Figure 4: Protection of the conversion of 5mC to 5hmC by PGC7 through H3K9me2-containing chromatin in early embryos.
From: PGC7 binds histone H3K9me2 to protect against conversion of 5mC to 5hmC in early embryos
a, 5mC and 5hmC status after the microinjection of Jhdm2a or RanBP5–mER mRNA. Zygotes were injected with Jhdm2a or RanBP5–mER mRNA and cultured for 4.5 h in KSOM. The 5mC and 5hmC states were analysed using anti-5mC and anti-5hmC antibodies (5mC, green; 5hmC, red; DAPI, blue). Scale bar, 20 μm. b, c, Flag-tagged Tet3 mRNA was injected into zygotes obtained from wild-type or PGC7−/−female mice with or without Jhdm2a or RanBP5–mER mRNA and cultured for 4.5 h in KSOM. After PT or TP treatment as described in the Fig. 2 legend, immunostaining was performed using an anti-Flag antibody. Tet3 is shown in green; nuclei were stained with DAPI (blue). Ten zygotes obtained from wild-type female mice injected with Flag-tagged Tet3 (Flag–Tet3) mRNA were stained with the anti-Flag antibody under PT conditions; 13 zygotes were injected with Flag–Tet3 mRNA alone and 16 zygotes were injected with Tet3 and Jhdm2a mRNA or with Tet3 and RanBP5–mER mRNA. These embryos were stained with anti-Flag antibody under TP conditions. Zygotes obtained from PGC7−/− female mice were injected with Flag–Tet3 mRNA. A total of 6 and 9 zygotes were stained with the anti-Flag antibody under PT and TP conditions, respectively (b) and the percentage of Flag staining in maternal (M) and paternal (P) pronuclei is shown (c). Scale bar, 20 μm. d, Effect of PGC7 on the chromatin binding of Tet3 in ES cells. Nuclei were isolated from PGC7−/− ES cells transfected with full-length PGC7, Flag–Tet3, PGC7 and Flag–Tet3, PGC7ΔC, and both PGC7ΔC and Flag–Tet3. These nuclei were treated with DNase I under various concentrations of NaCl (100, 200, 300, 400 and 500 mM) to separate nuclear extract from nuclear debris. Equivalent amount of aliquots were analysed by immunoblotting with anti-PGC7, anti-Flag and anti-H3 antibodies as described.
