Reestablishment of epigenetic patterns of histone modification after DNA replication and chromatin assembly in S. cerevisiae
Zusammenfassung der Projektergebnisse
The goal of this project was to determine the fate of epigenetic histone modifications during DNA replication and chromatin assembly using the yeast Saccharomyces cerevisiae as a model organism. Chromatin duplication encompasses not only duplication of the DNA sequence, but also the histones including their posttranslational modifications, raising the question of the fate of these modifications during chromatin assembly. Chromatin assembly is performed by specialized protein complexes, the chromatin assembly factors CAF-1 and Asf1. Since they incorporate newly synthesized histones into the chromatin that carry a different modification pattern than the parental nucleosomes, one can hypothesize that both histone acetylation and deacetylation are involved in adjusting acetylation levels to the parental state. Our previous work has suggested that the euchromatic mark H4 K16 acetylation may be placed in a replication-dependent fashion on the chromatin by the HAT complex SAS-I, because we identified an interaction between SAS-1 and the chromatin assembly factors CAF-I and Asf1. For cytoplasmic histone acetylation marks, one might expect an HDAC to interact with chromatin assembly factors. In this work, we have identified a physiologically relevant interaction between the HDAC Rpd3 and the chromatin assembly factor CAF-I, which suggests that Rpd3 is recruited to newly replicated chromatin to remove cytoplasmic histone acetylation marks. Furthermore, we investigated how histone acetylation marks depend on chromatin assembly factors, and how they fluctuate during DNA replication and chromatin assembly. We have shown that histone acetylation paterns for H4 K12 and K16 partially depend on the chromatin assembly factors CAF-I and Asf1. We now know that histone acetylation levels flutuate slightly during 26 DNA replication and chromatin assembly at the time of replication of the site, suggesting a fast reestablishment of these patterns after chromatin assembly. Furthermore, we have identified a synthetic genetic interaction between Rpd3 and Sas2 that suggests that the two enzymes have redundant functions in a cellular pathway. In summary, the information gained in this project is important in order to understand how daughter cells can be both genetically and epigenetically identical to the mother cell. We also encountered technical difficulties in this project. We have been unable to construct variants of chromatin modifying enzymes that can be switched off efficiently. These were the prerequisite for determining the dependence of the establishment of histone acetylation patterns on passage through the S phase of the cell cycle, an experiment that was suggested in the original experimental plan. Furthermore, we have spent considerable time and resources testing and evaluating commercially available anti-acetyl-lysine antibodies. Our experience has been that many of these antibodies do not meet the expectations in that they do not show the specificity that they are claimed to have by the manufacturers.
Projektbezogene Publikationen (Auswahl)
- Interactions within the mammalian DNA methyltransferase family. BMC Mol Biol. 2003 May 30;4:7
Margot JB, Ehrenhofer-Murray AE, Leonhardt H
- (2008) Das Schweigen der Gene - Wie Gene ein- und ausgeschaltet werden. Labor & more, 05/08
Ehrenhofer-Murray, Ann
