MELANIE: the quantitative analysis of methylation levels along DNA fibres

KILAR, Agata Magdalena, Michal FRANEK and Jiří FAJKUS. MELANIE: the quantitative analysis of methylation levels along DNA fibres. In Czech Plant Nucleus Workshop 2021. 2021.

Basic information

• Original name: MELANIE: the quantitative analysis of methylation levels along DNA fibres
• Name (in English): MELANIE: the quantitative analysis of methylation levels along DNA fibres
• Authors: KILAR, Agata Magdalena, Michal FRANEK and Jiří FAJKUS.
• Edition: Czech Plant Nucleus Workshop 2021, 2021.

Other information

• Type of outcome: Presentations at conferences
• Confidentiality degree: is not subject to a state or trade secret

Abstract (in English)

DNA fiber extension techniques are single-molecule methods based on the stretching of DNA on microscopic slides. Stretching of the template is followed by immunofluorescence and fluorescence in-situ hybridization detection to investigate replication dynamics, physical genome mapping or epigenetic profiles of repeats by microscopy. Fiber techniques are great tools to study arrangement of repetitive regions, such as telomeres, centromeres and pericentromeric regions, that are hard to map by genome-wide approaches. Thanks to our optimization of fiber extension protocol, we were able to obtain super-resolution microscopic images with separated fibers that could be subjected to the quantitative evaluation of DNA methylation levels using the image analysis approach. Thus, we created the Matlab-based tool - MELANIE, which uses various computational morphological operations on both, FISH signal and the 5-methylcytosine signal channels, to provide a quantitative analysis of methylation levels along DNA fibers. We applied our tool for methylation mapping on functional repeats, namely telomeric repeats in Nicotiana tabacum, ribosomal genes in Arabidopsis thaliana, and on centromeres in human embryonic stem cells before and after induced differentiation. With the tool, we demonstrate that DNA fiber extension is helpful to study individual clusters of repeats (e.g. a specific telomere or a single nucleolus organizer region) and their methylation profiles.