Molecular mechanisms of nucleosome positioning and DNA methylation in chromatin

摘要

In the eukaryotic cell nucleus DNA needs to be highly condensed. The initial level of DNA compaction is mediated by the wrapping of DNA around histone octamers to form nucleosomes. For efficient DNA metabolism, including DNA replication, transcription, repair and recombination, access to the required sequences must be granted. Hence, nucleosomes need to be highly dynamic. This is mediated by ATP-dependent chromatin remodeling complexes. It is still unclear to what extent these enzymes are influenced by local DNA sequences when shifting a nucleosome to different positions.udDuring my PhD thesis I studied ATP-dependent chromatin remodeling factors focusing on the molecular mechanisms of action in dependence on the underlying DNA sequence. I showed that each individual remodeling enzyme possesses distinct nucleosome translocation properties. The direction (outcome) of nucleosome translocation is determined by its underlying DNA sequence and is influenced by other remodeling complex subunits. I demonstrated that nucleosome positioning by two specific motor proteins is determined by the reduced affinity of the remodeling enzyme to the end product of the reaction.udIn the following, I characterized the kinetic properties of the DNA methyltransferase Dnmt1 in the context of chromatin. DNA methylation is an important epigenetic modification required for a variety of DNA associated processes. Dnmt1 is responsible for the maintenance of methylation patterns. In a second wave of DNA methylation following DNA replication, Dnmt1 needs to access nucleosomal DNA. Using an in vitro approach, I demonstrated that Dnmt1 requires a minimal length of DNA overhangs to bind to mononucleosomes. Furthermore, in vitro mapping of Dnmt1 interactions with its nucleosomal substrate suggests that Dnmt1 needs to contact flanking DNA as well as nucleosomal DNA for efficient binding. Finally, I could show that Dnmt1 methylation activity is inhibited within the nucleosomal core region. Interestingly, addition of recombinant ATP-dependent chromatin remodeling factors abolish the inhibitory effect of the nucleosome, most likely by rendering the nucleosomal DNA accessible to Dnmt1.udTaken together, these results suggest a major role for chromatin remodeling enzymes in nucleosome positioning which in turn might be crucial for epigenetic DNA modifications such as DNA methylation.ud