Schmid Manfred, Durussel Thérèse, Laemmli Ulrich K
Departments of Biochemistry and Molecular Biology, NCCR Frontiers in Genetics, University of Geneva, 30, Quai Ernest-Ansermet, CH1211, Geneva 4, Switzerland.
Mol Cell. 2004 Oct 8;16(1):147-57. doi: 10.1016/j.molcel.2004.09.007.
To map the genomic interaction sites of chromatin proteins, two related methods were developed and experimentally explored in Saccharomyces cerevisiae. The ChIC method (chromatin immunocleavage) consists of tethering a fusion protein (pA-MN) consisting of micrococcal nuclease (MN) and staphylococcal protein A to specifically bound antibodies. The nuclease is kept inactive during the tethering process (no Ca2+). The ChEC method (chromatin endogenous cleavage) consists of expressing fusion proteins in vivo, where MN is C-terminally fused to the proteins of interest. The specifically tethered nucleases are activated with Ca2+ ions to locally introduce double-stranded DNA breaks. We demonstrate that ChIC and ChEC map proteins with a 100-200 bp resolution and excellent specificity. One version of the method is applicable to formaldehyde-fixed nuclei, another to native cells with comparable results. Among various model experiments, these methods were used to address the conformation of yeast telomeres.
为了绘制染色质蛋白的基因组相互作用位点，人们开发了两种相关方法，并在酿酒酵母中进行了实验探索。ChIC方法（染色质免疫切割）包括将由微球菌核酸酶（MN）和葡萄球菌蛋白A组成的融合蛋白（pA-MN）与特异性结合的抗体相连。在连接过程中（无Ca2+），核酸酶保持无活性。ChEC方法（染色质内源切割）包括在体内表达融合蛋白，其中MN在C末端与感兴趣的蛋白质融合。特异性连接的核酸酶用Ca2+离子激活，以在局部引入双链DNA断裂。我们证明，ChIC和ChEC能够以100 - 200 bp的分辨率和出色的特异性绘制蛋白质图谱。该方法的一个版本适用于甲醛固定的细胞核，另一个版本适用于天然细胞，结果相当。在各种模型实验中，这些方法被用于研究酵母端粒的构象。
