Figure 5.

PU.1 has a direct role in leading DNA methylation changes at their targets. (A) Scheme depicting the two regions of the SPI1 gene (PU.1) (exon 2 and 3′UTR) targeted by the two siRNAs used in this study. (B) Effects of siRNA experiments on PU.1 levels at 1, 2, 4, and 6 days as analyzed by qRT-pCR. (C) Effects of siRNA experiments on PU.1 levels at 1, 2, 4, and 6 days as analyzed by western blot (D) Effects of PU.1 downregulation on expression and methylation of PU.1-target genes that become demethylated (ACP5, CTSK), genes that become hypermethylated (CX3CR1, NR4A2) and non-pU.1 target genes, PLA2G4E, which becomes also overexpressed and demethylated, and FSCN3, which is hypermethylated and does not undergo loss of methylation during osteoclastogenesis. (E) ChIP assays showing the effects of PU.1 downregulation in its recruitment, together with TET2 and DNMT3b binding to the same genes. Data were obtained at 0, 2, and 6 days after M-CSF /RANL stimulation. To simplify the representation negative control assays with IgG for each time point have been substracted to the experiments with each antibody. We have used the MYOD1 promoter as a negative control (data without substracting the background is presented in Additional file 10). The experiment was performed with three biological triplicates but only one experiment is shown. Error bars correspond to technical replicates. Some of them are smaller than the data point icon. T-student test was performed: * corresponds to P value <0.05; ** means P value <0.01; *** means P value <0.001.