Fig. 8

Summary of the effects of histone hyperacetylation on DNA replication timing. In untreated cells, (black box) showed typical marks for heterochromatin. Both heterochromatic blocks were hypoacetylated, but enriched for either H3K27me3 (X*, facultative heterochromatin) or for H3K9me3 (X, constitutive heterochromatin). These cells replicate in 9.4 h, where 1.9 h was required for the DNA replication of the H3K27me3 decorated X chromosome and 1.8 h for the duplication of the H3K9me3 decorated X chromosome. When cells were globally hyperacetylated with an HDAC inhibitor (red box), the histone acetylation level increases, but histone methylation marks decreased. The effect on DNA replication timing was dramatic, as the total S-phase duration was prolonged up to 12.6 h. The heterochromatic block of the X* was replicated in a time frame of 2.7 h, and constitutive heterochromatin of the X required 2.2 h for DNA duplication. This result, going hand in hand with a strong increase in DNA content in early S-phase, indicated a shift from facultative heterochromatin, which is normally replicated during mid-S-phase, toward early S-phase. The duration of early S-phase was also prolonged as a result of slower fork speed in treated samples. When the constitutive heterochromatin was targeted with a HBO1 (green box), we achieved histone hyperacetylation specifically at the H3K9me3 decorated X and, again, a loss of histone methylation marks. Our HAT targeting data confirmed the effect of histone hyperacetylation on DNA replication timing of genomic regions, as we also observed a prolongation of constitutive heterochromatin replication. That this approach is site specific was demonstrated by the fact that the DNA replication of the H3K27me3 decorated X chromosome was not affected by the targeting of HBO1 to the HP1 beta containing heterochromatin