Parp inhibition prevents ten-eleven translocase enzyme activation and hyperglycemia-induced DNA demethylation

Abstract

Studies from human cells, rats, and zebrafish have documented that hyperglycemia (HG) induces the demethylation of specific cytosines throughout the genome. We previously documented that a subset of these changes become permanent and may provide, in part, a mechanism for the persistence of complications referred to as the metabolic memory phenomenon. In this report, we present studies aimed at elucidating the molecular machinery that is responsible for the HG-induced DNA demethylation observed. To this end, RNA expression and enzymatic activity assays indicate that the ten-eleven translocation (Tet) family of enzymes are activated by HG. Furthermore, through the detection of intermediates generated via conversion of 5-methyl-cytosine back to the unmethylated form, the data were consistent with the use of the Tet-dependent iterative oxidation pathway. In addition, evidence is provided that the activity of the poly(ADP-ribose) polymerase (Parp) enzyme is required for activation of Tet activity because the use of a Parp inhibitor prevented demethylation of specific loci and the accumulation of Tet-induced intermediates. Remarkably, this inhibition was accompanied by a complete restoration of the tissue regeneration deficit that is also induced by HG. The ultimate goal of this work is to provide potential new avenues for therapeutic discovery.

Figure 1

Illustration shows the known activated DNA demethylation pathways intermediates and their supporting enzymatic machinery. BER, base excision repair.

Figure 2

HG induces gene expression changes in DNA demethylation machinery enzymes. Quantitative reverse transcriptase results (with SE) are presented graphically as the fold increase when compared with controls for weeks 1–3, respectively: Gaa (gadd45 αα): 2.01 ± 0.25, 2.35 ± 0.36, and 2.12 ± 0.34; Gab (gadd45 αβ): 1.98 ± 0.34, 4.82 ± 0.29, and 4.35 ± 0.35; A2a (apobec2a): 1.20 ± 0.11, 0.98 ± 0.23, and 0.90 ± 0.37; A2b (apobec2b): 0.85 ± 0.11, 1.13 ± 0.12, and 1.12 ± 0.24; Mbd4 (mbd4): 0.94 ± 0.26, 1.12 ± 0.13, and 0.98 ± 0.11; T1 (tet 1): 8.34 ± 0.22, 5.34 ± 0.41, and 5.12 ± 0.09; T2 (tet 2): 5.87 ± 0.34, 6.13 ± 0.12, and 6.23 ± 0.08; T3 (tet 3): 5.32 ± 0.26, 5.32 ± 0.36, and 5.45 ± 0.37; and Tdg (tdg): 1.01 ± 0.27, 3.46 ± 0.24, and 3.23 ± 0.34. Each gene at each time point was compared with the appropriate control, and a Student t test was performed to determine statistical significance. *P < 0.001 (indicating statistically significant changes existed).

Figure 3

HG induces the formation of Tet enzyme activity intermediates. A: Graphic representation of the 5hmC genomic content (ng/0.2 mg); control (C): 0.106 ± 0.023, W1 (week 1): 1.13 ± 0.19, W2 (week 2): 3.58 ± 0.19, W3 (W3): 3.64 ± 0.26 (n = 18 for C, W1, and W2; n = 16 for W3). B: Graphic representation showing the time course of 5fC formation (ng/0.5 mg) induced in fin tissue DNA. Control (C): 0.26 ± 0.13, W1: 1.25 ± 0.23, W2: 3.1 ± 0.45, and W3: 3.45 ± 0.22 (n = 18 for C, W1 and W2; n = 16 for W3). P < 0.0001. In both studies, a Student t test was performed to determine statistical significance. *P < 0.0001 (indicating statistically significant changes existed). In addition, a one-way ANOVA analysis revealed that week 1 was statistically different than weeks 2 and 3 (P < 1.0 E-5 for both assays).

Figure 4

Parp inhibition prevents both the accumulation of 5hmC and the HG-induced fin regeneration deficit. A: The administration of a Parp inhibitor (Parpi) prevents 5hmC formation (ng/0.2 mg). Control (C): 0.099 ± 0.033, DM: 3.51 ± 0.32, Parpi: 0.007 ± 0.003, and DM+Parpi: 00.073 ± 0.039 (n = 8). *P < 1.0 E-6 (indicating that only DM samples were statistically different from the other samples by one-way ANOVA analysis). B: Parp inhibition prevents the HG-induced impairment in fin regeneration. Control (C): 100 ± 1.3%, DM: 62.5 ± 4.7%, Parpi: 99.3 ± 5.2%, and DM+Parpi: 96.3 ± 6.3% (n = 18 for all groups). *P < 5.0 E-5 (indicating that the regeneration of only DM fish were statistically different from the other samples by a one-way ANOVA analysis).