FIGURE 3.

Binding of WDR5 and UTX is accompanied by H3K4me3 deposition, H3K27me3 demethylation, and transcriptional activation of the Runx2 gene in osteoblasts. C2C12 cells were differentiated to the osteoblast lineage by incubating with 300 ng/ml BMP-2. pC cells were infected with lentiviral particles coding for shRNAs against WDR5 (A, C, D, G, and H) and UTX (B, E, and F). Effective down-regulation of each chromatin modifier was confirmed by Western blot analyses 72 h post infection. TFIIB or RNA-polymerase II protein levels were used as loading controls. Runx2-II/p57 and osteocalcin (Oc/Bglap) mRNA levels were measured by qRT-PCR 72h after infection (and 48 h of differentiation) and normalized against GAPDH mRNA. ChIP assays were performed using antibodies against WDR5 and UTX proteins or against H3K4me3 and H3K27me3 histone marks. Results are expressed as % Input ± S.E., using normal IgG as specificity control. Statistical analyses were performed with respect to the cells infected with virus generated with the pLKO.1 empty vector (E.V). **p < 0.01; ***p < 0.001.