@conference{
author = "Tolić, Anja and Ninković, Niccoleta Aleksandra and Rajić, Jovana and Đorđević, Miloš and Đorđević, Marija and Uskoković, Aleksandra and Grdović, Nevena and Mihailović, Mirjana and Arambašić Jovanović, Jelena and Dinić, Svetlana and Okamoto, Akimitsu and Vidaković, Melita",
year = "2018",
abstract = "Objectives: This study aim to implement a novel method, methylation-specific fluorescence in situ hybridization (MeFISH), based on microscopic visualization of DNA methylation/hydroxymethylation status at specific DNA regions in individual nuclei after pancreatic cell treatment with different compounds that possess a pronounced DNA (de)methylation capacity.
Methods: The DNA (de)methylating properties of two selected compounds caffeine (Co) and azacitidine (A) were evaluated in a Rin-5F pancreatic beta-cell line. Rin-5F cells were spin down on microscopic slides and further processed for preparing HALOs (relaxed DNA with preserved contacts with non-soluble nuclear proteins). The fluorescent visualization was achieved using ICON probe that covers region of interest in the promoter of the CXCL12 gene and target C positioned on the +26 bp, osmium for methylated cytosine (5mC)-dependent crosslinking and Tyramide Signal Amplification Systems for enhanced fluorescent staining.
Results: In control and Rin-5F cells treated with Co we were able to detect clear, single fluorescent signal that correspond to 5mC positioned on the +26 bp within the promoter region of the CXCL12 gene using MeFISH. Confirmation for the in situ hybridization specificity was achieved by omitting the crosslinking reaction with osmium. We observed a clear difference between control and Co treated samples, indicating that Co acts as pronounced DNA methylating compound. Treatment of cells with A lead to the appearance of a specific signal in a limited number of HALO preparations confirming demethylating property of A.
Conclusions: The Co acts as a pronounced DNA methylating agent in contrast to A, which demethylates CXCL12 gene and subsequently promotes higher gene expression. Higher methylation of the CXCL12 gene after cell treatment with Co leads to suppression of the gene which was observed by RT-qPCR. The analysed C, positioned on the +26 bp, may represent one of the major sites whose methylation is important for the regulation of the CXCL12 gene expression in vivo.",
publisher = "Karger Publishers",
journal = "Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy",
title = "MeFISH: Fluorescent detection of Target Methylated Cytosines within the Genome",
doi = "10.1159/000490753",
pages = "16"
}
