Guillen-Ahlers Hector, Rao Prahlad K, Perumalla Danu S, Montoya Maria J, Jadhav Avinash Y L, Shortreed Michael R, Smith Lloyd M, Olivier Michael
Department of Genetics, Texas Biomedical Research Institute; Department of Internal Medicine-Molecular Medicine, Wake Forest University School of Medicine.
Department of Genetics, Texas Biomedical Research Institute.
J Vis Exp. 2018 Jun 1(136):57140. doi: 10.3791/57140.
The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.
用于蛋白质组学的染色质相关蛋白杂交捕获（HyCCAPP）技术最初是为了揭示酵母中新型DNA-蛋白质相互作用而开发的。它能够分析感兴趣的目标区域，而无需事先了解与该目标区域结合的可能蛋白质。从理论上讲，这使得HyCCAPP可用于分析任何感兴趣的基因组区域，并提供了足够的灵活性以在不同的细胞系统中开展工作。该方法并非用于研究已知转录因子的结合位点，这一任务更适合采用染色质免疫沉淀（ChIP）及类似ChIP的方法。HyCCAPP的优势在于其能够探索那些对与之结合的蛋白质了解有限或一无所知的DNA区域。它也是一种便捷的方法，可避免基于抗体的蛋白质染色质富集（在类似ChIP的方法中存在）所引入的偏差。HyCCAPP有可能成为揭示真正新型DNA-蛋白质相互作用的强大工具。迄今为止，该技术主要应用于酵母细胞或哺乳动物细胞中的高拷贝重复序列。为了成为我们所设想的强大工具，HyCCAPP方法需要进行优化，以有效捕获哺乳动物细胞中的单拷贝基因座。在此，我们展示了将最初的酵母HyCCAPP捕获方案应用于人类细胞系的过程，并表明通过这种改良方案能够有效分离单拷贝染色质区域。
