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

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.

Restricted TET2 Expression in Germinal Center Type B Cells Promotes Stringent Epstein-Barr Virus Latency

“…Although PU.1 is required at the first stage of B lymphoid development, and its expression level determines whether lymphoid progenitors differentiate into the myeloid or B cell lineage (44), PU.1 also functions as a tumor suppressor during later stages of B cell development and is often turned off in certain B cell malignancies (45,46). Given the previous findings that TET2 is required for efficient PU.1 activation of genes (39), loss of TET2 expression in B cell malignancies may serve as another mechanism to inhibit PU.1 activity.…”

“…Intriguingly, both PU.1 and EBF-1 were also recently shown to directly interact with TET2, but not the other TET enzymes (21). PU.1 targets TET2 to PU.1 sites in cellular promoters of genes required for osteoclast differentiation, and this interaction results in the promoters becoming 5hmC modified, and then demethylated, during osteoclast differentiation (39). Although the PU.1 DNA binding consensus motif does not contain a CpG motif, the interaction between TET2 and PU.1 results in demethylation of surrounding CpGs that inhibit promoter transcription when methylated (39).…”

“…PU.1 targets TET2 to PU.1 sites in cellular promoters of genes required for osteoclast differentiation, and this interaction results in the promoters becoming 5hmC modified, and then demethylated, during osteoclast differentiation (39). Although the PU.1 DNA binding consensus motif does not contain a CpG motif, the interaction between TET2 and PU.1 results in demethylation of surrounding CpGs that inhibit promoter transcription when methylated (39). Thus, it is likely that the PU.1/EBNA2 complex bound to the LMP1p induces activation of the promoter by recruiting TET2 and inducing demethylation of the promoter.…”

The Ground State of Innate Immune Responsiveness Is Determined at the Interface of Genetic, Epigenetic, and Environmental Influences

“…However, mature myeloid cells do not entirely fit a typical developmental model of progressive epigenetic silencing: human monocytes are remarkably fluid in epigenetic terms, given observations that loss of methylation occurs during differentiation to macrophages, dendritic cells (33), or osteoclasts, in a TET2-dependent manner ( Fig. 1) (34). Polarization of both mouse and human macrophages requires global loss of DNA methylation, as well as nucleosome remodeling at promoters and enhancers, to reactivate genes that would be otherwise silent.…”

Mutant IDH : A Targetable Driver of Leukemic Phenotypes Linking Metabolism, Epigenetics and Transcriptional Regulation

“…C/EBPa is a transcriptional activator of TET2, and transcriptional activation is associated with the acquisition of 5hmC and loss of 5mC at TET2 protein target gene promoters [58]. TET2 can directly interact with the transcription factors PU.1 [59] and WT1 [26,41] and likely cooperates to regulate their target genes. Thus, 2-HG inhibition of TET2 activity could result in aberrant gain of 5mC at both PU.1 and WT1 target gene promoters resulting in gene transcription repression.…”