Because it is implicated in the organization of chromatin structure and regulation of gene transcription
activity of DMNTs, a class of enzimes catalazying the transfer of a methyl moiety to cytosines principally in the CpG dinucleotide.
Annovi HRM and Epigenetics
Epigenetics and High ResolutionMelting-PCR7th European Elastin Meeting1st -4thseptember 2012Giulia Annovi, PhDUniversity of Modena and Reggio Emilia, ItalyIBCP Lyon, FranceMoulin L., Cenizo V., André V., Sommer P., Debret R.
HistonemodificationsDNAmethylationEpigenetic mechanisms are able to produce differentphenotypes from a single genotypeEffect of DNA methylation:# transcriptional repression# compact chromatinestructures# normal embryologicaldevelopment# X chromosome inactivation# imprinting controlO NH2CH3Cytosine
MethPrimer analysis of promoters : http://www.urogene.org/methprimer/index1.htmlNCBI Epigenomics: http://www.ncbi.nlm.nih.gov/epigenomics/view/genomeSize (Start - End)Island 1 401 bp (71 - 471)Island 2 261 bp (571 - 831)Predicted CpG islands
The bisulfite conversionHigh Resolution Melting (HRM)Tissue samplesfixed in paraffinCell culturess-DNADNA extraction
High Resolution Melting (HRM) - PCR=> Melting curves allow discrimination between methylated and unmethylatedsamples, because melting temperature is nucleotides composition dependent.
100% Methylated0% Methylated100% MethylatedUnconvertedQuantitation data forCycling:- converted ssDNA integrity- inter- and intra-samplecomparable amplification100% Methylated0% MethylatedHRM Data analysis
⇒Global Melt Curves analysis can’t give a clear result: cytosines methylation inhuman samples is unbalanced and depends on environnement.⇒ We have to analyse each curve independently to identify melting temperature foreach sample.100% Methylated0% Methylated100% Methylated
HRM is a powerful technique to identify methylation at specificpromoter very quickly and from different kind of samples.pRloxl1 pRfbn1 pRfbln5Predicted CpG islands-Moulin L. Romain D.-
HRM is a powerful technique to identify methylation at specificpromoter very quickly and from different kind of samples.74.9575.0575.1575.2575.3575.4575.55a-SMA promoter methylationCollagen5AZAMeltTemp=Methylationlevel00.511.522.533.544.55a SMA expression - qPCRFibro 5AZAmRNAaSMAexpressionFibroblasts Fibroblasts + 5 AZA