In recent years, ‘omic’ studies integrating transcriptomics (analysing RNA transcripts) with epigenetics (study of DNA methylation and histone modifications) have revolutionised our understanding of cellular processes and disease mechanisms. During the RANKL-mediated differentiation of monocytes into bone-digesting osteoclasts, epigenetic modifications regulate a unique transcriptional program that triggers cell-to-cell fusion and endows the resulting multinucleated progeny with the specialised machinery required for bone digestion.
This study aimed to analyse and integrate gene expression and methylation patterns during RANKL-mediated differentiation of human osteoclasts. Eight female participants aged 30-70 with European ancestry were selected. Peripheral blood monocytes (PBMCs) were collected and cultured to differentiate into osteoclast-like cells (OCs) in vitro. Bulk RNA-seq was performed on RNA samples extracted from PBMCs and OCs, with differential gene expression (DGE) analysis performed in RStudio using the edgeR and limma packages. In parallel, genomic DNA was subjected to DNA methylation profiling using an array chip, with differentially methylated probes (DMPs) and regions (DMRs) identified using the Minfi workflows.
DGE analysis identified significant upregulation and downregulation of genes in OCs compared to PBMCs. Our DGE analysis confirmed the upregulation of several well-established osteoclast-related genes, including CTSK, DCSTAMP, ACP5, SNX10, and ATPV0D2. Correspondingly, we observed significant methylation changes in the promoter regions of CTSK, ACP5, and SNX10, suggesting epigenetic regulation of these genes in OCs. Additionally, we noted methylation changes in DCSTAMP and ATPV0D2. These findings highlight the interplay between gene expression and methylation in regulating osteoclast-specific genes.
Pathway analysis of upregulated DGEs revealed association with metabolic pathways, lysosomes, and focal adhesion, with metabolic pathways most significantly associated with hypomethylation. Our study provides new insights into gene regulation and expression dynamics during RANKL-induced osteoclast differentiation. An integrated osteoclast transcriptome and methylome will be useful for informing future functional studies of genes related to osteoclast biology and bone pathology.