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标题：MPE-seq, a new method for the genome-wide analysis of chromatin structure
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作者：Haruhiko Ishii ; James T. Kadonaga ; Bing Ren 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：27
页码：E3457-E3465
DOI：10.1073/pnas.1424804112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe structure of chromatin is critical for processes such as transcription, DNA replication, and DNA repair. The most commonly used techniques for analyzing chromatin structure involve the use of enzymes such as micrococcal nuclease (MNase) and DNase I. These enzymes, however, have distinct characteristics that can at some times be an advantage but in other situations be a drawback. Here we describe methidiumpropyl-EDTA sequencing (MPE-seq), a method in which we use the chemical MPE-Fe(II) for the genome-wide analysis of chromatin structure. MPE-Fe(II) cleaves chromatin with minimal DNA sequence bias. Moreover, MPE-seq reveals noncanonical chromatin structures in active promoter regions that are not seen with standard MNase-seq conditions. MPE-seq provides insights into chromatin structure that complement the information gained from MNase-seq. 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.
关键词：MPE-Fe(II) ; chromatin ; genome-wide analysis ; promoter ; micrococcal nuclease