Footprinting of mammalian promoters: use of a CpG DNA methyltransferase revealing nucleosome positions at a single molecule level.
Footprinting of mammalian promoters: use of a CpG DNA methyltransferase revealing nucleosome positions at a single molecule level.
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哺乳动物启动子的足迹：使用CpG DNA甲基转移酶在单个分子水平上揭示了核小体位置。
DOI：
10.1093/nar/gni180
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发表时间：
2005-11-27
影响因子：
14.9
通讯作者：
Jones, PA
中科院分区：
文献类型：
--
作者：
Fatemi, M;Pao, MM;Jeong, S;Gal-Yam, EN;Egger, G;Weisenberger, DJ;Jones, PA
启动子是分子“模块”，作为个体实体进行控制，但通常通过核酸酶消化方法进行分析，这种方法先验地破坏了这种模块性。大约40%的哺乳动物基因在启动子和外显子区域含有CpG岛，通常是未甲基化的。我们开发了一种足迹策略，通过使用CpG特异性DNA甲基转移酶SssI （m.s SssI）处理分离细胞核来绘制未甲基化CpG岛的染色质结构，然后对单个后代DNA分子进行基因组亚硫酸盐测序。这给了启动子区域的单分子分辨率，并允许维持单个启动子分子上结合位点之间的物理连接。对表达p16基因25倍的两种人细胞系J82和LD419中p16启动子的比较表明，这两种细胞系在启动子区域上含有显著不同的、位置不均匀的核小体，用核酸酶的标准方法无法区分。我们的高分辨率方法给出了每个启动子的“数字化”可视化，提供了有关核小体占用的信息，并可用于定义转录因子结合和染色质重塑。
Promoters are molecular ‘modules’, which are controlled as individual entities yet are often analyzed by nuclease digestion methodologies which, a priori, destroy this modularity. About 40% of mammalian genes contain CpG islands in their promoters and exonic regions, which are normally unmethylated. We developed a footprinting strategy to map the chromatin structure at unmethylated CpG islands by treatment of isolated nuclei with the CpG-specific DNA methyltransferase SssI (M.SssI), followed by genomic bisulfite sequencing of individual progeny DNA molecules. This gave single molecule resolution over the promoter region and allowed for the physical linkage between binding sites on individual promoter molecules to be maintained. Comparison of the p16 promoters in two human cell lines, J82 and LD419, expressing the p16 gene at 25-fold different levels showed that the two cell lines contain remarkably different, heterogeneously positioned nucleosomes over the promoter region, which were not distinguishable by standard methods using nucleases. Our high resolution approach gives a ‘digitized’ visualization of each promoter providing information regarding nucleosome occupancy and may be utilized to define transcription factor binding and chromatin remodeling.
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影响因子：
14.9
作者：
Nguyen, CT;Gonzales, FA;Jones, PA
通讯作者：
Jones, PA
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FRAGOSO, G;JOHN, S;HAGER, GL
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