MPE-seq, a new method for the genome-wide analysis of chromatin structure

Abstract

The analysis of chromatin structure is essential for the understanding of transcriptional regulation in eukaryotes. Here we describe methidiumpropyl-EDTA sequencing (MPE-seq), a method for the genome-wide characterization of chromatin that involves the digestion of nuclei withMPE-Fe(II) followed by massively parallel sequencing. Like micrococcal nuclease (MNase), MPE-Fe(II) preferentially cleaves the linker DNA between nucleosomes. However, there are differences in the cleavage of nuclear chromatin by MPE-Fe(II) relative to MNase. Most notably, immediately upstream of the transcription start site of active promoters, we frequently observed nucleosome-sized (141-190 bp) and subnucleosome-sized (such as 101-140 bp) peaks of digested chromatin fragments with MPE-seq but not with MNase-seq. These peaks also correlate with the presence of core histones and could thus be due, at least in part, to noncanonical chromatin structures such as labile nucleosome-like particles that have been observed in other contexts. The subnucleosome-sized MPE-seq peaks exhibit a particularly distinct association with active promoters. In addition, unlike MNase, MPE-Fe(II) cleaves nuclear DNA with little sequence bias. In this regard, we found that DNA sequences at RNA splice sites are hypersensitive to digestion by MNase but not by MPE-Fe(II). This phenomenon may have affected the analysis of nucleosome occupancy over exons. These findings collectively indicate that MPE-seq provides a unique and straightforward means for the genome-wide analysis of chromatin structure with minimal DNA sequence bias. In particular, the combined use of MPE-seq and MNase-seq enables the identification of noncanonical chromatin structures that are likely to be important for the regulation of gene expression.n eukaryotes, transcription is regulated by the interplay between transcription factors and chromatin. The nucleosome, the basic building block of chromatin, can occlude the access of transcription factors and hinder transcription by RNA polymerases (1, 2). The positions of nucleosomes can, in turn, be influenced by the DNA sequence, transcription factors, ATP-driven chromatin remodelers, and RNA polymerases (see, for example, refs. 3, 4). Thus, the positions and properties of nucleosomes are important for the regulation of transcription in the chromatin landscape.Micrococcal nuclease (MNase) has been a useful reagent for mapping nucleosome positions because it preferentially cleaves the linker DNA between nucleosomes and can yield core particles upon extensive digestion (5). Analyses of MNase-generated fragments by hybridization to high-density DNA microarrays or by using massively parallel sequencing technologies have made it possible to map genome-wide positions of nucleosomes (6-14). However, MNase has a bias for AT-rich sequences (15, 16), and whether or not this sequence bias affects the interpretation of nucleosome positions and occupancies has been a matter of debate (17)(18)(19).To gain additional insights into chromatin structure, several altern...

Widespread changes in nucleosome accessibility without changes in nucleosome occupancy during a rapid transcriptional induction

“…The extent to which these changes are due to nucleosomes themselves as opposed to nonhistone proteins has been a matter of recent debate (Chereji et al 2017). In larger eukaryotes, fragile nucleosomes upstream of TSSs have been found in Drosophila (Chereji et al 2016, Mieczkowski et al 2016) and human embryonic stem cells (Ishii et al 2015;Voong et al 2016), but whether these seemingly ubiquitous features of eukaryotic promoters are true nucleosomes or other DNA-binding proteins remains to be determined. To address whether nucleosomes were responsible for protection in the work presented here, the experiments were all performed with a histone H3 immunoprecipitation step to indicate that all measured fragments contained nucleosomes.…”

The Pioneer Transcription Factor FoxA Maintains an Accessible Nucleosome Configuration at Enhancers for Tissue-Specific Gene Activation

“…A prior nucleosome mapping study, using relatively high levels of MNase digestion, reported no differences in nucleosome occupancy at selected sites in the absence of FoxA1 and FoxA2, possibly because nucleosomes at open chromatin regions had been missed (Li et al, 2011). A recent study, using substantially lower levels of chromatin digestion with methidiumpropyl-EDTA coupled with core histone ChIP, revealed more apparent nucleosome retention than had been revealed by prior high-level MNase studies (Ishii et al, 2015). In yeast, low levels of MNase digestion recovered DNA fragments within previously determined “nucleosome-free” regions (200 bp upstream of the TSS) in a subset of promoters in addition to within the −1 nucleosome position (upstream of the “nucleosome-free” region), and renamed them as “fragile nucleosomes” (Rhee et al, 2014; Weiner et al, 2010; Xi et al, 2011).…”

Prenucleosomes and Active Chromatin

“…Second, prenucleosome-sized DNA-containing particles with core histones are observed at the NDRs of active promoters in mouse cells (Fig. 7; also see Ishii et al 2015). Moreover, the occurrence of these particles correlates with the level of promoter activity.…”

“…To gain a different perspective on chromatin structure, we used the chemical reagent methidiumpropyl-EDTA-Fe(II) (MPE-Fe(II)) to cleave chromatin in nuclei, and then we analyzed the resulting DNA fragments by genome-wide paired-end sequencing (Ishii et al 2015). This method, termed MPE-seq, revealed that the upstream promoter regions of active genes in mouse cells contain chromatin particles with subnucleosome-sized DNA fragments.…”

“…The 61–100 bp data from Ishii et al (2015) were subjected to fragment positioning analysis as described in Ishii et al (2015). The fragment positioning index depicts the location of the central regions of the DNA fragments, which were identified by paired-end sequencing.…”