Abstract
BackgroundDNA methylation is a key epigenetic mechanism for driving and stabilizing cell-fate decisions. Local deposition and removal of DNA methylation are tightly coupled with transcription factor binding, although the relationship varies with the specific differentiation process. Conversion of monocytes to osteoclasts is a unique terminal differentiation process within the hematopoietic system. This differentiation model is relevant to autoimmune disease and cancer, and there is abundant knowledge on the sets of transcription factors involved.ResultsHere we focused on DNA methylation changes during osteoclastogenesis. Hypermethylation and hypomethylation changes took place in several thousand genes, including all relevant osteoclast differentiation and function categories. Hypomethylation occurred in association with changes in 5-hydroxymethylcytosine, a proposed intermediate toward demethylation. Transcription factor binding motif analysis revealed an over-representation of PU.1, NF-κB, and AP-1 (Jun/Fos) binding motifs in genes undergoing DNA methylation changes. Among these, only PU.1 motifs were significantly enriched in both hypermethylated and hypomethylated genes; ChIP-seq data analysis confirmed its association to both gene sets. Moreover, PU.1 interacts with both DNMT3b and TET2, suggesting its participation in driving hypermethylation and hydroxymethylation-mediated hypomethylation. Consistent with this, siRNA-mediated PU.1 knockdown in primary monocytes impaired the acquisition of DNA methylation and expression changes, and reduced the association of TET2 and DNMT3b at PU.1 targets during osteoclast differentiation.ConclusionsThe work described here identifies key changes in DNA methylation during monocyte-to-osteoclast differentiation and reveals novel roles for PU.1 in this process.
Highlights
DNA methylation is a key epigenetic mechanism for driving and stabilizing cell-fate decisions
Cell differentiation and fusion in osteoclastogenesis are accompanied by hypomethylation and hypermethylation of key functional pathways and genes To investigate the acquisition of DNA methylation changes during monocyte-to-osteoclast differentiation we first obtained three sets of matching samples corresponding to MOs (CD14+ cells) from peripheral blood and OCs derived from the same CD14+ cells, 21 days after the addition of M-CSF and RANKL
The quality of mature, bone-resorbing OCs obtained under these conditions was confirmed by several methods, including the presence of more than three nuclei in tartrate-resistant acid phosphatase (TRAP)-positive cells, the upregulation of OC markers, such as carbonic anhydrase 2 (CA2), cathepsin K (CTSK), ACP5/TRACP, and matrix metalloproteinase 9 (MMP9), and downregulation of the monocytic gene CX3CR1 (Additional file 1)
Summary
DNA methylation is a key epigenetic mechanism for driving and stabilizing cell-fate decisions. Signals end in the activation of NF-kB, MAPK, and c-Jun, leading to the activation of NFATc1 [15], the master transcription factor of osteoclastogenesis, together with PU. and MITF [16], which is already present in the progenitors. These transcription factors bind to the promoter and help upregulating OC markers such as dendritic cell-specific transmembrane protein (DC-STAMP/TM7SF4) [17], tartrate-resistant acid phosphatase (TRACP/ACP5) [18], cathepsin K (CTSK) [19], matrix metalloproteinase 9 (MMP9) [20], and carbonic anhydrase 2 (CA2)
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