Quantitative MNase-seq accurately maps nucleosome occupancy levels
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA528497 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8766790_1.fastq.gz | Fastqsanger.gz | |||
| SRR8766790_2.fastq.gz | Fastqsanger.gz | |||
| SRR8766800_1.fastq.gz | Fastqsanger.gz | |||
| SRR8766800_2.fastq.gz | Fastqsanger.gz | |||
| SRR8766791_1.fastq.gz | Fastqsanger.gz |
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Project description: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.
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Project description:Micrococcal nuclease (MNase) is widely used to map nucleosomes. However, its aggressive endo-/exo-nuclease activities make MNase-seq unreliable for determining nucleosome occupancies, because 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 with human cells, we observe no overall differences in nucleosome occupancies between Drosophila euchromatin and heterochromatin, which implies that heterochromatic compaction does not reduce MNase accessibility of linker DNA.
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Project description:MNase-Seq for nucleosome occupancy
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Project description:Chromatin accessibility plays a fundamental role in gene regulation. One mechanism to regulate accessibility is nucleosome placement, which is often measured by quantifying protection of DNA from enzymatic digestion. We introduce a metric that uses micrococcal nuclease (MNase) digestion in a novel manner to measure chromatin accessibility by combining information from several digests of increasing depths. This metric, MACC, quantifies the inherent heterogeneity of nucleosome accessibility in which some nucleosomes are seen preferentially at high MNase and some at low MNase. MACC interrogates each genomic locus, measuring both location of nucleosomes and accessibility to MNase in the same assay. MACC can be performed either with or without a histone immunoprecipitation step, and thereby compares behavior of nucleosomes to that of non-histone proteins. We find that enhancers, promoters and other regulatory regions have changes in accessibility that do not correlate with changes in nucleosome occupancy. Moreover, we show that high nucleosome occupancy does not necessarily preclude high accessibility, revealing novel principles of chromatin regulation.
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Project description:Embryonic stem cells (ESCs) and induced-pluripotent stem cells (iPSCs) self-renew and differentiate into an array of cell types in vitro and in vivo. A complex network of genetic and epigenetic pathways regulates the self-renewal and differentiation of these pluripotent cells, and the structure and covalent modifications of chromatin play a prominent role in this process. We examine nucleosome occupancy in mouse and human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), and differentiated cell types using MNase-seq. To address variability inherent in this technique, we developed a bioinformatic approach that enabled the identification of regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. The majority of changes in nucleosomal signatures that occur in differentiation are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of pluripotency and likely identify key regulatory regions that play a role in determining cell identity.
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