Epigenetic Regulation of Osteoclast Differentiation and Osteoclast-Specific Super-Enhancers

Objectives: We sought to identify key epigenetic regulators and super-enhancers (SEs) that drive transcription of genes important for osteoclast differentiation. Irf8, a negative regulator of osteoclastogenesis, whose genome-wide function is unclear, was used as a model to better understand changes in chromatin state during osteoclast differentiation process.
Methods: RNA-seq and ChIP-seq (H3K27ac) was performed in Irf8-WT and Irf8-KO bone marrow macrophages (BMMs) after RANKL treatment (day0=precursors, day4=pre-osteoclast, day8=osteoclast).
Results: RANKL treatment promoted osteoclast differentiation in both WT and KO BMMs, however, epigenetic silencing of Irf8 had profound effects on gene expression pattern (KOvsWT, fold-change>2, day4=1161 and day8=1560), which was underscored by robust osteoclast formation and deficient bone mass in Irf8-KO mice. RNA-seq corroborated many (e.g., Nfatc1;Ctsk;Oscar;Acp5;Mmp9;Ocstamp;Dcstamp;Mitf) previously established osteoclast markers in WT-Day8 (upregulated=138, downregulated=69) and KO-Day8 cultures (upregulated=209, downregulated=96), and revealed several novel genes associated with osteoclast differentiation. ChIP-seq identified unique class of SEs at Day0 (WT=769, KO=463), Day4 (WT=732, KO=584), and Day8 (WT=641, KO=661). Among the upregulated genes, 4.5% (92) of WT-Day4 vs 7.6% (168) of KO-Day4, and 3.9% (107) of WT-Day8 vs 6% (137) of KO-Day8 genes acquired SE architecture. In both groups, the dominant class of SEs enriched included osteoclast differentiation, signaling pathways for T/B-cell receptors, MAPK, chemokine, cytokine, TNF, cell-adhesion, and regulation of actin cytoskeleton. While some of the osteoclast-specific SEs (e.g., Nfatc1;Ctsk;JunB;;Acp5;Dnmt3a;Mmp9;Ocstamp;Dcstamp;Mitf;Fos) were enriched in both WT and KO, their enhancer stretches were significantly retained at Day8 in KOvsWT, suggesting Irf8 silencing shapes active enhancer landscape to promote osteoclastogenesis.
Conclusions: Irf8 contributes to the maintenance of stable repressive state as macrophages, and its silencing with RANKL-treatment or genetic ablation initiates transcriptional activation of several genes and acquisition of SEs that promote osteoclast differentiation. Targeting epigenetic chromatin regulators may offer promising interventional approaches for osteoclast-mediated bone disorders.