University of Arkansas for Medical Sciences, Department of Biochemistry and Molecular Biology, Little Rock, AR 72205, USA.
Cell Rep. 2012 Jul 26;2(1):198-205. doi: 10.1016/j.celrep.2012.06.019. Epub 2012 Jul 20.
The field of epigenomics has been transformed by chromatin immunoprecipitation approaches that provide for the localization of a defined protein or posttranslationally modified protein to specific chromosomal sites. While these approaches have helped us conceptualize epigenetic mechanisms, the field has been limited by the inability to define features such as the proteome and histone modifications at a specific genomic locus in an unbiased manner. We developed an unbiased approach whereby a unique native genomic locus was isolated, which was followed by high-resolution proteomic identification of specifically associated proteins and histone posttranslational modifications. This chromatin affinity purification with mass spectrometry (ChAP-MS) technique was used to specifically enrich a ~1,000 base pair section of GAL1 chromatin under transcriptionally active and repressive conditions, as well as to identify the specifically bound proteins and histone posttranslational modifications. ChAP-MS should yield insight into the regulatory mechanisms of transcription and help identify factors that epigenetically control chromatin function.
表观基因组学领域已经发生了转变，通过染色质免疫沉淀方法可以将特定的蛋白质或翻译后修饰的蛋白质定位到特定的染色体位点。虽然这些方法帮助我们理解了表观遗传机制，但该领域一直受到限制，无法以无偏倚的方式定义特定基因组位点的蛋白质组和组蛋白修饰等特征。我们开发了一种无偏倚的方法，可以分离独特的天然基因组位点，然后对特定相关的蛋白质和组蛋白翻译后修饰进行高分辨率蛋白质组学鉴定。这种通过质谱进行的染色质亲和纯化（ChAP-MS）技术可用于在转录活跃和抑制条件下特异性富集约 1000 个碱基对的 GAL1 染色质片段，以及鉴定特异性结合的蛋白质和组蛋白翻译后修饰。ChAP-MS 应该可以深入了解转录的调控机制，并帮助识别那些表观遗传控制染色质功能的因子。
