Identification of in vivo DNA targets of chromatin proteins using tethered Dam methyltransferase
Identification of in vivo DNA targets of chromatin proteins using tethered Dam methyltransferase
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DOI：
10.1038/74487
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发表时间：
2000-04-01
影响因子：
46.9
通讯作者：
Henikoff, S
中科院分区：
文献类型：
--
作者：
van Steensel, B;Henikoff, S
我们已经开发了一种新的技术，命名为Dam1D，用于鉴定在体内与真核生物中的特定核蛋白相互作用的DNA位点。通过将大肠杆菌DNA腺嘌呤甲基转移酶（Dam）拴系到染色质蛋白上，Dam可以在体内靶向该蛋白的天然结合位点，导致局部DNA甲基化。随后可以使用甲基化特异性限制酶或抗体来定位甲基化位点。我们证明了Dam1D在果蝇细胞培养和整个果蝇中的成功应用。当Dam与GAL4的DNA结合结构域连接时，靶向甲基化仅限于GAL4结合序列周围的几个DNA酶区域。使用Dam1D，我们确定了一些预期和意想不到的果蝇异染色质蛋白1的靶位点。Dam1D具有在各种真核生物中染色质蛋白的体内靶点的全基因组映射的潜力。
We have developed a novel technique, named Dam1D, for the identification of DNA loci that interact in vivo with specific nuclear proteins in eukaryotes. By tethering Escherichia coli DNA adenine methyltransferase (Dam) to a chromatin protein, Dam can be targeted in vivo to native binding sites of this protein, resulting in local DNA methylation. Sites of methylation can subsequently be mapped using methylation-specific restriction enzymes or antibodies. We demonstrate the successful application of Dam1D both in Drosophila cell cultures and in whole flies. When Dam is tethered to the DNA-binding domain of GAL4, targeted methylation is limited to a region of a few kilobases surrounding a GAL4 binding sequence. Using Dam1D, we identified a number of expected and unexpected target loci for Drosophila heterochromatin protein 1. Dam1D has potential for genome-wide mapping of in vivo targets of chromatin proteins in various eukaryotes.