Genome binding/occupancy profiling by high throughput sequencing
Summary
Arrays of regularly spaced nucleosomes dominate chromatin and are often phased, i.e., aligned at reference sites like active promoters. How distances between nucleosomes and distances between phasing sites and nucleosomes are determined remained unclear, specifically, the role of ATP dependent chromatin remodelers in it. Here, we used a genome-wide reconstitution system to probe how yeast remodelers generate phased nucleosome arrays. We find that remodelers bear a structural element named the ‘ruler’ that sets nucleosome spacing, in the order Chd1 < ISW1a < ISW2 < INO80. Structure-based mutagenesis confirmed the functional significance of the ruler element in INO80. Differences in the ruler elements of different remodelers explain the observed nucleosome array features. More generally, we propose that remodelers use their rulers to regulate the direction of nucleosome sliding in response to nucleosome density and environment, leading to nucleosome positioning relative to other nucleosomes, DNA bound factors or DNA sequence elements.
Overall design
We used our genome-wide in vitro reconstitution system (Krietenstein et al. 2016, Cell) to investigate the effect of varying nucleosome densities, different barriers (Reb1 and DNA double strand breaks) and underlying DNA sequences from different genomes (S. cerevisiae, S. pombe and E. coli) on all S. cerevisiae remodelers with spacing activity (INO80, ISW2, ISW1a and Chd1). Additionally, we used structural-guided INO80 mutans to find insights into INO80s' spacing mechanism.
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