Byrum Stephanie D, Taverna Sean D, Tackett Alan J
University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, Arkansas 72205, USA.
Johns Hopkins School of Medicine, 855 North Wolfe Street, Baltimore, Maryland 21205, USA.
J Clin Bioinforma. 2011 Jul 7;1(1):17. doi: 10.1186/2043-9113-1-17.
Genome-wide studies use techniques, like chromatin immunoprecipitation, to purify small chromatin sections so that protein-protein and protein-DNA interactions can be analyzed for their roles in modulating gene transcription. Histone post-translational modifications (PTMs) are key regulators of gene transcription and are therefore prime targets for these types of studies. Chromatin purification protocols vary in the amount of chemical cross-linking used to preserve in vivo interactions. A balanced level of chemical cross-linking is required to preserve the native chromatin state during purification, while still allowing for solubility and interaction with affinity reagents.
We previously used an isotopic labeling technique combining affinity purification and mass spectrometry called transient isotopic differentiation of interactions as random or targeted (transient I-DIRT) to identify the amounts of chemical cross-linking required to prevent histone exchange during chromatin purification. New bioinformatic analyses reported here reveal that histones containing transcription activating PTMs exchange more rapidly relative to bulk histones and therefore require a higher level of cross-linking to preserve the in vivo chromatin structure.
The bioinformatic approach described here is widely applicable to other studies requiring the analysis and purification of cognate histones and their modifications. Histones containing PTMs correlated to active gene transcription exchange more readily than bulk histones; therefore, it is necessary to use more rigorous in vivo chemical cross-linking to stabilize these marks during chromatin purification.
全基因组研究使用染色质免疫沉淀等技术来纯化小的染色质片段，以便分析蛋白质-蛋白质和蛋白质-DNA相互作用在调节基因转录中的作用。组蛋白翻译后修饰（PTMs）是基因转录的关键调节因子，因此是这类研究的主要目标。染色质纯化方案在用于保存体内相互作用的化学交联量方面各不相同。在纯化过程中需要平衡的化学交联水平来维持天然染色质状态，同时仍要保证其溶解性以及与亲和试剂的相互作用。
我们之前使用了一种结合亲和纯化和质谱的同位素标记技术，称为相互作用的瞬时同位素分化作为随机或靶向（瞬时I-DIRT），以确定在染色质纯化过程中防止组蛋白交换所需的化学交联量。此处报道的新生物信息学分析表明，含有转录激活PTMs的组蛋白相对于整体组蛋白交换更快，因此需要更高水平的交联来维持体内染色质结构。
本文所述的生物信息学方法广泛适用于其他需要分析和纯化同源组蛋白及其修饰的研究。与活跃基因转录相关的含有PTMs的组蛋白比整体组蛋白更容易交换；因此，在染色质纯化过程中需要使用更严格的体内化学交联来稳定这些标记。
