Visualising individual sequence-specific protein-DNA interactions in situ

Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, SE-75185 Uppsala, Sweden.
New Biotechnology (Impact Factor: 2.9). 08/2011; 29(5):589-98. DOI: 10.1016/j.nbt.2011.08.002
Source: PubMed


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.

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Available from: Ulf Landegren, Jan 19, 2015
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    • "In accordance, modulation of ANRIL expression led to the epigenetic regulation of target genes expression in cis[66,67] and in trans[59▪,64,73]. We have recently shown that trans-regulation was dependent on an Alu-DEIN motif [74,75], which marked the promoters of ANRIL target genes and was mirrored in ANRIL RNA transcripts (Fig. 3). The functional relevance of Alu motifs in ANRIL was confirmed by deletion and mutagenesis, reversing trans-regulation and restoring normal cellular functions [59▪]. "
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    ABSTRACT: Since 2007, genome-wide association studies (GWAS) have led to the identification of numerous loci of atherosclerotic cardiovascular disease. The majority of these loci harbor genes previously not known to be involved in atherogenesis. In this review, we summarize the recent progress in understanding the pathophysiology of genetic variants in atherosclerosis. Fifty-eight loci with P < 10 have been identified in GWAS for coronary heart disease and myocardial infarction. Of these, 23 loci (40%) overlap with GWAS loci of classical risk factors such as lipids, blood pressure, and diabetes mellitus, suggesting a potential causal relation. The vast majority of the remaining 35 loci (60%) are at genomic regions where the mechanism in atherogenesis is unclear. Loci most frequently found in independent GWAS were at Chr9p21.3 (ANRIL/CDKN2B-AS1), Chr6p24.1 (PHACTR1), and Chr1p13.3 (CELSR2, PSRC1, MYBPHL, SORT1). Recent work suggests that Chr9p21.3 exerts its effects through epigenetic regulation of target genes, whereas mechanisms at Chr6p24.1 remain obscure, and Chr1p13.3 affects plasma LDL cholesterol. Novel GWAS loci indicate that our understanding of atherosclerosis is limited and implicate a role of hitherto unknown mechanisms, such as epigenetic gene regulation in atherogenesis.
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    • "Both padlock probes and in situ PLA will very likely have a substantial role in elucidating the mechanisms involved in the cellular communications in tumors, something that then can be adopted for personalized medicine to predict disease progression, and in development and selection of therapy to restore the tumor microenvironment to a state that does not promote cancer growth. Albeit in situ PLA analysis of epigenetic alterations of individual promoters is not feasible at the moment, improvement in efficiency of the padlock-based in situ PLA [55] might soon provide a tool to investigate epigenetic reprogramming of cancer cells during transitions between epithelial and mesenchymal phenotypes. "
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    ABSTRACT: A tumor does not consist of a homogenous population of cancer cells. Therefore, to understand cancer, the tumor microenvironment and the interplay between the different cell types present in the tumor has to be taken into account, and how this regulates the growth and survival of the cancer cells. To achieve a full picture of this complex interplay, analysis of tumor tissue should ideally be performed with cellular resolution, providing activity status of individual cells in this heterogeneous population of different cell-types. In addition, in situ analysis provides information on the architecture of the tissue wherein the cancer cells thrive, providing information of the identity of neighboring cells that can be used to understand cell-cell communication. Herein we describe how padlock probes and in situ PLA can be used for visualization of nucleic acids and protein activity, respectively, directly in tissue sections, and their potential future role in personalized medicine.
    EPMA Journal, The 12/2012; 3(1):7. DOI:10.1007/s13167-012-0140-3
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    • "The requirement for recognition by two affinity reagents in proximity in order to generate a reporter molecule, followed by amplification by PCR or rolling-circle amplification (RCA), provides for highly sensitive assays to detect low amounts of proteins. The assays can also be used to study interactions among proteins [9]–[11] or between protein and DNA sequences [12], [13]. PLA has been used to detect potential biomarkers in plasma, serum, cerebrospinal fluid, and cell lysates, both single proteins, protein aggregates (Aβ protofibrils) [14], and interacting proteins have been targeted [10], [11]. "
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