These experiments constitute some of the most highly time resolved analyses of histone methylation formation and turnover across the cell cycle and test the hypotheses that histone methylation is a replication-coupled or replication-independent process. Methylation at H4K20 is largely restricted to specific cell cycle stages, while methylation at H3K36 is generally independent of the cell cycle. H3K9 methylation and H3K27 methylation are formed similarly to one another, despite the initial hypotheses that H3K9 trimethylation would be replication coupled (41
) while H3K27 trimethylation would be postreplication coupled. In particular, we provide evidence that trimethylation of H3K9 and H3K27 begins as early as S phase but is restored to steady-state levels only by the G1
phase of the next cell cycle ().
Fig 7 Model summarizing the origin and formation of histone methylation across the cell cycle for H3K9 and H3K27. Preexisting histone modifications (open circles) are diluted following new histone synthesis (shaded squares) during S phase. For simplicity, only (more ...)
An important discovery is that the preexisting dimethylated states for both residues contribute to the new trimethylated states following S phase (). De novo formation of H3K9 and H3K27 trimethylation from the unmodified state during the S and G2 phases (i.e., H3K9me3:3 and H3K27me3:3) reaches approximately 50% of the newly synthesized histone H3 levels. The remaining 50% is accounted for by the conversion of preexisting dimethylated residues, which acquire an additional methyl group during G1 phase to become new trimethylated residues (i.e., me3:1). Thus, new dimethylation must occur in excess of newly synthesized histone H3 proteins in order to replace the fraction of preexisting dimethylated histones that is converted to the trimethylated state.
The sum of all new H3K9 and H3K27 dimethylation events exceeds the levels of newly synthesized histone H3 proteins by >25% (i.e., 1.25 × 40% = 50%) ( and ) before M phase and by approximately 50% during the subsequent G1 phase (i.e., 1.5 × 40% = 60%) (data not shown). The excess production of newly dimethylated H3K9 and H3K27 proteins may replace the fraction of preexisting dimethylated histones (i.e., H3K9me2:0 and H3K27me2:0) that is being converted to the trimethylated pool (i.e., H3K9me3:1 and H3K27me3:1). Total H3K9me2 and H3K27me2 peptides occur at relative abundances approximately 1.3 and 2 times greater than those of total H3K9me3 and H3K27me3 peptides, respectively ( and ). Therefore, allocation of 50% of the preexisting dimethylated histones would be sufficient (i.e., 50% × 1.3 = 0.65 [>0.5]) to reach the steady-state trimethylated levels that otherwise could not be achieved with trimethylation from the unmodified state alone.
The two distinct trimethylation pathways suggested by our data are consistent with ChIP-Seq (high-throughput sequencing in combination with chromatin immunoprecipitation) experiments that have mapped the genomic localization of H3K9 and H3K27 methylation (5
). In particular, regions enriched in H3K9 and H3K27 monomethylation generally do not overlap regions enriched in H3K9 and H3K27 trimethylation. This mutual exclusion is logical given that H3K9 monomethylation and H3K27 monomethylation are generally considered euchromatic marks, while H3K9 trimethylation and H3K27 trimethylation are classically heterochromatic marks. ChIP-Seq reveals a much closer overall distribution between dimethylation and trimethylation at both residues, supporting our results suggesting that the preexisting dimethylated H3K9 and H3K27 pools do contribute to trimethylated H3K9 and H3K27 formation.
A second compelling finding is that H3K9 and H3K27 trimethylation is fully established much later than replication-dependent histone synthesis, during the G1
phase of the next cell cycle. Such a model has been predicted before on the basis of numerous other studies (45
). Elegant immunofluorescence experiments have shown that Polycomb repressive complexes 1 and 2, which coordinate Polycomb silencing, are disrupted during mitosis. The complexes are re-formed on chromatin predominantly during mid- to late-G1
phase in Drosophila melanogaster
) and U2OS cells (2
). These experiments support and complement our experiments on the timing and formation of H3K27me3 itself. In contrast, another series of experiments was unable to detect the complete synthesis of H3K9me2, H3K9me3, or H3K27me3 to match the newly synthesized histone H3 levels (51
). As stated previously, this would imply a serial dilution of those histone modifications over successive cell cycles and would be difficult to reconcile with stable maintenance of epigenetic information across cell generations.
One possible mechanism for the completion of H3K9 trimethylation in the next cell cycle is that SUV39H1 methyltransferase activity does not fully restore H3K9me3 levels, due to antagonism from the mitotic occurrence of H3S10 phosphorylation. In particular, the surge in H3S10ph levels may prevent H3K9me3 from being fully synthesized by M phase. HP1 association with chromatin is diffuse in D. melanogaster
embryos during metaphase and anaphase, and HP1 relocalizes on chromatin during the next interphase (30
). This disruption in HP1 localization may lead to a disruption in SUV39H1 methyltransferase recruitment and activity, and thus in H3K9 trimethylation, during mitosis. A similar event may occur for H3K27me3, where another cell cycle-specific PTM on a nearby residue may occlude EZH2 binding. Neither SUV39H1 nor EZH2 mRNA levels were observed to be upregulated during G1
. Thus, a change in methyltransferase protein levels or modulation of methyltransferase activity by other histone modifications or by modifications on the enzymes themselves could account for the latency of H3K9 and H3K27 trimethylation. The absence of appreciable turnover at these residues is similar to the lack of H4K20me3 turnover across the cell cycle (39
), where H4K20me3 was initially implicated in position effect variegation silencing, such as that for H3K9me3, but has recently been questioned as an epigenetic silencing mark (44
). Furthermore, ectopic overexpression of EZH2, and presumably increased H3K27 trimethylation, accelerates G1
-phase progression and leads to increased accumulation in S phase (8
). Thus, it is possible that the complete re-formation of H3K9me3 and H3K27me3 may provide a rate-limiting step restraining cells from beginning another round of histone synthesis and DNA replication.