No abstract

Diabetes elevates matrix metalloproteinase-9 (MMP-9) in the retina and its capillary cells, and activated MMP-9 damages mitochondria, accelerating retinal capillary cell apoptosis, a phenomenon which precedes the development of retinopathy. Diabetes also favors epigenetic modifications regulating expression of many genes. DNA methylation is maintained by methylating-hydroxymethylating enzymes, and retinal DNA methyltransferase (Dnmt) is activated in diabetes. Our aim is to investigate the role of DNA methylation in MMP-9 regulation. Effect of high glucose on 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and binding of Dnmt1 and hydroxymethylating enzyme (Tet2) on MMP-9 promoter were quantified in retinal endothelial cells. Specific role of Tet2 in MMP-9 activation was validated using Tet2-siRNA. The results were confirmed in the retina from streptozotocin-induced diabetic mouse. Although glucose increased Dnmt1 binding at MMP-9 promoter, it decreased 5mC levels. At the same promoter site, Tet2 binding and 5hmC levels were elevated. Tet2-siRNA ameliorated increase in 5hmC and MMP-9 transcription, and protected mitochondrial damage. Diabetic mice also presented similar dynamic DNA methylation changes in the retinal MMP-9 promoter. Thus, in diabetes transcription of retinal MMP-9 is maintained, in part, by an active DNA methylation-hydroxymethylation process, and regulation of this machinery should help maintain mitochondrial homeostasis and inhibit the development/ progression of diabetic retinopathy.

Hoogsteen (HG) base pairs (bps) provide an alternative pairing geometry to Watson–Crick (WC) bps and can play unique functional roles in duplex DNA. Here, we use structural features unique to HG bps (syn purine base, HG hydrogen bonds and constricted C1′–C1′ distance across the bp) to search for HG bps in X-ray structures of DNA duplexes in the Protein Data Bank. The survey identifies 106 A•T and 34 G•C HG bps in DNA duplexes, many of which are undocumented in the literature. It also uncovers HG-like bps with syn purines lacking HG hydrogen bonds or constricted C1′–C1′ distances that are analogous to conformations that have been proposed to populate the WC-to-HG transition pathway. The survey reveals HG preferences similar to those observed for transient HG bps in solution by nuclear magnetic resonance, including stronger preferences for A•T versus G•C bps, TA versus GG steps, and also suggests enrichment at terminal ends with a preference for 5′-purine. HG bps induce small local perturbations in neighboring bps and, surprisingly, a small but significant degree of DNA bending (∼14°) directed toward the major groove. The survey provides insights into the preferences and structural consequences of HG bps in duplex DNA.

Conformational changes in nucleic acids play a key role in the way genetic information is stored, transferred, and processed in living cells. Here, we describe new approaches that employ a broad range of experimental data, including NMR derived chemical shifts and residual dipolar couplings, small angle X-ray scattering, and computational approaches such as molecular dynamics simulations, to determine ensembles of DNA and RNA at atomic resolution. We review the complimentary information that can be obtained from diverse sets of data and the various methods that have been developed to combine these data with computational methods to construct ensembles and assess their uncertainty. We conclude by surveying RNA and DNA ensembles determined using these methods, highlighting the unique physical and functional insights that have been obtained so far.