Fig. 5: ARID1A activates Nfatc1 through constructing condensates with coactivator BRD4/lineage-specifying TF PU.1 at SE.
From: ARID1A safeguards the canalization of the cell fate decision during osteoclastogenesis
a TRAP staining of JQ1 or vector treated RANKL-induced BMCs from control and LysM-Cre;Arid1afl/fl group. Scale bar, 100 μm. b Mmp9, Acp5, Ctsk, and Dcstamp mRNA and c MMP9 and CTSK protein expressions in JQ1 or vector treated RANKL-induced BMCs from control and LysM-Cre;Arid1afl/fl group. n = 3 biologically independent samples. d Heatmap representation of ChIP-seq against ARID1A and BRD4 in ± 3 kb around the center of the ARID1A peak. e Nfatc1 mRNA expression in JQ1 or vector treated RANKL-induced BMCs from control and LysM-Cre;Arid1afl/fl group. n = 3 biologically independent samples. f BRD4 and ARID1A immunofluorescence in RANKL-induced BMCs. Yellow arrows indicate BRD4/ARID1A nuclear puncta. Scale bar, 5 μm. g Gene tracks of ARID1A, BRD4 and PU.1 ChIP-seq occupancy at Nfatc1 super enhancer in BMCs from control and LysM-Cre;Arid1afl/fl group before or after RANKL-induction. Gray box indicates the promoter region (Pro). The orange shadows highlight the regions containing enhancer elements. h Top 10 motifs enriched in ARID1A binding sites and ARID1A-dependent BRD4 binding sites which loss after ARID1A depletion. i PU.1 and ARID1A immunofluorescence in BMCs after RANKL-induction. Yellow arrows indicate PU.1/ARID1A nuclear puncta. Scale bar, 5 μm. j Co-IP assay with ARID1A antibody (or IgG) in BMCs during osteoclastic induction, followed by immunoblotting of ARID1A, BRD4, and PU.1. k ChIP-qPCR with PU.1 antibody (or IgG) in RANKL-induced BMCs from LysM-Cre;Arid1afl/fl (n = 3) and control mice (n = 6). The positions of the sets of primers used for the ChIP–qPCR are denoted in g. l Immunofluorescence and co-localization analysis of PU.1 and BRD4 in RANKL-induced BMCs from control and LysM-Cre;Arid1afl/fl group. Yellow arrows indicate PU.1/BRD4 nuclear puncta. Scale bar, 5 μm. All data in this figure are represented as mean ± SD. One-way ANOVA with Tukey’s multiple comparisons test for b and e. The one-tailed Fisher’s Exact test for h. Two-tailed Student’s t-test for k. All experiments were performed in triplicates unless otherwise stated. Source data and exact p values are provided in the Source data file.
