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. 2017 Jan 31;8:14272. doi: 10.1038/ncomms14272

Figure 6. HDAC2-dependent de-repression by JMJD2C and KDM3A.

Figure 6

(a,d) Relative luciferase activity of the full Oct6 SCE, the ΔHR1 (constructs #3 and #19, ref. 35) and the ΔHR2 SCE showing that overexpression of HDAC1, HDAC2, JMJD2C or KDM3A increases the activation of the full Oct6 SCE or the ΔHR1 SCE, but not of the ΔHR2 SCE, compared with control GFP (=1) in RSCs cultured under dedifferentiating conditions. The experiment carried out in the presence of the HDAC1/2 inhibitor Mocetinostat or its vehicle indicates that activation induced by JMJD2C or KDM3A is prevented by HDAC1/2 inhibition. (i) Similarly, activation of the Krox20 MSE induced by JMJD2C or KDM3A overexpression in RSCs cultured under redifferentiating conditions is prevented by HDAC1/2 inhibition. Chromatin immunoprecipitation of (b) HDAC2 and Flag (Neg=1) in unlesioned (no lesion) nerves or at 1 dpl, of (e) JMJD2C, KDM3A and Neg (GFP or flag) in control or dKO nerves at 1 dpl, of (f,g) H3K9me3, H3K9me2 and Neg (GFP or Flag) in unlesioned nerves and at 1 dpl in control (f) and dKO (g) on the Oct6 SCE HR2, and of (h) HDAC2, JMJD2C, KDM3A, H3K9me3, H3K9me2 and Neg on the Krox20 MSE in unlesioned nerves or at 12 dpl. (c) Oct6 mRNA fold increase in RSCs transfected with GFP (=1), HDAC2, JMJD2C or KDM3A expression constructs and cultured under dedifferentiating conditions. Asterisks show significance compared with no lesion (b,f–h), to control (e) or to GFP (a,c,d,i), and crosses compared with vehicle (d,i). One-tailed (grey asterisks) or two-tailed (black asterisks or crosses) Student's t-tests, unpaired (d,i: HDAC1/2 inhibitor versus Vehicle; b,e–h) or paired (d,i: JMJD2C or KDM3A versus GFP; a,c), P values: *P<0.05, +P<0.05, **P<0.01, ***P<0.001. Values, mean; error bars, s.e.m. Sample size: (a) full Oct6 SCE, n=12; ΔHR1 and ΔHR2 SCE, n=4; (b) 3 animals per group no lesion and 1 dpl; (c) HDAC2, JMJD2C, n=3; GFP, KDM3A, n=6; (d) n=4; (e) n=3 animals per group control and dKO; (f,g) 3 animals per group no lesion and 1 dpl; (h) 3 animals per group no lesion and 12 dpl; (i) n=3.