Abstract
DNA methylation and hydroxymethylation represent important epigenetic modifications involved in cell differentiation. DNA hydroxymethylation can be used to classify independent biological samples by tissue type. Relatively little is known regarding the genomic abundance and function of 5-hydroxymethylcytosine (5-hmC) in ocular tissues. The choroid supplies oxygen and nutrients to the outer retina through its dense network of blood vessels. This connective tissue is mainly composed of pigmented melanocytes, and stromal fibroblasts. Since DNA hydroxymethylation level is relatively high in cutaneous melanocytes, we investigated the presence of 5-hmC in choroidal melanocytes, as well as the expression of ten-eleven translocation methylcytosine dioxygenases (TETs) and isocitrate dehydrogenases (IDHs) implicated in this DNA demethylation pathway. Immunofluorescence, DNA slot blots and liquid chromatography coupled to tandem mass spectrometry performed with choroidal tissues and melanocytes within these tissues revealed that they have a relatively high level of 5-hmC. We also examined the expression of TET1/2 and IDH1/2 in choroidal melanocytes by gene expression profiling, qPCR and Western blotting. In addition, we detected decreased levels of 5-hmC when choroidal melanocytes were exposed to a lower concentration of oxygen. Our study therefore demonstrates that DNA hydroxymethylation is present in choroidal melanocytes, and that the abundance of this epigenetic mark is impacted by hypoxia.