Effects of non-CpG site methylation on DNA thermal stability: a fluorescence study.
Cytosine methylation is a widespread epigenetic regulation mechanism. In healthy mature cells, methylation occurs at CpG dinucleotides within promoters, where it primarily silences gene expression by modifying the binding affinity of transcription factors to the promoters. Conversely, a recent study showed that in stem cells and cancer cell precursors, methylation also occurs at non-CpG pairs and involves introns and even gene bodies. The epigenetic role of such methylations and the molecular mechanisms by which they induce gene regulation remain elusive. The topology of both physiological and aberrant non-CpG methylation patterns still has to be detailed and could be revealed by using the differential stability of the duplexes formed between site-specific oligonucleotide probes and the corresponding methylated regions of genomic DNA. Here, we present a systematic study of the thermal stability of a DNA oligonucleotide sequence as a function of the number and position of non-CpG methylation sites. The melting temperatures were determined by monitoring the fluorescence of donor-acceptor dual-labelled oligonucleotides at various temperatures. An empirical model that estimates the methylation-induced variations in the standard values of hybridization entropy and enthalpy was developed.
Department of Health Sciences, University of Milano Bicocca, Via Cadore 48, Monza, MB 20900, Italy luca.nardo@unimib.it.
Luca
L
Nardo
Department of Science and High Technology, University of Insubria, Via Valleggio 11, Como, CO 22100, Italy.
Marco
M
Lamperti
Department of Health Sciences, University of Milano Bicocca, Via Cadore 48, Monza, MB 20900, Italy.
Domenico
D
Salerno
Department of Health Sciences, University of Milano Bicocca, Via Cadore 48, Monza, MB 20900, Italy.
Valeria
V
Cassina
Department of Health Sciences, University of Milano Bicocca, Via Cadore 48, Monza, MB 20900, Italy.
Natalia
N
Missana
Institute for Photonics and Nanotechnology, National Research Council, Via Valleggio 11, Como, CO 22100, Italy.
Maria
M
Bondani
LENS-Department of Physics and Astronomy, University of Firenze, Via Sansone 1, Sesto Fiorentino, FI 50019, Italy.
Alessia
A
Tempestini
Department of Health Sciences, University of Milano Bicocca, Via Cadore 48, Monza, MB 20900, Italy.
Francesco
F
Mantegazza
D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D005470:Fluorometry; D009691:Nucleic Acid Denaturation; D015345:Oligonucleotide Probes; D013696:Temperature; D013816:Thermodynamics
D015345:Oligonucleotide Probes; D004247:DNA