Ishii Haruhiko, Kadonaga James T, Ren Bing
Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA 92093-0653;
Section of Molecular Biology, University of California, San Diego, La Jolla, CA 92093-0347;
Proc Natl Acad Sci U S A. 2015 Jul 7;112(27):E3457-65. doi: 10.1073/pnas.1424804112. Epub 2015 Jun 15.
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.
染色质结构分析对于理解真核生物中的转录调控至关重要。在此，我们描述了甲磺酸丙酯 - 乙二胺四乙酸测序（MPE-seq），这是一种用于全基因组染色质特征分析的方法，该方法涉及用MPE-Fe(II)消化细胞核，然后进行大规模平行测序。与微球菌核酸酶（MNase）一样，MPE-Fe(II)优先切割核小体之间的连接DNA。然而，相对于MNase，MPE-Fe(II)对核染色质的切割存在差异。最值得注意的是，在活跃启动子的转录起始位点上游紧邻处，我们经常观察到用MPE-seq而非MNase-seq检测到的消化染色质片段的核小体大小（141 - 190 bp）和亚核小体大小（如101 - 140 bp）的峰。这些峰也与核心组蛋白的存在相关，因此至少部分可能归因于非规范的染色质结构，如在其他情况下已观察到的不稳定核小体样颗粒。亚核小体大小的MPE-seq峰与活跃启动子表现出特别明显的关联。此外，与MNase不同，MPE-Fe(II)切割核DNA时几乎没有序列偏好。在这方面，我们发现RNA剪接位点处的DNA序列对MNase消化高度敏感，但对MPE-Fe(II)不敏感。这种现象可能影响了外显子上核小体占有率的分析。这些发现共同表明，MPE-seq为全基因组染色质结构分析提供了一种独特且直接的方法，且DNA序列偏差最小。特别是，MPE-seq和MNase-seq的联合使用能够鉴定可能对基因表达调控很重要的非规范染色质结构。
