McManus Kirk J, Hendzel Michael J
Department of Oncology, University of Alberta, Cross Cancer Institute, Edmonton, Alta., Canada T6G 1Z2.
Methods. 2005 Aug;36(4):351-61. doi: 10.1016/j.ymeth.2005.03.010.
Until recently, identifying the specificities of enzymes that post-translationally modify core histones was performed in vitro using synthetic peptides, purified mononucleosomes or short nucleosome arrays. Unfortunately, the variable results obtained for identical enzymes are often dependent on the in vitro conditions employed. These results are consistent with the conclusion that the manner in which histone tails are presented to the modifying enzymes dramatically affects specificity. Because traditional in vitro biochemical approaches do not accurately recapitulate higher-order chromatin structure or consider the influences that additional chromatin binding proteins may have on determining the specificity of modifying enzymes, the development of new and innovative approaches is warranted. Here, we describe a novel in situ microscopy approach that accurately assesses enzyme substrate specificities through single cell measurements performed under physiologically relevant conditions. This approach couples the spatial resolving power of microscopy with robust statistical analyses to determine the substrate specificities of transiently expressed enzymes using histone modification- and residue-specific antibodies. This methodology can also be applied to measuring changes in the abundance of histone modifications as cells traverse the cell cycle.
直到最近，确定对核心组蛋白进行翻译后修饰的酶的特异性都是在体外使用合成肽、纯化的单核小体或短核小体阵列来进行的。不幸的是，对于相同的酶所获得的可变结果往往取决于所采用的体外条件。这些结果与以下结论一致，即组蛋白尾巴呈现给修饰酶的方式会显著影响特异性。由于传统的体外生化方法不能准确重现高阶染色质结构，也没有考虑其他染色质结合蛋白对确定修饰酶特异性可能产生的影响，因此有必要开发新的创新方法。在这里，我们描述了一种新颖的原位显微镜方法，该方法通过在生理相关条件下进行的单细胞测量来准确评估酶底物特异性。这种方法将显微镜的空间分辨能力与强大的统计分析相结合，以使用组蛋白修饰和残基特异性抗体来确定瞬时表达酶的底物特异性。这种方法还可应用于测量细胞在细胞周期中组蛋白修饰丰度的变化。
