A single fiber view of the nucleosome organization of eukaryotic chromatin

Nucleosomes arrange into extended arrays, much like beads on a string. They are often phased at genomic landmarks and are thought to be evenly spaced. Here we tested to what extent this stereotypic organization describes the nucleosome landscape in Saccharomyces cerevisiae using a long-read nucleosome-sequencing technique called Fiber-Seq. Fiber-Seq maps the nucleosome pattern on individual chromatin fibers. As such, it is ideally suited to measure the density of nucleosomes per read and quantitate the nucleosome occupancy throughout the genome. We document substantial deviations from the stereotypical nucleosome organization, with unexpectedly long linker DNAs between individual nucleosomes, genomic regions lacking entire nucleosomes, heterogeneous phasing of arrays, truly irregular spacing of arrays and read-to-read variation in nucleosome densities. We exploited the technology to test mechanistic models for the biogenesis of nucleosome arrays. We can rule out transcription elongation playing a decisive role in array formation and detect signatures for a clamping activity of remodelers of the ISWI and CHD1 families after acute nucleosome depletion in vivo. Given that nucleosomes are cis-regulatory elements, the cell-to-cell heterogeneity that Fiber-Seq uncovers provides much needed information to understand chromatin structure and function. Overall design: Methylation profiling of S. cerevisiae strains using the Oxford Nanopore MinION and R9.4.1 version chemistry. Genomic DNA was treated by M.SssI, extracted and Nanopore sequencing was applied to detect DNA cytosine modification.