Extended Data Fig. 7: OSK-induced axon regeneration and survival require non-global active DNA demethylation through thymine DNA glycosylase.
From: Reprogramming to recover youthful epigenetic information and restore vision
a, Representative images of retinal wholemounts transduced with sh-Scr-H2B-GFP or sh-TDG-H2B-GFP AAV2 s for 4 weeks, demonstrating that knockdown of thymine DNA glycosylase (TDG) increased levels of 5-hydroxymethylcytosine (5-hmC). n = 4 retinas for each condition. b, Representative retinal wholemount images and images of longitudinal sections through the optic nerve showing CTB-labelled regenerative axons in WT mice, 16 dpc after an intravitreal injection of AAV2-tTA ;TRE-OSK in combination with AAV2-sh-Scr (sh-Scr) or AAV2-sh-TDG (sh-TDG) at titre ratio 5:5:1. n = 4 retinas for each condition. c, d, Quantification of regenerating axons (c) and RGC survival (d) in OSK-treated mice 16 dpc with AAVs carrying sh-Scr or sh-TDG (n = 4 nerves for each condition). e, Representative image of retinal wholemounts transduced with AAV2-tTA;TRE-OSK. Retinal wholemounts were immunostained for 5-methylcytosine (5-mC) and Klf4, showing a lack of global demethylation in OSK expressing RGCs. n = 3 retinas. f, Representative images of retinal wholemounts transduced with AAV2 vectors encoding the HA-TET1 catalytic ___domain (TET1-CD) or its catalytic mutant (TET1-mCD) for 4 weeks, demonstrating that overexpression of TET1-CD decreases global 5-mC levels. n = 3 retinas for each condition. g, h, Quantification of axon regeneration (g) and RGC survival (h) at 2 wpc in retinas transduced without or with AAV2 vectors encoding HA-TET1 CD mutant or HA-TET1 CD (n = 3, 4 and 3 eyes). i, A schematic diagram illustrating passive demethylation and TDG-dependent active DNA demethylation. Scale bars (a, b, e, f), 100 μm. One-way ANOVA with Bonferroni’s multiple comparison test in c, g, h; unpaired two-tailed Student’s t-test in d. There was no difference between the groups in g and h using two-way ANOVA and one-way ANOVA, respectively. All data are mean ± s.e.m.
