Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.
Mol Cell Biol. 2012 Jun;32(12):2170-82. doi: 10.1128/MCB.00010-12. Epub 2012 Apr 9.
The specific set of proteins bound at each genomic locus contributes decisively to regulatory processes and to the identity of a cell. Understanding of the function of a particular locus requires the knowledge of what factors interact with that locus and how the protein composition changes in different cell types or during the response to internal and external signals. Proteomic analysis of isolated chromatin segments (PICh) was developed as a tool to target, purify, and identify proteins associated with a defined locus and was shown to allow the purification of human telomeric chromatin. Here we have developed this method to identify proteins that interact with the Drosophila telomere-associated sequence (TAS) repeats. Several of the purified factors were validated as novel TAS-bound proteins by chromatin immunoprecipitation, and the Brahma complex was confirmed as a dominant modifier of telomeric position effect through the use of a genetic test. These results offer information on the efficacy of applying the PICh protocol to loci with sequence more complex than that found at human telomeres and identify proteins that bind to the TAS repeats, which might contribute to TAS biology and chromatin silencing.
特定的蛋白质组结合在每个基因组位置，对调控过程和细胞的身份起决定性作用。了解特定基因座的功能需要知道哪些因素与该基因座相互作用，以及在不同的细胞类型或对内部和外部信号的反应中，蛋白质组成如何变化。分离染色质片段的蛋白质组学分析（PICh）被开发为一种靶向、纯化和鉴定与特定基因座相关的蛋白质的工具，并已被证明可用于纯化人类端粒染色质。在这里，我们开发了这种方法来鉴定与果蝇端粒相关序列（TAS）重复序列相互作用的蛋白质。通过染色质免疫沉淀验证了几种纯化的因子是新的 TAS 结合蛋白，并且通过遗传测试证实了 Brahma 复合物是端粒位置效应的主要修饰因子。这些结果提供了有关将 PICh 方案应用于比人类端粒中发现的序列更复杂的基因座的有效性的信息，并鉴定了与 TAS 重复序列结合的蛋白质，这些蛋白质可能有助于 TAS 生物学和染色质沉默。
