title: Visualising individual sequence-specific protein-DNA interactions in situ.
abstract: 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.
authors: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, SE-75185 Uppsala, Sweden. irene_weibrecht@gmx.de
authors: Irene
authors: I
authors: Weibrecht
authors: Milan
authors: M
authors: Gavrilovic
authors: Lena
authors: L
authors: Lindbom
authors: Ulf
authors: U
authors: Landegren
authors: Carolina
authors: C
authors: Wählby
authors: Ola
authors: O
authors: Söderberg
mesh_terms: D020087:Alu Elements / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay / Q000379:methods*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015894:Genome, Human / Q000235:genetics; D006657:Histones / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D051379:Mice; D041681:NIH 3T3 Cells; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000378:metabolism; D011485:Protein Binding; D013698:Templates, Genetic
chemical_list: D004268:DNA-Binding Proteins; D006657:Histones; D009841:Oligonucleotides; D004247:DNA