Functional characterisation of the fission yeast Ino80 complex

INOsitol-requiring 80 (Ino80) is a catalytic ATP-dependent nucleosome remodelling enzyme of the Ino80 complex which is involved in transcription, replication and the DNA damage response. In this thesis, I characterise for the first time the Ino80 complex from Schizosaccharomyces pombe. Purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein, lec1, with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Krüppel family of proteins. Deletion of this lec1 protein or the Ino80 complex subunits: Arp8, les6 or les2 causes defects in DNA damage repair, response to replication stress and nucleotide metabolism. I demonstrate that lec1 is important for the correct expression of genes involved in nucleotide metabolism such as ribonucleotide reductase subunit cdc22. Ino80 is recruited to a large number of promoter regions upon phosphate starvation, including those of phosphate and adenine responsive genes that depend on lec1 for correct expression. lec1 is required for binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes. this suggests that the lec1-Ino80 complex promotes transcription through nucleosome eviction. My study of the Ino80 complex subunits lec1 and Arp8 shows that they act together or oppose one another, depending on the target locus. These subunits affect gene expression, histone density and deposition of histone marks: H3K4me3, H3K9me3 and H3K36me3. Arp8 affects iec1 gene expression and regulates the presence of lec1 at target genes. These results reveal the modular nature of the Ino80 complex. Finally, I show that deletion of Ino80 subunits leads to sensitivity to drugs that inhibit transcription elongation.