Figure 4.

Interactions between PU.1 and DNTM3b and TET2 and association with promoters undergoing DNA methylation changes. (A) Quantitative RT-PCR analysis for CTSK, PU.1, p65 NF-kB, TET1, TET2, and DNMT3B during osteoclastogenesis. (B) Western blot for the same factors indicated above. (C) Immunoprecipitation experiment of p65 and PU.1 with DNMT3B and TET2 at 0, 2, and 4 days after RANKL and M-CSF stimulation. IgG used as a negative control. Reciprocal immunoprecipitation experiments in the bottom panel. (D) Quantitative ChIP assays showing PU.1, TET2, and DNMT3b binding to hypomethylated genes (ACP5, TM7SF4, TM4SF19) and hypermethylated genes (CX3CR1, NR4A2), all direct PU.1 targets, and a negative control (MYOD1 promoter) without PU.1 target sites. The experiment was performed with three biological triplicates but only one experiment is shown. T-student test comparing binding of each antibody between 0 days vs. 2 days was performed: * corresponds to P value <0.05; ** means P value <0.01; *** means P value <0.001. (E) Examples showing PU.1 binding (from ChIPseq data, GSE31621) to the region neighboring hypo- and hypermethylated CpGs. The PU.1 binding motif location is presented as a horizontal blue dot and the CpG displaying differential methylation (Illumina probe) between MO and OC is marked with a red bar. (F) Analysis of ChIPseq analysis for PU.1 and comparison to TRANSFAC predictions. Top panel: proportion of the CpG-containing probes displaying DNA methylation changes that have peaks for PU.1 binding in the same 500-bp window. Diagrams are separated in the hypo- and hypermethylated sets and in promoter and distal regions (gene bodies, 3′UTR, and intergenic regions). Bottom panel, Venn diagrams showing the overlap of PU.1 targets from ChIPseq data (GEO accession number: GSE31621) in MOs and TRANSFAC prediction for PU.1, both using a window of 500 pb centered by the CpG displaying significant methylation changes.