Ophthalmology, Visual and Anatomical Sciences, Kresge Eye Institute, Wayne State University, Detroit, MI, 48201, USA.
Sci Rep. 2021 Jul 8;11(1):14097. doi: 10.1038/s41598-021-93420-4.
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 via active 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.
细胞溶质 ROS，由 NADPH 氧化酶 2（Nox2）在糖尿病中产生，损害视网膜线粒体，导致视网膜病变的发展。Rac1 是一种小分子 G 蛋白，是 Nox2 激活所必需的，也通过活性 DNA 甲基化-羟甲基化转录激活。DNA 甲基化是一个动态过程，也可以通过组蛋白修饰来调节；糖尿病改变了视网膜组蛋白甲基化机制。我们的目的是研究 Rac1 启动子组蛋白甲基化（H3K9me3）在动态 DNA 甲基化-转录激活中的作用。在 20mM D-葡萄糖中使用人视网膜内皮细胞，通过染色质免疫沉淀技术定量 Rac1 启动子处的 H3K9me3。在转染了组蛋白三甲基转移酶、Suv39H1 或 Dnmt1 的 siRNA 的细胞中，研究了 H3K9me3 与 DNA 甲基化之间的串扰，这些细胞暴露于高葡萄糖中。在链脲佐菌素诱导的糖尿病小鼠的视网膜微血管中，通过玻璃体内给予 Suv39H1-siRNA 或 Dnmt1-siRNA 来确认关键参数。与正常葡萄糖中的细胞相比，高葡萄糖增加了 Rac1 启动子处的 H3K9me3 和 Suv39H1 结合，并且 Suv39H1-siRNA 阻止了葡萄糖诱导的 5-羟甲基胞嘧啶（5hmC）和 Rac1 mRNA 的增加。同样，在糖尿病小鼠中，Suv39H1-siRNA 减弱了 5hmC 和 Rac1 mRNA 的增加。因此，Rac1 启动子处的 H3K9me3 有助于活性 DNA 甲基化-羟甲基化，激活 Rac1 转录。Suv39H1-H3K9 三甲基化的调节可以防止进一步的表观遗传修饰，并预防糖尿病性视网膜病变。
