Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, SE-75185 Uppsala, Sweden.
N Biotechnol. 2012 Jun 15;29(5):589-98. doi: 10.1016/j.nbt.2011.08.002. Epub 2011 Aug 31.
Gene expression - a key feature for modulating cell fate-is regulated in part by histone modifications, which modulate accessibility of the chromatin to transcription factors. Until now, protein-DNA interactions (PDIs) have mostly been studied in bulk without retrieving spatial information from the sample or with poor sequence resolution. New tools are needed to reveal proteins interacting with specific DNA sequences in situ for further understanding of the orchestration of transcriptional control within the nucleus. We present herein an approach to visualise individual PDIs within cells, based on the in situ proximity ligation assay (PLA). This assay, previously used for the detection of protein-protein interactions in situ, was adapted for analysis of target PDIs, using padlock probes to identify unique DNA sequences in complex genomes. As a proof-of-principle we detected histone H3 interacting with a 26 bp consensus sequence of the Alu-repeat abundantly expressed in the human genome, but absent in mice. However, the mouse genome contains a highly similar sequence, providing a model system to analyse the selectivity of the developed methods. Although efficiency of detection currently is limiting, we conclude that in situ PLA can be used to achieve a highly selective analysis of PDIs in single cells.
基因表达是调节细胞命运的关键特征，部分受组蛋白修饰调控，组蛋白修饰调节染色质对转录因子的可及性。到目前为止，蛋白质-DNA 相互作用（PDI）主要在没有从样本中获取空间信息或序列分辨率较差的情况下进行整体研究。需要新的工具来揭示与特定 DNA 序列在原位相互作用的蛋白质，以进一步了解核内转录控制的协调。我们在此介绍了一种基于原位邻近连接分析（PLA）在细胞内可视化单个 PDI 的方法。该检测方法之前用于原位检测蛋白质-蛋白质相互作用，通过使用发夹探针来识别复杂基因组中的独特 DNA 序列，从而适用于靶 PDI 的分析。作为原理验证，我们检测到组蛋白 H3 与人类基因组中大量表达的 Alu 重复 26 个碱基的共有序列相互作用，但在小鼠中不存在。然而，小鼠基因组中含有高度相似的序列，为分析所开发方法的选择性提供了模型系统。尽管目前检测效率有限，但我们得出结论，原位 PLA 可用于在单个细胞中实现对 PDI 的高度选择性分析。
