Mitotic-specific methylation of histone H4 Lys 20 follows increased PR-Set7 expression and its localization to mitotic chromosomes

Abstract

We describe distinct patterns of histone methylation during human cell cycle progression. Histone H4 methyltransferase activity was found to be cell cycle-regulated, consistent with increased H4 Lys 20 methylation at mitosis. This increase closely followed the cell cycle-regulated expression of the H4 Lys 20 methyltransferase, PR-Set7. Localization of PR-Set7 to mitotic chromosomes and subsequent increase in H4 Lys 20 methylation were inversely correlated to transient H4 Lys 16 acetylation in early S-phase. These data suggest that H4 Lys 20 methylation by PR-Set7 during mitosis acts to antagonize H4 Lys 16 acetylation and to establish a mechanism by which this mark is epigenetically transmitted.

Figure 1

Increased histone H3 and H4 methyltransferase activity during human cell cycle progression. (A) In nucleo assay in synchronized HeLa cells. Cells were released from G₁ arrest and analyzed every 2.5 h. The cell cycle phase was confirmed by FACS. Autoradiography indicates the HMT activity specific for histones H3 and H4 at distinct phases in the cell cycle (top panel). Coomassie-stained core histones represent the loading control (bottom panel). (B) Quantitative assessment of HMT activity during the cell cycle. The changes in HMT activity relative to G₁ were determined (see text for details). The X-axis is the time in hours after release from G₁ arrest, and the Y-axis represents the fold increase in HMT activity relative to G₁ for both histone H3 (left) and H4 (right). This experiment was performed twice with similar results as shown by the error bars.

Figure 2

Histone H4 Lys 20 methylation decreases at mid-S-phase and increases during mitosis. (A) Analysis of histone H4 modifications in synchronized HeLa cells. Cells were released from G₁ arrest and analyzed every 2.5 h. The cell cycle phase was confirmed by FACS. The relative protein levels of histone H4 arginine 3-methyl, H4 Lys 20-methyl, H4 Lys 16-acetyl, and the H3 serine 28-phos modifications were determined by Western blot. Ponceau-stained core histones represent the loading control (bottom panel). This experiment was performed ≥2 times for each antibody. (B) Immunofluorescence staining of histone H4 Lys 20 methylation (red) in Drosophila embryos. The cell cycle phase was determined by DAPI staining (blue).

Figure 3

Histone H4 Lys 20 methylation occurs prior to or during metaphase. Immunofluorescence staining of histone H4 Lys 20 methylation (red) in mitotic and interphase HeLa cells. The cell cycle phase was determined by DAPI staining (blue).

Figure 4

PR-Set7 localizes to mitotic chromosomes and is cell cycle regulated. (A) Analysis of PR-Set7 RNA and protein expression in synchronized HeLa cells. Cells were released from G₁ arrest and analyzed every 2.5 h. The cell cycle phase was confirmed by FACS. Northern analysis was performed with a probe generated against the PR-Set7 open reading frame. The relative protein levels of PR-Set7, phosphorylated histone H3 serine 28, and actin (loading control) were determined by Western blot. The shift in molecular weight of the recombinant PR-Set7 protein is owing to its fusion product. This experiment was performed ≥2 times for each antibody. (B) Immunofluorescence staining of PR-Set7 (red) in HeLa cells. The cell cycle phase was determined by DAPI staining (blue). The green arrow indicates prometaphase, the white arrow indicates metaphase, and the yellow arrow indicates anaphase.