Abstract
Ascorbate requiring Fe2+/2-oxoglutarate-dependent dioxygenases located in the nucleoplasm have been shown to participate in epigenetic regulation of gene expression via histone and DNA demethylation. Transport of dehydroascorbic acid is impaired in the endomembranes of fibroblasts from arterial tortuosity syndrome (ATS) patients, due to the mutation in the gene coding for glucose transporter GLUT10. We hypothesized that altered nuclear ascorbate concentration might be present in ATS fibroblasts, affecting dioxygenase activity and DNA demethylation. Therefore, our aim was to characterize the subcellular distribution of vitamin C, the global and site-specific changes in 5-methylcytosine and 5-hydroxymethylcytosine levels, and the effect of ascorbate supplementation in control and ATS fibroblast cultures. Diminished nuclear accumulation of ascorbate was found in ATS fibroblasts upon ascorbate or dehydroascorbic acid addition. Analyzing DNA samples of cultured fibroblasts from controls and ATS patients, a lower global 5-hydroxymethylcytosine level was found in ATS fibroblasts, which could not be significantly modified by ascorbate addition. Investigation of the (hydroxy)methylation status of specific regions in six candidate genes related to ascorbate metabolism and function showed that ascorbate addition could stimulate hydroxymethylation and active DNA demethylation at the PPAR-γ gene region in control fibroblasts only. The altered DNA hydroxymethylation patterns in patient cells both at the global level and at specific gene regions accompanied with decreased nuclear accumulation of ascorbate suggests the epigenetic role of vitamin C in the pathomechanism of ATS. The present findings represent the first example for the role of vitamin C transport in epigenetic regulation suggesting that ATS is a compartmentalization disease.
Highlights
Beyond its antioxidant properties, ascorbic acid (AA) [1]—the reduced form of vitamin C—is required for the proper functioning of several enzymes, including Fe2+/2-oxoglutarate-dependent dioxygenases [2] present in various subcellular compartments
Oxidative Medicine and Cellular Longevity methylcytosine dioxygenases [4] promote the demethylation of histones and DNA, respectively, both mechanisms being involved in the epigenetic regulation of transcription [5]. 5-Methylcytosine (5-mC) demethylation occurs via consecutive oxidative steps catalyzed by the TET dioxygenases through a 5-hydroxymethylcytosine (5-hmC) intermediate [6]
While compartmentalization of AA is well established in plants, the compartment-specific levels of the vitamin are still unclear in mammalian cells
Summary
Ascorbic acid (AA) [1]—the reduced form of vitamin C—is required for the proper functioning of several enzymes, including Fe2+/2-oxoglutarate-dependent dioxygenases [2] present in various subcellular compartments. Fe2+/2-oxoglutarate-dependent dioxygenases catalyze diverse oxidative reactions including the posttranslational modification of proteins in the endoplasmic reticulum (ER) and demethylations related to epigenetic regulation in the nucleus. Oxidative Medicine and Cellular Longevity (ten-eleven translocation) methylcytosine dioxygenases [4] promote the demethylation of histones and DNA, respectively, both mechanisms being involved in the epigenetic regulation of transcription [5]. 5-Methylcytosine (5-mC) demethylation occurs via consecutive oxidative steps catalyzed by the TET dioxygenases through a 5-hydroxymethylcytosine (5-hmC) intermediate [6]. To ensure the AA supply of these AA-dependent enzymes in various subcellular compartments, transporters are needed in the membrane of organelles. The occurrence and functioning of these transporters have been hardly elucidated in the endomembranes [9]
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