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PU.1 target genes undergo Tet2-coupled demethylation and DNMT3b-mediated methylation in monocyte-to-osteoclast differentiation

Lorenzo de la Rica1, Javier Rodríguez-Ubreva1, Mireia García2, Abul BMMK Islam34, José M Urquiza1, Henar Hernando1, Jesper Christensen5, Kristian Helin5, Carmen Gómez-Vaquero2 and Esteban Ballestar1*

Author Affiliations

1 Chromatin and Disease Group, Cancer Epigenetics and Biology Programme (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Barcelona 08908, Spain

2 Rheumatology Service, Bellvitge University Hospital (HUB), L’Hospitalet de Llobregat, Barcelona 08908, Spain

3 Department of Experimental and Health Sciences, Barcelona Biomedical Research Park, Universitat Pompeu Fabra (UPF), Barcelona 08003, Spain

4 Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka 1000, Bangladesh

5 Biotech Research and Innovation Center (BRIC), Center for Epigenetics University of Copenhagen, Ole Maaløes Vej 5, Copenhagen 2200, Denmark

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Genome Biology 2013, 14:R99  doi:10.1186/gb-2013-14-9-r99

Published: 12 September 2013



DNA 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.


Here 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.


The work described here identifies key changes in DNA methylation during monocyte-to-osteoclast differentiation and reveals novel roles for PU.1 in this process.