DNA topoisomerases and nucleosome dynamics in fission yeast

Abstract

The organization of DNA and associated proteins into chromatin allows for compaction and protection of large eukaryotic genomes. However, it also poses a challenge to fundamental cellular processes such as DNA replication, recombination, repair and transcription. In order to orchestrate and regulate these processes chromatin structure needs to be both variable and dynamic. The basic unit of chromatin is the nucleosome. ATP dependent chromatin remodelers and DNA topoisomerases have emerged as important factors in regulating nucleosome transactions. The studies presented in this thesis further our understanding of the roles of such enzymes in nucleosome organization using high- resolution genome-wide techniques in the model organism Schizosaccharomyces pombe.

We find that the DNA topoisomerases Top1 and Top2 play overlapping, yet distinct, roles in relieving supercoiling during transcription. Top1 removes negative supercoiling behind the RNA polymerase, helping to maintain the nucleosome depleted region (NDR). This is particularly important for sustaining successive rounds of initiation and elongation at highly transcribed genes. Both Top1 and Top2 also relieve positive supercoiling ahead of the RNA polymerase, thereby preventing stalling of the polymerase during elongation, with Top2 being particularly important at long genes. We also identify a new role for Top3 in maintaining normal levels of the centromere specific histone H3 variant CENP-A. This is largely independent of the role of Top3 in homologous recombination and we suggest that it reflects a role for Top3 in regulating supercoiling at centromeres, thereby affecting CENP-A nucleosome dynamics and perhaps structure.

Furthermore, we find that the fission yeast CHD1-type chromatin remodelers Hrp1 and Hrp3 have an important role in maintaining the characteristic pattern of nucleosome positioning at transcribed genes. We demonstrate that Hrp1 and Hrp3 have nucleosome assembly and spacing activity in vitro, and are required for linking regular nucleosomal arrays to the 5̍ end of genes, thereby preventing cryptic transcription.

Last, we present the first genome-wide map of replication-independent nucleosome turnover in fission yeast, and show that successive mono-, di-, and trimethylation of H4K20 can be used as a proxy marker for nucleosome age. We find that transcription at low and intermediate levels promotes conservation of old nucleosomes in gene bodies and suggest that this reflect efficient recycling of histones behind the RNA polymerase. Moreover, we show that transcription promotes incorporation of newly synthesized nucleosomes at the borders of genes.

Overall, these studies support a model in which nucleosome dynamics are dependent on a large number of factors, including the cooperation between DNA topoisomerases, chromatin remodelers and histone chaperones, and DNA-dependent processes, such as transcription.