BACKGROUND: Bone disease, a common source of morbidity in multiple myeloma (MM), is caused by RANKL-induced aberrant activation of osteoclasts (OC). RANKL-induced OC lineage commitment requires repression of an Irf-8 dependent macrophage inflammatory transcriptional programme commensurate with activation of an OC lineage-specific programme. Functional data have shown the requirement for the histone acetylation readers Brd2-4 BET proteins and of cMyc for OC lineage development. However, how Brd2-4 and Myc co-operate genome-wide to regulate transcriptome changes that underpin the very early stages of RANKL-induced OC lineage commitment has not been defined.
METHODS: The OC progenitor-like murine RAW264.7 cell line was used for osteoclastogenesis. OC were assayed by TRAP staining. We performed RNA-seq for transcriptome analysis and ChIP-seq against Brd2-4, cMyc, and H3K27Ac mark for epigenomic profiling. The pan-Bet inhibitor IBET151 was used alone or in combination with RANKL. ChIP-seq/RNA-seq data were processed using standard bioinformatics pipelines; downstream analyses (pathway and motif enrichment, factor differential binding) were performed by various tools including EnrichR, R packages ChIPpeakAnno/DiffBind, Rose.
RESULTS: Transcriptomic profiling of OC progenitors at 0, 4, 14 and 24h post-RANKL treatment identified 12 distinct clusters of expression trends. The 4h activated cluster includes OC master transcription factors (TFs; cMyc, Nfatc1, Fosl), and is enriched in OC-defining pathways. Notably, by 14h the majority of the genes required for mature OC formation and activation are already highly expressed (e.g. Ctsk, Mmp9). The downregulated clusters include monocyte defining TFs (e.g. Irf8, Mafb and Bcl6). These RANKL-dependent transcriptome changes are completely abrogated by iBET151, highlighting the critical role of Brd2-4 in osteoclastogenesis.
Differential chromatin binding analysis upon RANKL induction revealed an overall enhanced Brd2-4 binding at already existing or de novo gained sites. This was more pronounced for Brd2&4 and much less for Brd3, with differentially binding sites (DBS) comprising 50% and 20% respectively of all binding sites in RANKL-treated cells. For Brd2&3, DBS were primarily distributed at promoters and for Brd4 at intergenic, candidate enhancers regions. Notably, nearly all gained DBS were sensitive to and abrogated by iBET151.
Combinatorial profiling of Brd2 and Brd4 showed that almost half of Brd2 DBS peaks overlap with Brd4 (47%; 897/1896), while only 24% (766/3234) of Brd4 DBS peaks are co-occupied by Brd2. Transcriptome and Brd2&4 DBS integration in combination with motif enrichment analysis, identified genes that are predicted to be regulated by Brd2 and/or Brd4. EnrichR analysis suggests that enhanced binding of Brd2&4, singly or in combination, is required for activation of the critical OC lineage-specific and repression of the macrophage-defining transcriptional programs highlighting the non-redundant roles of Brd2&4 in OC development.
Cell lineage commitment often requires 'commissioning' of cell-specific super-enhancers (SE). Combined analysis of genome-wide Brd4/H3K27ac profiles identified 678 RANKL-induced SE and their respective target genes. Further, 110 of these SE showed enhanced Brd4 binding in 2 peaks: 20/110 were linked to significantly up- and 90/100 to down-regulated genes. The repressed genes were significantly enriched to previously described Irf8, MafB and RunX1 targets, suggesting a critical role of SE in the repression of the monocyte/macrophage inflammatory programme during OC lineage commitment.
Strikingly, among top hits, we detected a SE linked to the regulation of cMyc. To further investigate its role in OC development, we obtained the cistrome of cMyc after RANKL induction. We identified 560 binding sites which were highly enriched in cMyc, Max, Fli1, Fosl2 and Irf8 motifs. Cistrome-transcriptome integration suggested direct activation of 141 and repression of 52 genes by cMyc in response to RANKL; these are enriched in ribosome biogenesis pathways and Irf8-dependent targets respectively.
CONCLUSIONS: Myc and Brd4 mark SE that repress an Irf8-dependent transcriptional programme, a requirement for OC lineage commitment. The non-redundant roles of Brd2&4 suggest that selective targeting of either could inhibit aberrant OC activation associated with MM.
Disclosures
Caputo: GSK: Research Funding. Auner:Amgen: Other: Consultancy and Research Funding; Takeda: Consultancy; Karyopharm: Consultancy. Karadimitris:GSK: Research Funding.
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