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

为了建立一种研究大基因组区域DNA甲基化和染色质结构的方法，我们将甲基化敏感的限制性内切酶分析和核酸酶敏感分析与场翻转凝胶电泳法相结合。在模型实验中分析了小鼠7号染色体的远端区域，该区域包含印记的ins2/lgf2/h19基因。当用这种方法扫描100kb的DNasel超敏位点时，鉴定出三个新的超敏位点簇。研究大于25kb的片段是可能的。同样，用HpaII和HhaI部分消化后，可以确定大于25kb的基因组区域的甲基化状态。通过用常规的短程方法检查相同的区域，证实了这些方法的可靠性。我们还发现，用HpaII和HHAI进行部分消食对于识别和定位天然基因组和克隆DNA中的CpG岛是有用的。该方法已经在包含各种插入片段的lambda、Cosmida和P1克隆上得到了验证，并且可以一次分析长达85kb的片段。这些方法简单可靠，可用于基因组和染色质结构的远程分析。

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.