Chromatin is composed of DNA and histones, which provide a unified platform for regulating DNA-related processes, mostly through their post translational modification. What happens to this epigenetic landscape during DNA replication?
The epigenetic landscape is largely perturbed during DNA replication. First, specific modifications are used to define replication origins, or are deposited during the course of DNA polymerase progression. In addition, histones must be removed from the DNA template to enable replication fork progression and then restored in the wake of the fork.
In addition, during replication, DNA content increases, requiring additional histones for its wrapping (I). Newly synthesized histones are used for wrapping the newly replicated DNA (II). These new histones lack position-specific modifications, but instead are generally modified on multiple residues. Therefore, the chromatin landscape must also be reproduced on the newly replicated DNA.
We studied how epigenetic information is transmitted during the cell cycle by looking on dynamics. The dynamics of retrieval may reveal functional information – For example, if modifications are recovered after a very long delay, it may suggest that other processes (not coupled to DNA replication) are involved in the recovery.
We followed the genome-wide pattern of multiple histone modifications during and after replication. We synchronized budding yeast to the beginning of S-phase, and measured histone modifications using ChIP-Seq. In addition we also measured expression levels (with RNA-Seq), and the replication profile using DNA-Seq. In the right graph, a chromosome is shown. The x axis is location on the chromosome, along time (y-axis). This is the duration of S-phase in this experiment. It is evident that some regions have already replicated, as HU arrests cells after replication has begun. These orange peaks from which replication begins correspond to known origins of replication in yeast.
We asked – when does the modification increase, compared to the time of replication of the same region. We saw that some modifications appeared on replicated DNA quite immediately (e.g. H4K16ac, H3K4me1), whereas all tri-methylation were deposited with a significant delay that extended for over 20 min.
We next examined the effect of transcription-related parameters on this delay. We started by separating genes that replicate at the same time but have different levels of expression (top). We saw that this delayed deposition was largely correlated with gene transcription strength and gene promoter structure (bottom), so that actively transcribing genes were tri-methylated significantly faster than low-expressing ones.
Therefore we conclude that replication-independent processes, rather than active memory, account for the recovery of histone tri-methylation pattern following DNA replication.
When examining the delays, we were intrigued by one modification in particular. H3K9ac is a modification extensively studied in the context of transcription. It correlates with transcription levels, and is found mainly in the beginning of genes. However, in our cell-cycle experiment, H3K9ac, was deposited ahead of the replication fork in a distance of 5kb from the fork (therefore – a negative delay), everywhere in the genome.
We next asked which enzyme is responsible for this deposition ahead of the fork. Two enzymes acetylate H3K9: Gcn5 and Rtt109. We deleted either and repeated the same experiment (also for WT) by a different synchronization method (alpha-factor). We observed that when Rtt109 the deposition of H3K9 ahead of the fork disappears. In fact, it followed replication.
We examined the raw H3K9ac pattern along the chromosomes. In the slide, see Chr V. Each box represents the chromosome in a different strain (WT, gcn5-deleted, rtt109-deleted), along time from top to bottom. When looking on the raw H3K9ac in the different strains we observed a nice separation of the pattern in the mutants: When only Rtt109 is present (middle), a replication-like pattern is observed. In contrast, when only Gcn5 is functional, we observe lines that are not changing throughout time, and these are the beginning of genes. It is evident that the pattern of H3K9ac in wild type combines the replication- and transcription-associated patterns.
Therefore we have two different enzyme (Gcn5, Rtt109) responsible for one histone mark (K9ac), that act in different context – Gcn5 in transcription, in the beginning of genes, and Rtt109 in replication, everywhere in the genome during replication.