Hyperglycemia induces a dynamic cooperativity of histone methylase and demethylase enzymes associated with gene-activating epigenetic marks that coexist on the lysine tail

Abstract

Objective: Results from the Diabetes Control Complications Trial (DCCT) and the subsequent Epidemiology of Diabetes Interventions and Complications (EDIC) Study and more recently from the U.K. Prospective Diabetes Study (UKPDS) have revealed that the deleterious end-organ effects that occurred in both conventional and more aggressively treated subjects continued to operate >5 years after the patients had returned to usual glycemic control and is interpreted as a legacy of past glycemia known as "hyperglycemic memory." We have hypothesized that transient hyperglycemia mediates persistent gene-activating events attributed to changes in epigenetic information.

Research design and methods: Models of transient hyperglycemia were used to link NFkappaB-p65 gene expression with H3K4 and H3K9 modifications mediated by the histone methyltransferases (Set7 and SuV39h1) and the lysine-specific demethylase (LSD1) by the immunopurification of soluble NFkappaB-p65 chromatin.

Results: The sustained upregulation of the NFkappaB-p65 gene as a result of ambient or prior hyperglycemia was associated with increased H3K4m1 but not H3K4m2 or H3K4m3. Furthermore, glucose was shown to have other epigenetic effects, including the suppression of H3K9m2 and H3K9m3 methylation on the p65 promoter. Finally, there was increased recruitment of the recently identified histone demethylase LSD1 to the p65 promoter as a result of prior hyperglycemia.

Conclusions: These studies indicate that the active transcriptional state of the NFkappaB-p65 gene is linked with persisting epigenetic marks such as enhanced H3K4 and reduced H3K9 methylation, which appear to occur as a result of effects of the methyl-writing and methyl-erasing histone enzymes.

FIG. 1.

Ambient and prior hyperglycemia sustains increased activating H3K4m1-associated Set7 enrichment on the NFkB-p65 gene in aortic endothelial cells. A: Ambient and prior hyperglycemia sustains increased NFkB-p65 gene expression in bovine aortic endothelial cells. RNA was extracted, and NFκB-p65 mRNA levels were quantified by real-time RT-PCR (qRT-PCR) and normalized to the level of 18S. *P < 0.05 vs. low glucose (LG) 16 h group. Mono-methylation is the predominant H3K4 mark after transient hyperglycemia. Bovine aortic endothelial cells were exposed to glucose and soluble chromatin was immunopurified at the indicated times using H3K4m1 (B), H3K4m2 (C), and H3K4m3 (D) antibodies. qPCR was used to measure the level of enrichment on the NFκB-p65 promoter (−400 bp from the +1 transcription start site). Error bars represent SE. Samples were analyzed in triplicate, and data are presented as means ± SE. *P < 0.05 vs. LG 16 h group, **P < 0.01 vs. LG 16 h group. E: Set7 associated histone methyltransferase activity of human FLAG tagged Set7 (Flag-Set7) overexpressed in human endothelial cells and immunoprecipitated using anti-flag antibody. The substrate used to determine methyltransferase activity was recombinant histone H3. F: Lysine 4 of histone H3 (H3K4) is a major methylation site for the Set7 enzyme. Set7 was immunoprecipitated by anti-FLAG antibody in overexpressed Set7 or control pCX4neo human endothelial cells. Substrates used to determine methyltransferase activity were synthesized histone H3K4 and mutant H3R4 peptides. G: Aortic endothelial cells were exposed to glucose and soluble chromatin were immunopurified at the indicated times with Set7 antibody, and qPCR was used to measure the level of enrichment on the NFκB-p65 promoter. *P < 0.01 vs. LG 16 h group. H: The osmolyte mannitol does not increase NFκB-p65 mRNA levels. Samples were analyzed in triplicate, and data are presented as means ± SE. I: Mannitol does not increase Set7 association with the NFκB-p65 promoter. Samples were analyzed in triplicate, and data are presented as means ± SE.

FIG. 2.

Erasure of the activating H3K4m1 mark on the NFκB-p65 promoter in Set7 KD cells despite transient hyperglycemia. A: Protein blot of Set7 knockdown in HMECs exposed to ambient and prior hyperglycemia for the indicated times. B: Ambient and prior hyperglycemia does not sustain increased NFkB-p65 gene expression in Set7 KD cells. RNA was extracted, and NFκB-p65 mRNA levels were quantified by real-time RT-PCR (qRT-PCR) and normalized to the level of 18S. C: Knockdown of the Set7 enzyme rescinds H3K4m1 mark on the NFκB-p65 promoter. Samples were analyzed in triplicate, and data are presented as means ± SE. *P < 0.05 vs. LG 16 h group. D: Set7 KD does not change the H3K4m2 mark on the NFκB-p65 promoter. HMECs were exposed to glucose, and soluble chromatin was immunopurified at the indicated times for SuV39h1 (E), H3K9m1 (F), H3K9m2 (G), H3K9m3 (H), and LSD1 (I) antibodies. qPCR was used to measure the level of enrichment on the NFκB-p65 promoter. Error bars represent SE. Samples were analyzed in triplicate, and data are presented as means ± SE. *P < 0.05 vs. LG 16 h group.

FIG. 3.

Plaque area remained increased in the previously hyperglycemic mice. Atherosclerotic plaques in aorta staining in red with picro sirus red solution in control apoE KO (A), diabetic apoE KO (B), and previously hyperglycemic apoE KO mice (C). ABDOM, abdominal; THOR, thoracic. (A high-quality digital representation of this figure is available in the online issue.)