Fig 2: Clinical implications of modulating the DOT1L network in cartilage.(a–c) Inactivation of SIRT1 protects against DOT1L inhibitor-induced osteoarthritis: (a) C57/Bl6 wild-type mouse knees were injected with DOT1L inhibitor EPZ-5676 (5 mg kg-1) and SIRT1 inhibitor EX527 (1.25 mg kg–1), or vehicle (V) and killed after 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O (a). Scale bar, 200 µm. Cartilage damage was scored (see Methods section) and is shown in (b). One experiment was performed with n=3 (vehicle), 8 (EPZ) and 10 (EPZ+EX527). *P<0.05 by one-way ANOVA. Error bars indicate mean±s.e.m. (c) Immunohistochemistry of TCF1 in the indicated groups. TCF1 levels are increased after EPZ treatment and normalized by additional EX527 treatment. The images are representative of three different animals. Scale bar, 200 µm. (d,e) Loss of DOT1L function causes severe growth retardation as demonstrated by skeletal staining (d) and histology of the growth plate (e) of 4-week-old Dot1lfl/fl;Col2-Cre-/- (Cre-neg) and Dot1lfl/fl;Col2-Cre+/- (Dot1lCart-KO) mice. (f,g) Increased TCF1 levels in Dot1lCart-KO mice as shown by immunohistochemistry in the indicated mice strains in the articular cartilage (f) and growth plate (g). The images are representative of three different animals. Scale bar, 200 and 100 µm.

Fig 3: miR-216 Targets Dot1l in the Retina during Photoreceptor Regeneration(A) For post-mitotic MG isolation, GFP+ cells were sorted from dark adapted undamaged Tg(gfap:gfp) retinas. For dedifferentiated MG isolation, GFP+ cells were isolated from 45-h light-lesioned Tg(1016tuba1a:GFP) retinas.(B) Fold changes in dot1l levels in FACS-purified MG were determined by qPCR. After 45 h of light damage, dot1l is upregulated in dedifferentiated MG (GFP+) in Tg(1016tuba1a:gfp) fish. Dot1l expression did not change in non-MG cells (GFP-) during regeneration. Data represent the means ± SEMs from 15 undamaged fish, and dedifferentiated MG were purified from 18 light-damaged fish.(C) Experimental scheme to test the effects of miR-216a overexpression on dot1l levels. Wild-type adult zebrafish were dark adapted and then either control miRNA or miR-216a was injected and electroporated into the left eyes before intense light exposure. After 24 h of light exposure, retinas were dissected for RNA isolation.(D) Fold changes in miR-216a levels in control miRNA (small interfering RNA [siRNA] against luciferase [siLuc]) or miR-216a mimic electroporated retinas were quantified by qPCR. miR-216a levels were upregulated by 15-fold in miR-216a mimic-injected retinas compared to controls. Data represent the means ± SEMs from 6 retinas.(E) Fold changes in dot1l levels in siLuc or miR-216 mimic electroporated retinas were quantified by qPCR. After 24 h of light damage, dot1l was downregulated in miR-216a-overexpressing retinas ~20-fold. Data represent means ± SEMs from 3 independent experiments. Six retinas were pooled for RNA isolation in each experiment.**p < 0.01 (Student’s t test), p = 0.0093.Scale bars, 50 µm.See also Figures S1 and S2.

Fig 4: DOT1L protects against osteoarthritis by preventing Wnt hyper-activation.(a) Wnt antagonist XAV-939 (XAV) rescues gene expression changes in ACAN, COL2A1 and COL1A1 induced by EPZ-5676 in healthy human articular chondrocytes. Data are from one experiment with three technical replicates. (b,c) C57/Bl6 wild-type mouse knees were injected with EPZ (5 mg kg-1), XAV (0.1 (low dose—L) or 0.5 (high dose—H) mg kg-1) or vehicle and killed after 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O (b). Scale bar, 200 µm. Cartilage damage was scored (see Methods section) and is shown in (c). One experiment was performed with n=3 (vehicle, EPZ and XAV-939 only) and n=10 (EPZ+XAV9393 groups). **P<0.01 (two-tailed t-test) compared to EPZ treatment only. All error bars indicate mean±s.e.m.

Fig 5: miR-216a and Dot1l Regulate Retinal Regeneration through the Wnt/ß-Catenin Pathway(A) Control MOs or Dot1l MOs were injected and electroporated into the left eyes of wild-type zebrafish, followed by either 4% DMSO or GSK-ß-inhibitor (1 mM) before intense light exposure (0 h). Eyes were collected after 51 h of intense light lesioning and immunostained using antibodies against PCNA. Nuclei were counterstained with TOPRO (blue).(B) dot1l MOs significantly decreased the number of PCNA+ cells in the INL, while there was no significant difference between dotl1l MO + GSK-3ß-inhibitor co-injected eyes and control eyes.(C) Quantification of PCNA+ proliferating progenitors in MO-ctl + DMSO, MO-dot1l + DMSO, MO-ctl + GSK-3ß-inhibitor, and MO-dot1l + GSK-3ß-inhibitor electroporated retinas. Activation of Wnt signaling rescued the decrease in the number of proliferating progenitors upon dot1l knockdown after 51 h of intense light lesioning. Data represent means ± SEMs; n = 9–11 fish. *p < 0.05, p = 0.0167 (MO-ctl + DMSO versus MO-dot1l + DMSO) by 1-way ANOVA with Dunnett’s multiple comparisons test.(D) GSK-3ß-inhibitor alone, Dot1l inhibitor (iDot1l) alone, or the combination was injected into the left eyes of Tg(1016tuba1a:GFP) zebrafish; DMSO alone was used as a control. At 51 h post-injection, eyes were collected for PCNA immunostaining. GSK-3ß-inhibitor alone induced MG proliferation in undamaged eyes while co-injection of the Dot1l inhibitor led to no significant changes in number of proliferating MG. Data represent means ± SEMs. Each data point represents an individual fish. ****p < 0.0001, by 1-way ANOVA with Dunnett’s multiple comparisons test.(E) Control MO or miR-216a MO was injected and electroporated into the left eyes of Tg(1016tuba1a:GFP) zebrafish, followed by either 4% DMSO or XAV939 (10 µM). Eyes were collected at 51 h post-injection and immunostained using antibodies against GFP for dedifferentiated MG and PCNA for proliferating progenitors. Nuclei were counterstained with TOPRO (blue).(F) Suppression of miR-216a by MO-216a injection stimulates MG proliferation; however, upon co-injection with XAV939, no significant increase in the number of proliferating progenitors was detected.(G) Quantification of PCNA+ proliferating progenitors in MO-ctl + DMSO, MO-216a + DMSO, MO-ctl + XAV939, and MO-216a + XAV939 electroporated retinas. Inhibition of Wnt signaling reversed the increase in the number of progenitors upon miR-216a knockdown after 51 h of intense light lesioning. Data represent means ± SEMs; n = 18–21 fish. **p < 0.01, p = 0.0089 (MO-ctl + DMSO versus MO-216 + DMSO) by 1-way ANOVA with Dunnett’s multiple comparisons test.GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars, 50 µm.See also Figure S4.