Abstract
Cytosolic ROS, generated by NADPH oxidase 2 (Nox2) in diabetes, damage retinal mitochondria, which leads to the development of retinopathy. A small molecular weight G-protein essential for Nox2 activation, Rac1, is also transcriptionally activated viaactive DNA methylation-hydroxymethylation. DNA methylation is a dynamic process, and can also be regulated by histone modifications; diabetes alters retinal histone methylation machinery. Our aim is to investigate the role of histone methylation (H3K9me3) of Rac1 promoter in dynamic DNA methylation- transcriptional activation. Using human retinal endothelial cells in 20 mM D-glucose, H3K9me3 at Rac1 promoter was quantified by chromatin-Immunoprecipitation technique. Crosstalk between H3K9me3 and DNA methylation was examined in cells transfected with siRNA of histone trimethyl-transferase, Suv39H1, or Dnmt1, exposed to high glucose. Key parameters were confirmed in retinal microvessels from streptozotocin-induced diabetic mice, with intravitreally administered Suv39H1-siRNA or Dnmt1-siRNA. Compared to cells in normal glucose, high glucose increased H3K9me3 and Suv39H1 binding at Rac1 promoter, and Suv39H1-siRNA prevented glucose-induced increase 5 hydroxy methyl cytosine (5hmC) and Rac1 mRNA. Similarly, in diabetic mice, Suv39H1-siRNA attenuated increase in 5hmC and Rac1 mRNA. Thus, H3K9me3 at Rac1 promoter assists in active DNA methylation-hydroxymethylation, activating Rac1 transcription. Regulation of Suv39H1-H3K9 trimethylation could prevent further epigenetic modifications, and prevent diabetic retinopathy.
Highlights
Cytosolic reactive oxygen species (ROS), generated by NADPH oxidase 2 (Nox2) in diabetes, damage retinal mitochondria, which leads to the development of retinopathy
Using human retinal endothelial cells (HRECs), we have investigated the effect of high glucose on H3K9 methylation on Rac[1] promoter
Cells incubated in 20 mM L-glucose, instead of 20 mM D-glucose, did not show any alterations in H3K9 methylation and Suv39H1 binding at Rac[1] promoter, and the values obtained from cells in 20 mM L-glucose were not different from cells in 5 mM D-glucose, but were significantly different from those obtained from cells in 20 mM D-glucose
Summary
Cytosolic ROS, generated by NADPH oxidase 2 (Nox2) in diabetes, damage retinal mitochondria, which leads to the development of retinopathy. Our aim is to investigate the role of histone methylation (H3K9me3) of Rac[1] promoter in dynamic DNA methylation- transcriptional activation. Rac[1] is transcriptionally activated in the retina and its vasculature, and its promoter undergoes active DNA methylation-hydroxymethylation; while DNA methyl transferase 1 (Dnmt1) methylates cytosine forming 5 methyl cytosine (5mC), concomitant activation of Tet[2] hydroxymethylates 5mC, forming 5 hydroxymethyl cytosine (5hmC) This opens up the chromatin for the binding of the transcription factor, and upregulates Rac1 transcription[9,12]. Key parameters were confirmed in an in vivo model using retinal microvessels from mice, that were administered Suv39H1-siRNA or Dnmt1-siRNA (intravitreally) soon after induction of streptozotocin-induced diabetes
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