Super-resolution microscopy of chromatin fibers and quantitative DNA methylation analysis of DNA fiber preparations
Language English Country Great Britain, England Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
34350964
DOI
10.1242/jcs.258374
PII: 271206
Knihovny.cz E-resources
- Keywords
- Chromatin, DNA, Fiber, Methylation, Microscopy,
- MeSH
- Chromatin genetics MeSH
- Chromatin Immunoprecipitation MeSH
- DNA genetics MeSH
- DNA Methylation * genetics MeSH
- Microscopy * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Chromatin MeSH
- DNA MeSH
Analysis of histone variants and epigenetic marks is dominated by genome-wide approaches in the form of chromatin immunoprecipitation-sequencing (ChIP-seq) and related methods. Although uncontested in their value for single-copy genes, mapping the chromatin of DNA repeats is problematic for biochemical techniques that involve averaging of cell populations or analysis of clusters of tandem repeats in a single-cell analysis. Extending chromatin and DNA fibers allows us to study the epigenetics of individual repeats in their specific chromosomal context, and thus constitutes an important tool for gaining a complete understanding of the epigenetic organization of genomes. We report that using an optimized fiber extension protocol is essential in order to obtain more reproducible data and to minimize the clustering of fibers. We also demonstrate that the use of super-resolution microscopy is important for reliable evaluation of the distribution of histone modifications on individual fibers. Furthermore, we introduce a custom script for the analysis of methylation levels on DNA fibers and apply it to map the methylation of telomeres, ribosomal genes and centromeres.
Department of Biology Faculty of Medicine Masaryk University Kamenice 5 CZ 62500 Brno Czech Republic
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