Loss of H3K27me3 in ASXL1-deficient osteoclasts is mediated by Jmjd3. (A) H3K27me3 peaks at individual loci (highlighted blue bar) on Jmjd3 promoter; WT (ASXL1^flox/flox) and KO (ASXL1^cKO). (B) ASXL1^flox/flox and ASXL1^cKO BMMs were cultured in the presence of M-CSF and RANKL (25 ng/mL). RNA was harvested on days 1 and 2 of RANKL stimulation, and histone H3 Lys 27 (H3K27) demethylase Jmjd3 mRNA abundance was determined by qPCR. Unpaired nonparametric Student t test was used for statistical analysis. Error bars represent + SD; **P < .01, ***P < .001. (C-D) ASXL1^cKO BMMs, transduced with scr or Jmjd3 short hairpin RNA (shRNA), were exposed to M-CSF and RANKL (25 ng/mL) for 2 days. H3K27me3 binding to NFATc1 promoter (C) and Blimp1 promoter (D) was determined by ChIP assay. IgG served as control. n = 2 independent experiments from 10- to 12-week-old male mice. Two-way ANOVA was used for statistical analysis. Error bars represent + standard error of the mean; ***P < .001. (E) ASXL1^cKO BMMs, transduced with scr or Jmjd3 shRNA, were exposed to M-CSF and + RANKL for 3 days. Expression of osteoclast differentiation proteins NFATc1 and β3 integrin was determined by immunoblot (left) and Blimp1 by qPCR (right). n = 3 independent experiments from 8-week-old male mice. One-way ANOVA was used for statistical analysis. Error bars represent + SD; *P < .05, **P < .01. (F) ASXL1^cKO BMMs, transduced with scr or shRNA for Jmjd3, were exposed to M-CSF and + RANKL for 5 days and stained for TRAP activity. Images were captured on Nikon Eclipse E400. Scale bar represents 400 μm.