Development and application of a method to study DNA methylation and chromatin structure at the chromosome domain level

染色体结构域水平DNA甲基化和染色质结构研究方法的开发与应用

• 批准号: 06680669
• 负责人: SASAKI Hiroyuki
• 金额: $ 1.47万
• 依托单位: Kyushu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06680669/
• 关键词: DNA methylation; Chromatin; CpG island

In order to develop a method to study DNA methylation and chromtin structure of large genomic regions, we combined the methylation-sensitive restriction assay and nuclease-sensitivity assay with field-inversion gel electrophoresis (FIGE). The distal region of mouse chromosome 7, which comprises the imprinted ins2/lgf2/H19 genes, was analyzed in model expeniments. When 100 kb of the region was scanned by this method for DNasel hypersensitive sites, three new clusters of hypersensitive sites were identified. It was possible to study fragments larger than 25 kb. Similarly, it was possible to determine the methylation status of genomic regions larger than 25 kb after partial digestion with HpaII and HhaI.The reliability of these methods was comfirmed by examining the same regions by the conventional, short-range methods. We also found that partial degestion with HpaII and HhaI was useful for identifying and mapping CpG islands in native genomic and cloned DNA.The usefulness of the method has been shown with lambda, cosmid and P1 clones containing various inserts and a fragment as large as 85 kb could be analyzed at once. These methods are quite simple and reliable and thus can be applied to the long-range analysis of genome and chromatin structure.

为了开发一种研究大基因组区域的 DNA 甲基化和染色质结构的方法，我们将甲基化敏感限制性分析和核酸酶敏感分析与场反转凝胶电泳 (FIGE) 结合起来。在模型实验中对小鼠 7 号染色体的远端区域（包含印记 ins2/lgf2/H19 基因）进行了分析。当用这种方法扫描该区域的 100 kb 的 DNAsel 超敏感位点时，鉴定出了三个新的超敏感位点簇。研究大于 25 kb 的片段是可能的。同样，在用 HpaII 和 HhaI 部分消化后，可以确定大于 25 kb 的基因组区域的甲基化状态。通过用传统的短程方法检查相同区域，证实了这些方法的可靠性。我们还发现，用 HpaII 和 HhaI 进行部分消化对于识别和定位天然基因组和克隆 DNA 中的 CpG 岛非常有用。该方法的实用性已在包含各种插入片段的 lambda、粘粒和 P1 克隆中得到证明，并且可以一次分析大至 85 kb 的片段。这些方法非常简单可靠，可应用于基因组和染色质结构的远程分析。

项目成果

Sasaki, H.et al.: "Temporal and spatial regulation of H19 imprinting in normal and uniparental embryos." Development. 121. 4195-4202 (1995)

Sasaki, H.et al.：“正常和单亲胚胎中 H19 印记的时间和空间调节。”

Sasaki, H. et al.: "Temporal and spatial regulation of H19 imprinting in normal and uniparental embryos." Development. 121. 4195-4202 (1995)

Sasaki, H. 等人：“正常和单亲胚胎中 H19 印记的时间和空间调节。”

Surani, M. A. et al.: "Genomic imprinting : causes and consequences" Cambridge University Press (eds. Ohlsson, R., Hall, K. & Ritzen, M.), 374 (1995)

Surani, M. A. 等人：“基因组印记：原因和后果”剑桥大学出版社（Ohlsson, R.、Hall, K. 编辑）

Surani, M.A.et al.: "Imprinting of H19 and Xist in uniparental embryos. In : Genomic imprinting : causesand consequences." Cambridge University Press (eds.Ohlsson, R., Hall, K.& Ritzen, M.). 142-156 (1995)

Surani, M.A.等人：“单亲胚胎中 H19 和 Xist 的印记。见：基因组印记：原因和后果。”