Fig 2: Depletion of KDM4A/B/C increases trimethylation of H3K9 leading to a decrease in HIF-1α in intermittent hypoxia. MCF7 cells were transfected with combined siKDM4A, siKDM4B, and siKDM4C (referred to as siKDM4) or siCONTROL followed by exposure to normoxia, chronic hypoxia (2% v/v), and intermittent hypoxia (5 min/5 min) over 18 h. mRNA expression of (A) KDM4A, (B) KDM4B, (C) KDM4C, (E) HIF1A, (F) HK2, and (G) PLOD2. All values are normalized to normoxia (Log2 scale). Results are the mean ± SD n ≥ 4 independent experiments. ns = not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. D, nuclear extracts of KDM4A, KDM4B, KDM4C, HIF-1α, H3K9me3, and histone H3 (loading control), and cytoplasmic extracts of HK2 and β-actin (loading control).

Fig 3: HIF-1 activity and H3K9me3 levels in a 3D spheroid model of chronic and intermittent hypoxia.A, HCT116 cells were stably transfected with a GFP reporter linked to the hypoxia response element (HRE). Cells grown as spheroids and imaged on day 0, 3, 7, and 10. B, protein expression of HIF-1α, KDM4B, KDM4C, HK2, Glut1, LDHA, and H3K9me3 in HCT116 cells grown as a monolayer or as spheroids grown over 3, 7, or 10 days. Total histone H3 is used as a loading control. C, schematic illustration of spheroid exposure to oxygen conditions using oxygen-permeable membranes and schematic illustration demonstrating how confocal microscopy is used to visualize the spheroid through multiple transverse planes. D and E, confocal images of HCT116 cells grown as spheroids over 3 days, transferred onto oxygen-permeable membranes for 24 h, and then exposed to normoxia or intermittent hypoxia over a further 18 h. D, live cell fluorescence images of spheroids expressing GFP linked to the HRE in four different transverse planes (plane 4 = membrane level). Magenta = NucRed live stain; green = GFP. E, bright field and fluorescence images of spheroids that were fixed, permeabilized, and probed with antibodies. Cyan = Histone H3 protein expression; red = H3K9me3 protein expression. Images from other transverse planes are shown in Fig. S7. F, examples of single cell analysis from individual cells attached to the membrane as a monolayer and from the periphery and core of spheroids (Zoomed in images taken from (E)). The scale bar = represents 300 μm for (A, D, and E), 50 μm for (F). G, fluorescence of H3K9me3:Histone H3 analysis from (E and F). Results are the mean ± SD of n = 20 cells. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Complete statistical analysis comparing fluorescence between the monolayer versus periphery versus core is presented in Table S2.

Fig 4: Proposed mechanism of HIF-1 activation in intermittent versus chronic hypoxia. In normoxia, HIF1A is constitutively transcribed and translated into HIF-1α but HIF-1α is posttranslationally degraded. In chronic hypoxia, HIF-1α is stabilized, increasing HIF-1 transcriptional activity and the expression of HIF target genes, KDM4B and KDM4C. Despite increased enzyme levels, KDM4A, KDM4B, and KDM4C are largely inactive due to limited amounts of oxygen, which are required for KDM activity. This leads to an increase in H3K9me3, including at the HIF1A locus, which ultimately decreases the amount of HIF1A mRNA being transcribed. In intermittent hypoxia, HIF-1α increases as compared to normoxia. KDM4B and KDM4C expression levels increase somewhat but not to the same level as chronic hypoxia. However, in contrast to chronic hypoxia, KDM4A, KDM4B, and KDM4C activity increases due to sufficient oxygen and higher levels of demethylases, leading to higher levels of H3K9 demethylation at the HIF1A gene when compared to normoxia or chronic hypoxia. This results in increased HIF1A mRNA production.