Dissection of nucleosome positioning and spacing mechanisms in Saccharomyces cerevisiae

Eukaryotic genomes are packaged as chromatin that contains an ever-repeating succession of nucleosomes like beads-on-a-string. Nucleosomes, together with linker histones, promote packaging of genome into higher-order chromatin structure. Nucleosomes regulate access to the underlying DNA as well as all nuclear processes, including transcription, replication, and double-strand break repair. Defects in these processes lead to genome instability and diseases. Therefore, it is essential to understand the fundamental mechanisms which regulate nucleosome organization. Nucleosomes attain a stereotypical organization near major regulatory sites in the genome. At these regions, a nucleosome-free region is followed by well-positioned nucleosomes which are phased to a known genomic point, such as transcription start sites in S. cerevisiae and at CCCTC-binding sites in higher organisms. The average distance between these nucleosomes, called nucleosome repeat length (NRL), is surprisingly constant and lead to a regular nucleosome array. How this primary structure of chromatin is established is not completely clear. Several nuclear factors, like transcription, ATP-dependent nucleosome remodelers, barrier factors and DNA sequence are known to contribute to this process. Many of these factors act in a redundant manner in the cell. Therefore, it is difficult to understand the mechanism behind regular arrays and the role of individual factor towards it. In this thesis, we employed the baker’s yeast (S. cerevisiae) to dissect the mechanism of regular array formation. We made use of a yeast strain lacking bona fide spacing remodelers of the ISWI- and CHD- families to cleanly dissect the role of cellular factors and function of regular arrays. In chapter 2.1, we show that the RNA Pol II-dependent transcription destroys regular nucleosome arrays and overrides effects of the spacing remodelers. By inhibiting transcription in cells, we identify residual spacing activity and assign it to the INO80 nucleosome remodeler. Several orthogonal approaches establish INO80 as a bona fide spacing remodeler in vivo. We dissected the spacing mechanism of INO80 and show that the Arp8 module determines NRL by INO80, while the Nhp10 module is dispensable. We determine the interplay of histone amounts and remodelers to show that the spacing remodelers critically depend on high histone density to establish the WT-like nucleosome array. ISWI and Chd1 remodelers possess “clamping” activity to establish regular arrays, but this activity is rather weak in vivo. Finally, we find that the DNA sequence co-determines NRL in most part of the genome and spacing remodelers override DNA sequence-influenced extremely short- or long- NRLs in the genome. We show that the regular arrays established by the spacing remodelers protect the genome from genotoxic stress such as DNA damage and ectopic recombination, and regulate chromatin accessibility in the gene body. We propose a four-step model for the establishment of regular nucleosome arrays and suggest that it has evolved to regulate as well as to protect the genome. In chapter 2.2 and 2.3, we investigated the regulatory mechanism of the ISW1 and ISW2 spacing remodelers in vivo. We find that ISW2 requires the N-terminus of its accessory subunit Itc1 to position and space nucleosomes. Unlike ISW1 and Chd1, ISW2 resolves dinucleosomes in the gene body. We present a genome-wide cell-type specific nucleosome architecture analysis and dissect the role of ISW2 in this process. Lastly, we investigated the role of individual domains and subunits in the ISW1 remodeler towards nucleosome positioning and spacing. We show that ISW1 depends on the N-terminus and NegC motif within its ATPase subunit for nucleosome positioning. Surprisingly, the AutoN motif is not required for ISW1 spacing mechanism. Overall, this study establishes a unifying model of the biogenesis of regular nucleosome arrays and provides a basis for future investigation of the interplay of transcription and spacing remodelers towards establishing the primary structure of chromatin