title: PU.1 target genes undergo Tet2-coupled  demethylation and DNMT3b-mediated  methylation in monocyte-to-osteoclast  differentiation.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: de la Rica
authors.forename: Javier
authors.initials: J
authors.lastname: Rodríguez-Ubreva
authors.forename: Mireia
authors.initials: M
authors.lastname: García
authors.forename: Abul B M M K
authors.initials: AB
authors.lastname: Islam
authors.forename: José M
authors.initials: JM
authors.lastname: Urquiza
authors.forename: Henar
authors.initials: H
authors.lastname: Hernando
authors.forename: Jesper
authors.initials: J
authors.lastname: Christensen
authors.forename: Kristian
authors.initials: K
authors.lastname: Helin
authors.forename: Carmen
authors.initials: C
authors.lastname: Gómez-Vaquero
authors.forename: Esteban
authors.initials: E
authors.lastname: Ballestar
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D001665:Binding Sites; D002454:Cell Differentiation; D018899:CpG Islands; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D049308:Dioxygenases; D044127:Epigenesis, Genetic; D006801:Humans; D009000:Monocytes / Q000166:cytology / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D000097584:DNA Methyltransferase 3B; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D044503:5-Methylcytosine; D003596:Cytosine; D004248:DNA (Cytosine-5-)-Methyltransferases; D004268:DNA-Binding Proteins; D049308:Dioxygenases; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D000097584:DNA Methyltransferase 3B; D000099232:Proto-Oncogene Protein Spi-1; C011865:5-hydroxymethylcytosine; C541081:TET2 protein, human
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