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. 2025 Sep 28;40(10):1127-1143.
doi: 10.1093/jbmr/zjaf084.

Integrative single-cell RNA-seq and ATAC-seq identifies transcriptional and epigenetic blueprint guiding osteoclastogenic trajectory

Amitabh Das  1 , Keita Saeki  2 , Stefania Dell'Orso  3 , Xiaobei Wang  1 , Kim C Mansky  4 , Joseph Stains  5 , Keiko Ozato  2 , Hong-Wen Deng  6 , Vivek Thumbigere-Math  1
Affiliations

Integrative single-cell RNA-seq and ATAC-seq identifies transcriptional and epigenetic blueprint guiding osteoclastogenic trajectory

Amitabh Das et al. J Bone Miner Res. .

Abstract

Osteoclasts (OCs) are multinucleated bone resorbing cells essential for skeletal development and remodeling. In adulthood, OCs originate from the serial fusion of monocytes, yet the transcriptional and epigenetic mechanisms shaping their osteoclastogenic potential at a single-cell resolution remain poorly understood. Here, we present an integrative multi-omics analysis, combining single-cell (sc) RNA-seq, scATAC-seq, bulk RNA-seq, and Chromatin Immunoprecipitation (ChIP)-seq, to comprehensively define the regulatory landscape of osteoclastogenesis in WT and interferon regulatory factor 8 (IRF8) conditional KO mice. We uncovered a highly structured and sequential differentiation trajectory from hematopoietic stem and progenitor cells to common monocyte progenitors (cMoPs) to mature monocytes, with each stage exhibiting distinct transcriptional and epigenetic signatures. cMoPs and monocytes are the critical stages when OC lineage priming occurs, characterized by transcriptional and epigenetic activation of cytoskeletal, immune, and cell migration pathways. This priming is tightly regulated to prevent premature OC differentiation and IRF8 acts as a negative regulator of osteoclastogenesis by maintaining monocyte identity and restricting chromatin accessibility at osteoclastogenic loci. IRF8 deficiency disrupts this balance, leading to chromatin reprogramming characterized by increased accessibility at OC-promoting loci (Nfatc1, Cebpe) and reduced accessibility at monocyte-specific genes (Mafb, Klf4), thereby priming precursors towards pre-mature osteoclastogenesis. Just as NFATc1 is recognized as a master activator of osteoclastogenesis, our findings position IRF8 as a master negative regulator of osteoclastogenesis, maintaining the delicate balance required for proper bone homeostasis. Collectively, this study provides unprecedented resolution into the molecular mechanisms shaping OC precursor identity and offers novel insights into potential therapeutic targets for osteolytic disorders.

Keywords: ChIP-seq; IRF8; chromatin accessibility; monocytes; osteoclasts; single-cell ATAC-seq; single-cell RNA-seq; transcriptional regulation.

Plain language summary

This study reveals how monocytes, a type of immune cells, gradually transform into bone-eating osteoclasts (OCs) through tightly regulated changes in gene activity and DNA structure. The research highlights the gene IRF8 acts as a key brake in this process, preventing premature osteoclast formation by maintaining monocyte identity and controlling DNA accessibility. Loss of IRF8 disrupts this balance, reprogramming cells to become OCs too early, which may contribute to bone loss in diseases like osteoporosis and points to IRF8 as a promising target for more selective and effective bone disease therapies.

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