitoring incorporation of new H3-T7 on act1⁺, ade6⁺, sib1⁺ vps1302⁺ and cen-dg repeats. Data are mean ±SD (n=3). H3 turnover on sib1⁺ and vps1302⁺ was significantly lower than on act1⁺ and ade6⁺ P<0.05 (t-test). (D and E) sib1⁺ and vps1302⁺ lose H3K9me2 after TetR-Clr4* release. Position of tetO sites within sib1 and vps1302. own promoters were replaced with ura4⁺ (no) or swapped to low, medium (med), or high adh1 promoter versions (20). Dumbbells indicate primers. qChIP of H3K9me2 levels, at time points relative to AHT addition, on sib1:4xtetO (D) and vps1302:1xtetO (E) with no or indicted promoters. Data are mean ±SD (n=3), P<0.05 (t-test). H3K9me2 level within sib1 carrying its own promoter is decreased with a probability of respectively P=0.068 and P=0.051, 3h and 6h following TetR-Clr4* release.

Fig. 3. H3K9 methylation rapidly declines through the cell cycle and in non-cycling cells

Regimes for release of TetR-Clr4* from 4xtetO-ade6⁺ following AHT addition to cdc25-22 G2 synchronised cultures (A) or double-blocked cdc25-22 G2 cells (B). Synchrony was assessed by septation index. qChIP time course of H3K9me2 or FLAG-TetR-Clr4* levels on 4xtetO-ade6⁺ using indicated primers. Data are mean ±SD (n=3).

Fig. 4. epe1 mutants retain heterochromatin without tethered Clr4 methyltransferase through multiple cell divisions and meiosis

(A) wild-type, epe1Δ, epe1-K314A and epe1-H297A cells carrying 4xtetO-ade6⁺ and expressing TetR-Clr4*, were grown −/+ AHT. Colony colour assay to assess 4xtetO-ade6⁺ silencing (red-pink colonies; % of total indicated) and H3K9me2 qChIP on 4xtetO-ade6⁺ with (−AHT) or without (+AHT) tethered TetR-Clr4* Data are mean ±SD (n=3), P<0.05 (t-test). (B) TetR-Clr4* was completely removed from F0 epe1Δ 4xtetO-ade6⁺ tetR-Clr4* cells by crossing to epe1Δ lacking TetR-Clr4* and 4xtetO-ade6⁺. F1 progeny were crossed to epe1Δ cells, generating epe1Δ F2 progeny. epe1⁺ F2xwt progeny were produced by crossing epe1⁺ into epe1Δ 4xtetO-ade6⁺ F2 cells. Naïve epe1Δ 4xtetO-ade6⁺ cells never expressed TetR-Clr4*. Colony colour, qRT-PCR and qChIP assays to assess silencing and transcription of 4xtetO-ade6⁺, and H3K9me2 levels on 4xtetO-ade6⁺ in indicated cell types. Data are mean ±SD (n=3). 4xtetO-ade6⁺ RNA levels are significantly reduced in F0, F1 and F2 compared to wild-type cells without TetR-Clr4*; P<0.05 (t-test).