Figure 3

JMJD3 regulates RUNX2 transcriptional activity during osteoblast differentiation.

(A) RT-qPCR to determine the expression levels of Jmjd3, Runx2, Atf4, Sp7, Spp1, Bglap, Col1a1 and Fgf18 relative to GAPDH in parietal osteoblasts of E18.5 WT and Jmjd3^−/− embryos. *p < 0.05, Error bar represents the SE of three independent experiments. (B) ChIP-qPCRs assays with antibodies specific for JMJD3 (a,b) or H3K27me3 (c,d) at the promoters of Runx2 (a,c) or Bglap (b,d) genes in WT and Jmjd3^−/− primary parietal osteoblasts. Signals were shown as a percentage of the input. *p < 0.05, Error bar represents the SE of three independent experiments. (C) The total level of H3K27me3 in primary osteoblast of E18.5 WT and Jmjd3^−/− parietal bones was examined by western blot assays. (D) Endogenous interaction of JMJD3 and RUNX2 was examined by Co-IP assays in the primary osteoblasts from E18.5 parietal bone. IP was performed as indicated. (E) Immunofluorescence under confocal microscope with anti-JMJD3 (green) and anti-RUNX2 (red) antibodies on the slides of primary osteoblasts of E18.5 embryos. Scale bar: 20 μm. (F) Luciferase reporter assays in HEK293 cells, which were transfected with the pGL3-basic reporter containing 2 kb Bglap promoter with or without expression plasmids encoding Runx2 (100 ng) , Jmjd3 (100 ng) or sh-Jmjd3 in increasing amounts (50, 100 ng) as indicated. The basal luciferase activity for each reporter was calculated as 1 in y axis. *p < 0.05, **p < 0.01, Error bar represents the SE of three independent experiments. (G) ChIP-qPCRs assays with antibodies specific for JMJD3 (a,b) or H3K27me3 (c,d) at the promoters of Runx2 (a,c) or Bglap (b,d) genes in WT and Runx2^−/− primary parietal osteoblasts. Signals were shown as a percentage of the input. *p < 0.05, Error bar represents the SE of three independent experiments.