Studies of histone modification systems in Schizosaccharomyces pombe

Abstract

The genetic information in every cell is carried in the DNA. In eukaryotes the DNA is wrapped twice around proteins called histones forming a repeating unit called the nucleosome, in fibers known as chromatin. Nucleosomes are folded into higher order structures forming the chromosomes. The chromatin can be more or less compacted, controlling the accessibility of the DNA to ensure correct gene expression. Histones have long N-terminal tails, which can be covalently modified by different modifying enzymes. Different types of histone modifications are linked to the different functional properties of chromatin. In this thesis we have studied three histone modification systems in the model organism Schizosaccharomyces pombe (fission yeast).

In paper I, the Rpb7 subunit of the RNA Pol II polymerase function is analyzed in cells with a missense mutation in the rpb7 gene (rpb7-G150D). Chromatin Immunoprecipitation showed decreased levels of methylation of histone H3 at lysine 9 (H3K9me2), heterochromatin protein Swi6 and cohesin Rad21, which is the cause of lagging chromosomes and defects in chromosome segregation in rpb7-G150D. Analysis of centromeric forward and reverse pre-siRNA transcripts showed accumulation in the dcr1delta mutant. In rpb7-G150D the reverse transcripts decreased to 25% of wild type levels. In the double mutant dcr1delta/rpb7-G150D the reverse transcript levels remained low indicating that Rpb7 functions upstream of Dcr1 in the RNAi pathway that maintains the H3K9me2 in heterochromatin. More specifically, we show that Rpb7 is required for initiation of pre-siRNA transcription in this pathway.

In paper II, we show that the amino-oxidase family demethylases Lsd1 and Lsd2 from fission yeast are able to demethylate H3K9 but not H3K4 in vitro. Microarray studies showed increased H3K9me2 in 8,2% of the genes both in promoter and coding regions in the lsd1delta knock-out and 3,8% of the genes also showed increased H3K4me2 in promoter regions. These genes also tend to become upregulated in lsd1delta. Conversely, the down-regulated genes in lsd1delta showed increased levels of H3K9me2, consistent with the notion that H3K9me2 is needed for gene repression and silencing. Our data also suggest that Lsd1 and the HDAC Clr6 cooperate to repression of genes. Thus, Lsd1 seems to have demethylase activity at both H3K4 and K9 in vivo but in vitro only K9me activity was detected, suggesting that additional factors are directing Lsd1 specificity in vivo.

In paper III, we found that Pst3, one of the three Sin3 homologs in fission yeast, is localized to the entire nuclear space, including the nucleolar core. The deletion of the pst3+ gene affects genome stability, and causes sporulation defects, altered nucleolar structure and chomosome mis-segregation. Our data indicate that genome stability requires an established heterochromatic environment at rDNA repeats maintained by the Clr6 HDAC and Pst3. Pst3 co-purifies with two different Clr6 multi-protein complexes, suggesting that Clr6 HDAC complexes are dynamic. We show that Pst3 is associated with rDNA chromatin and is involved in rDNA silencing. Interestingly, Pst3 is specifically required for repression of endogenous Pol II mediated non-coding RNA transcripts within the rDNA spacer region.