van Steensel B, Henikoff S
Fred Hutchinson Cancer Research Center and Howard Hughes Medical Institute, Seattle, WA 98109, USA.
Nat Biotechnol. 2000 Apr;18(4):424-8. doi: 10.1038/74487.
We have developed a novel technique, named DamID, 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 DamID 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 DamID, we identified a number of expected and unexpected target loci for Drosophila heterochromatin protein 1. DamID has potential for genome-wide mapping of in vivo targets of chromatin proteins in various eukaryotes.
我们开发了一种名为DamID的新技术，用于鉴定真核生物中在体内与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）与一种染色质蛋白相连，Dam在体内可被靶向至该蛋白的天然结合位点，从而导致局部DNA甲基化。随后可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DamID在果蝇细胞培养物和整个果蝇中的成功应用。当Dam与GAL4的DNA结合结构域相连时，靶向甲基化仅限于围绕GAL4结合序列的几千碱基的区域。使用DamID，我们鉴定出了果蝇异染色质蛋白1的一些预期和意外的靶位点。DamID有潜力用于全基因组绘制各种真核生物中染色质蛋白的体内靶标。
