title: 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.
authors.affiliation: Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA 92093-0653;
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Ishii
authors.affiliation: Section of Molecular Biology, University of California, San Diego, La Jolla, CA 92093-0347; jkadonaga@ucsd.edu biren@ucsd.edu.
authors.forename: James T
authors.initials: JT
authors.lastname: Kadonaga
authors.affiliation: Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA 92093-0653; Department of Cellular and Molecular Medicine, Institute of Genome Medicine, Moores Cancer Center, University of California, San Diego, School of Medicine, La Jolla, CA 92093-0653 jkadonaga@ucsd.edu biren@ucsd.edu.
authors.forename: Bing
authors.initials: B
authors.lastname: Ren
mesh_terms: D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D053595:Embryonic Stem Cells / Q000378:metabolism; D005091:Exons / Q000235:genetics; D016678:Genome / Q000235:genetics*; D007438:Introns / Q000235:genetics; D051379:Mice; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D002843:Chromatin; D009707:Nucleosomes; C038200:methidiumpropyl-EDTA-iron(II); D004247:DNA; D004492:Edetic Acid
keywords: MPE-Fe(II); chromatin; genome-wide analysis; micrococcal nuclease; promoter