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Quantitative MNase-seq accurately maps nucleosome occupancy levels

ABSTRACT: Chromatin mapping using micrococcal nuclease (MNase) has been the standard tool for mapping nucleosomes for >40 years. When coupled with DNA sequencing, MNase-seq can provide base-pair-resolution nucleosome maps. However, determining nucleosome occupancy using MNase-seq has been hampered by its aggressive endo-/exo-nuclease activities, whereby cleavages within linker regions produce oligo- and mono-nucleosomes whereas cleavages within nucleosomes destroy them. Here we introduce a theoretical framework for predicting nucleosome occupancies and an experimental protocol with appropriate spike-in normalization that confirms our theory and provides accurate occupancy levels over an MNase digestion time-course. As expected, DNaseI hypersensitive sites and transcription units are digested by MNase at elevated rates, and the apparent deficiency of nucleosomes at 3’ ends of Drosophila genes is an artifact of MNase preference for AT-rich DNA. Surprisingly, we observed no overall differences between Drosophila euchromatin and heterochromatin, which implies that heterochromatin compaction does not render nucleosomal DNA less accessible than euchromatin.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE128689 | GEO | 2019/08/27

REPOSITORIES: GEO

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Project description:Maintenance of the correct level and organization of nucleosomes is crucial for genome function. Here we uncover a role for a conserved bromodomain AAA-ATPase, Abo1, in maintenance of nucleosome architecture in fission yeast. Cells lacking abo1+ experience both a reduction and mis-positioning of nucleosomes at transcribed sequences in addition to increased intragenic transcription, phenotypes that are hallmarks of defective chromatin re-establishment behind RNA polymerase II. Abo1 is recruited to gene sequences and associates with histone H3 and the histone chaperone FACT. Furthermore, the distribution of Abo1 on chromatin is disturbed by impaired FACT function. The role of Abo1 extends to some promoters and also to silent heterochromatin. Abo1 is recruited to pericentromeric heterochromatin independently of the HP1 ortholog, Swi6, where it enforces proper nucleosome occupancy. Consequently, loss of Abo1 alleviates silencing and causes elevated chromosome mis-segregation. We suggest that Abo1 provides a histone chaperone function that maintains nucleosome architecture genome-wide. A chromatin-seq/MNase-seq approach called Chromatin Particle Spectrum Analysis (Kent et al., (2011) Nucleic Acids Res. 39:e26) was used to map and compare nucleosome position in wild-type (strain 972) and isogenic abo1 knock-out (strain HM463) fission yeast cells. For each strain, three independent in vivo MNase digest bio-reps were performed and the purified DNA pooled. CPSA was performed using paired-end mode Illumina technology with pooled samples multiplexed over two HiSeq2000 lanes. Each CPSA paired read describes a microcococcal nuclease (MNase) resistant DNA species from chromatin, with the insert-size equivalent to the size of DNA protection. For each strain type, two analysed data sets are provided here: one listing the genomic distribution of MNase-protected DNAs of 150bp (â150bp CPSA size classâ) and corresponding to mono-nucleosomes; the other listing the genomic distribution of MNase-protected DNAs of 300bp (â300bp CPSA size classâ) and corresponding to di-nucleosomes.

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Quantitative MNase-seq accurately maps nucleosome occupancy levels

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