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Seth Fortmann, Maria B Grant; Retinal Ischemia Induces Rapid and Reversible Histone Hypermethylation. Invest. Ophthalmol. Vis. Sci. 2022;63(7):772 – F0331.
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Hypoxia-induced VEGF expression is a well-characterized disease mechanism that underlies ischemic retinopathies and requires stabilization of hypoxia-inducible factors (HIFs). However, less is known about HIF-independent hypoxia-sensitive pathways, which may alter the epigenetic landscape of hypoxic retinal cells and thereby promote the transcriptional activity of HIFs.
The oxygen-induced retinopathy (OIR) mouse model (75% O2 from P7-P12) was used to investigate histone modifications during acute retinal ischemia following hyperoxia exposure. Immunofluorescence (IF) and western blotting (WB) were performed at various timepoints in OIR mice (time post-hyperoxia exposure: 0 hours (h; baseline), 2h, 4h, 1 day (d), 3d, and 5d) to probe the spatiotemporal modifications that occur on histones following acute retinal ischemia, including H3K4me3, H3K9me3, H3K27me3, H3K36me2, and H3K36me3. The dynamicity of hypoxia-induced histone hypermethylation was examined using reoxygenation experiments in the OIR mouse model, where mice were removed from hyperoxia for 2h and then returned to hyperoxia (reoxygenation) for 2h or 4h. Lastly, IF, WB, and single-cell RNA-sequencing data (scRNA-seq) were used to examine the retina for expression of the hypoxia-sensitive histone demethylases, KDM5A and KDM6A.
WB on whole retinal lysates showed that the hypoxia-sensitive histone demethylases, KDM5A and KDM6A, were strongly expressed in the mouse retina. IF and scRNA-seq showed that KDM5A and KDM6A were globally expressed in the retina, with the strongest expression in inner nuclear layer cells. WB on whole retinal lysates from OIR mice showed that H3K4me3 was induced as early as 2 hours following the onset of hypoxia, which mirrored the simultaneous stabilization of HIF1α. Reoxygenation experiments in OIR mice showed that the induction of H3K4me3 was rapidly reversible and that H3K4me3 levels returned to baseline within 2 hours of reoxygenation.
These findings reveal a novel, rapidly induced, and reversible hypoxia sensitive pathway in the ischemic retina that acts on histone modifications. H3K4me3 is a promoter-specific histone modification that influences transcription factor docking and thus, may play a role in instructing the genomic binding of HIFs during retinal ischemia.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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