FIG. 10.

siRNA knockdown of Hoxa10 decreases histone acetylation and H3K4 methylation of osteogenic genes. (A) MC3T3 cells were treated with Hoxa10 siRNA and nonspecific control (NS) for 72 h. The knockdown effect is shown by Western blotting. (B) DNA samples from ChIP with HOXA10, nonspecific IgG, methylated histone K4 (H3K4), and acetylated histone H4 (ACH4) were amplified by gene-specific (Sp) and 3′ control UTR primers (Table 1) for the indicated gene promoters. The left panel for each gene shows the effect of Hoxa10 siRNA on HOXA10 recruitment in ChIP assay of the indicated promoter. The right panel shows the status of H3K4 methylation or H4 acetylation of the chromatin modification by Hoxa10-specific knockdown. (C) ChIP-reChIP assays were performed to identify the association of coregulatory factors p300 and CBP with HOXA10 on the Runx2 and OC promoters. A HOXA10 ChIP was performed with HOXA10 antibody, and the resulting chromatin immunoprecipitate (second input) was subjected to reChIP with the indicated antibodies (α) (secondary pull-down). Normal IgG and green fluorescent protein (GFP) antibodies provided controls. The DNA fragments for Runx2 and OC were amplified as described in Materials and Methods. (D) Schematic illustration to show how HOXA10 belongs to an epigenetic coregulatory complex for remodeling chromatin to induce transcription of osteogenic genes. We propose that HOXA10 may be among the earliest factors recruited to bone promoters. HOXA10 recruitment is followed by the coordinated occupancy of other bone-related transcription factors for maximal expression of individual genes during stages of differentiation. HD, homeodomain.