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Loic Blanchon, Corinne BELVILLE, Nicolas BONNIN, Vincent Sapin, Frederic Chiambaretta; Dna Methytransferases Are Differentially Expressed In Human Eyes. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5130.
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Epigenetic marks, such as methylation of DNA, miRNA expression or post-translational histone modifications, are involved in the control of genes expression. Epigenetic remaniements are involved in several human pathologies but poorly described in ophthalmology ones. For this purpose, we focus on DNA methylation and particularly on the expression level of DNA methyltransferases (DNMT) implicated in CpG methylation pattern. The aim of our work was to extensively check the presence of DNMT (mRNA and protein levels) and quantify their expressions in various ocular zones.
mRNAs were extracted from human cornea, conjunctiva, capsule, trabeculum and from related cell lines (HCE, HTCE, CHANG and TM). Quality controls were done on Bioanalyzer 2100 Agilent. cDNAs were generated by reverse transcription, quantified by amplification (LightCycler LC480-Roche) with specific primers for DNMT1, 3A, 3B and 3L and normalized to the housekeeping gene 36B4. Positive and negative controls were done with cDNAs from placenta already known to express DNMTs or in absence of cDNA. For immunohistochemistry, normal human corneas were provided by the regional corneal graft bank. All proteins were detected by specific primary antibody for DNMT1, 3A, 3B and 3L (Santa-Cruz®), negative control were done with a rabbit IgG antibody as primary one. Secondary antibody Cy3-coupled was used.
All DNMT are expressed in the human eyes but at different levels. Furthermore, DNMT3L, not directly implied in the DNA methylation looks like to be the weakly expressed. All tested celllines also expressed DNMTs. Quantitative RT-PCR demonstrated strong differences between members and zones of expression of these DNA methyltransferases. Our transcripts results were confirmed by immunohistochemistry experiments on cornea sections.
For the first time, an inventory of the DNMT expression in tissues and cells lines from human ocular sphere was performed. Future studies will certainly permit to demonstrate the presence of a direct link between a deregulation of the CpG methylation pattern and the already "well known" eye pathologies.
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