Figure 5: H3K64me3 is important for maintaining PH integrity.

(a) NIH3T3 cells transfected with K64-non-methylatable flagha-hmg.H3K64R mutant show (compared with flagha-hmg.H3 wild-type (wt) transfected cells) impaired PH integrity as revealed by perturbed enrichment of the heterochromatin hallmark H3K9me3. Graph shows the percentage of cells displaying normal enrichment (grey bars: present) or absence (black bars: absent) of this hallmark of heterochromatin after transfection and specific expression of fagha-hmg, flagha-hmgH3 wt or flagha-hmgH3K64R mutant at PH. Means of two replicates from two independent experiments are represented. Error bars represent range. (b) The same transfection experiment shows impaired PH integrity as revealed by perturbed enrichment of two H3K9me3-dependent heterochromatin hallmarks, HP1 (upper graph) and H4K20me3 (lower graph). Means of two independent experiments are represented. Error bars represent range. Note that only the flagha-hmgH3K64R mutant markedly compromises heterochromatin integrity, leading to an increase of, in certain cases, over 20% of cells, in which hallmarks disappeared from PH foci.(c) Model of heterochromatinization events at the pericentromere. Suv39 enzymes are targeted by a yet unknown mechanism to repeat-rich pericentromeric chromatin, resulting in enrichment of H3K9me3. Consequently, chromatin binding and modifying enzymes are recruited to these regions, involving HP1 proteins and, downstream thereof, Dnmts and Suv4-20 enzymes. H3K9me3 also promotes H3K64me3 deposition, leading potentially to structural stabilization of chromatin by strengthening DNA–histone octamer interactions. H3K64me3 itself could furthermore reinforce heterochromatin integrity by locally stabilizing H3K9me3, HP1 and H4K20me3. Thus, trimethylation of H3 at K64 seems important for the formation of a robust heterochromatin at pericentromeric sites, characterized by high levels of H3K9me3, H3K64me3, CpG methylation and H4K20me3. See text for details.