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  • Epigenetic modifications the modifications that regulate gen


    Epigenetic modifications, the modifications that regulate gene expression without affecting DNA sequence, have been implicated in the development of diabetic retinopathy [19]. Methylation of cytosine in DNA, a repressive modification, by DNA methyltransferases, is a key component of the epigenetic machinery, and altered patterns of DNA methylation are observed in many diseases [49]. DNA methylation enzyme machinery is activated in the retina and its vasculature in diabetes, and while the levels of methylated cytosine are increased at the promoter of polymerase gamma (an enzyme responsible for replication of mtDNA), that of hydroxymethylated cytosine are increased at the promoters of MMP-9 and Rac1, resulting in their gene suppression and activation respectively [5,31]. Mfn2 promoter (human and rats) is rich in CpG islands [50], and here our results show it is hypermethylated in diabetes; 5mC levels are elevated at its promoter, and the binding of Dnmt1, the only isoform of the Dnmt family upregulated in hyperglycemic milieu [23], is increased. Regulation of Mfn2 expression-DNA methylation by pharmacological and molecular inhibitors of Dnmts, further supports the role of DNA methylation in Mfn2 expression. Binding of the transcription factor SP1 is considered essential for normal transcription of Mfn2, and this TATA-less human MFN2 promoter has multiple SP1 ATPγS tetralithium salt [51]. We show that high glucose also decreases binding of SP1 at Mfn2 promoter, and this can be regulated by both azacytidine and Dnmt1-siRNA, further supporting the role of DNA methylation in transcriptional activation of Mfn2. The role of DNA methylation in Mfn2 regulation is further confirmed by increased 5mC levels and Dnmt1 binding in the retinal microvessels from diabetic rats, and also from human donors with diabetic retinopathy. Mfn2 promoter has at least six transcription start sites [51], and our results show that in rat Mfn2 promoter, −245 to −84 region had the highest level of 5mC and Dnmt1 binding (and the lowest SP1 binding) compared to other regions, suggesting that this ~150 bp promoter region (upstream of the transcription start site) is important in regulation of Mfn2 expression in diabetes. Consistent with this, others have shown that the promoter activity in rat vascular smooth muscle cells is suppressed by deletion of the region between −229 and −54, suggesting the importance of this region of the core Mfn2 promoter [51]. Our study is focused on the role of Mfn2 in mitochondrial homeostasis, and we recognize that both fusion and fission are critical in maintaining mitochondrial structural and functional homeostasis. In addition, many epigenetic modifications including histone modifications and DNA methylation can work in concordance to regulate a single gene expression [24,52]. The role of fission in mitochondrial homeostasis, and possibility of other epigenetic modifications and micro RNAs regulating Mfn2 expression in diabetes, cannot be ruled out. Furthermore, this study focused on mitochondrial fission in retinal microvasculature, however, photoreceptors are very rich in mitochondria, and are considered to play a major source of ROS production in diabetic retinopathy [53]; similar mechanism operating in photoreceptors remains to be investigated. In conclusion, this is the first report showing the role of mitofusin in diabetic retinopathy; decreased levels of Mfn2 in retinal vasculature in diabetes, in addition to affecting the mitochondrial morphology, also contribute to mitochondrial ROS accumulation, mtDNA damage and a dysfunctional electron transport chain. Regulation of Mfn2 protects mitochondrial structural and functional abnormalities, and prevents electron transport chain damage and accumulation of free radicals, experienced in hyperglycemic milieu. Our results also imply the role of epigenetics in transcriptional regulation of Mfn2 in diabetes, and show DNA ATPγS tetralithium salt hypermethylation of its promoter. Mitochondrial dysfunction is intimately associated in the development of diabetic retinopathy; our study provides a new and ‘untested’ avenue to regulate mitochondrial homeostasis, and prevent possible loss of vision in diabetic patients.