Figure 4. RANK signaling induces NCoR/HDAC3/PGC1β interaction required for H3K27 acetylation.
(A) Expression of Ppargc1a and Ppargc1b in WT cells as a function of time following RANKL treatment.
(B) The overlap between IDR-defined PGC1β ChIP-seq peaks at Day0 and at Day4 is shown by Venn diagram.
(C) The overlaps of ATAC-defined gained H3K27ac peaks in the presence of RANKL (n=1525 in Figure S2E) with NCoR, HDAC3 and/or PGC1β ChIP-seq peaks at Day4 are shown by pie chart.
(D) BMDMs were treated with or without RANKL for 6 hours in the presence or absence of RGFP966, and then the whole-cell lysates (WCL) were subjected to immunoprecipitation (IP) using anti-PGC1β antibody and immunoblot (IB) analysis with anti-acetylated lysine, PGC1β, HDAC3 or NCoR antibody.
(E) Histone acetyltransferase (HAT) activity of immunoprecipitated PGC1β protein in whole-cell lysates from BMDMs treated with or without RANKL for 6 hours was measured in the presence of acetyl-CoA and histone H3 substrate. Data are mean ± s.d. (n=3 biological replicates). Student’s t-test was performed for comparisons. ***p < 0.001.
(F) 10–30% sucrose density gradient centrifugation was performed on nuclear fractions from BMDMs treated with or without RANKL for 6 hours. All fractions (1–24, top to bottom) were subjected to IB analysis with anti-PGC1β, NCoR, HDAC3 or TBL1 antibody. The molecular weight standards are indicated at the top of the panel; 66 kDa, bovine serum albumin; 200 kDa, β-amylase; 669 kDa, thyroglobulin.
(G) Normalized distribution of H3K27ac ChIP-seq tag density in control (Ad-RFP) and PGC1β KO (Ad-Cre) at the vicinity of NCoR and HDAC3-associated gained H3K27ac peaks in WT at Day4 after RANKL treatment (n=2757 in Figure 2C).
(H) Genome browser tracks of H3K27ac, PGC1β, NCoR and HDAC3 ChIP-seq peaks in the vicinity of Acp5 and Ocstamp loci. Yellow shading: lost H3K27ac by PGC1β KO at RANKL-induced NCoR, HDAC3 and PGC1β binding regions.
(I) Bar plots for expression of Acp5 and Ocstamp in control (Ad-RFP) and PGC1β KO (Ad-Cre) at Day0 and Day4 after RANKL treatment. ***p-adj < 0.001.
See also Figure S4.