Fig 2: KDM4B and CCAR1 are colocalized near TSSs and enhance the gene expression of a set of osteoclast-related genes. a Immunoblots of nuclear fractions of BMMs after RANKL treatment. b KDM4B stabilization by RANKL or MG132. c Heatmap of normalized tag densities on KDM4B and CCAR1 peaks (left panels). Quantifications of tag counts are shown in the box plots (right panels). The boxes enclose the 25th to 75th percentile values. The whiskers extend to the 10th and 90th percentiles. The central horizontal bar indicates the median. ****P < 0.000 1 (Kolmogorov–Smirnov test). d Genomic locations of binding sites within each cluster are expressed as percentages of the total binding sites. e Gene Ontology (GO) enrichment analysis of Cluster III. f Representative UCSC Genome Browser tracks showing co-occupancy of KDM4B and CCAR1 at Fosl2 and Tpm1. g mRNA expression levels of Fosl2 and Tpm1 in cells expressing control shRNA, Kdm4b shRNA, or Ccar1 shRNA1. The data are presented as the mean ± SD of three independent experiments (two-way ANOVA). **P < 0.01; ***P < 0.001. See also Supplementary Fig. 3

Fig 3: The KDM4B–CCAR1–MED1 axis is required for osteoclastogenesis. a, b MED1 expression during osteoclast differentiation. qRT-PCR (a) and immunoblot (b) results. c TRAP staining of BMMs expressing either control shRNA or Med1 shRNA. Scale bar, 75 μm. d Heatmap of normalized tag densities on KDM4B, CCAR1, and MED1 peaks (2 kb up/downstream of the peak centers) (left panels). Quantifications of MED1 tag counts are shown in the box plots (right panel). The boxes enclose the 25th to 75th percentile values. The whiskers extend to the 10th and 90th percentiles. The central horizontal bar indicates the median. ****P < 0.000 1 (Kolmogorov–Smirnov test). e Representative UCSC Genome Browser tracks showing co-occupancy of KDM4B, CCAR1, and MED1 at Fosl2 and Tpm1. f mRNA expression levels of Fosl2 and Tpm1 in cells expressing control shRNA or Med1 shRNA. The data are presented as the mean ± SD values of three independent experiments [one-way ANOVA in (a, c)]. *P < 0.05; **P < 0.01; ***P < 0.001. See also Supplementary Fig. 4

Fig 4: Association of p65 and KDM4B is critical for osteoclastogenesis. a The most significantly enriched transcription factor (TF) motifs at KDM4B binding regions, as identified by de novo motif analysis using HOMER. b KDM4B interaction with p65. c, d Mapping the binding domain of KDM4B and p65. RHD Rel homology domain, TAD transactivation domain. e Heatmap of normalized tag densities on KDM4B, CCAR1, MED1, and p65 peaks (left panels). Quantifications of p65 tag counts are shown in the box plots (right panel). The boxes enclose the 25th to 75th percentile values. The whiskers extend to the 10th and 90th percentiles. The central horizontal bar indicates the median. ****P < 0.000 1 (Kolmogorov–Smirnov test). f ChIP assay of KDM4B occupancy at the Fosl2 or Tpm1 promoter in p65-depleted cells. g ChIP assay of p65 occupancy at the Fosl2 or Tpm1 promoter in KDM4B- or CCAR1-depleted cells. The data are presented as the mean ± SD values of three independent experiments [two-way ANOVA in (f, g)]. **P < 0.01; ***P < 0.001. See also Supplementary Fig. 6

Fig 5: KDM4B associates with CCAR1. a Schematic diagram of the method used to purify the KDM4B-interacting complex from FLAG-KDM4B-expressing cells. Lane 1, control preparation from the nuclear extract of normal HeLa cells; lane 2, KDM4B-interacting complex from the nuclear extract of KDM4B-expressing HeLa cells. b KDM4B-interacting complexes. c TRAP staining of BMMs expressing either control shRNA or Ccar11 shRNA1. Scale bar, 75 μm. d, e CCAR1 expression during osteoclast differentiation. qRT-PCR (d) and immunoblot (e) results. f Coimmunoprecipitation of FLAG-KDM4B and HA-CCAR1. g Endogenous interaction of KDM4B and CCAR1 in BMMs. h, i Mapping the binding domain of KDM4B and CCAR1. JmjN N-terminal jumonji domain, JmjC jumonji C-terminal domain, PHD plant homeodomain finger domain, Tudor Tudor domain. SAP, SAF-A/B, Acinus, and PIAS DNA/RNA binding motif. The data are presented as the mean ± SD values of three independent experiments [one-way ANOVA in (c, d)]. *P < 0.05; **P < 0.01; ***P < 0.001. See also Supplementary Fig. 2