Epigenetic regulation of early osteogenesis and mineralized tissue formation by a HOXA10-PBX1-associated complex

Abstract

Homeodomain-containing (HOX) factors such as the abdominal class homeodomain protein HOXA10 and the TALE-family protein PBX1 form coregulatory complexes and are potent transcriptional and epigenetic regulators of tissue morphogenesis. We have identified that HOXA10 and PBX1 are expressed in osteoprogenitors; however, their role in osteogenesis has not been established. To determine the mechanism of HOXA10-PBX-mediated regulation of osteoblast commitment and the related gene expression, PBX1 or HOX10 were depleted (shRNA or genetic deletion, respectively) or exogenously expressed in C3H10T1/2, bone marrow stromal progenitors, and MC3T3-E1 (preosteoblast) cells. Overexpression of HOXA10 increased the expression of osteoblast-related genes, osteoblast differentiation and mineralization; expression of PBX1 impaired osteogenic commitment of pluripotent cells and the differentiation of osteoblasts. In contrast, the targeted depletion of PBX1 by shRNA increased the expression of bone marker genes (osterix, alkaline phosphatase, BSP, and osteocalcin). Chromatin-associated PBX1 and HOXA10 were present at osteoblast-related gene promoters preceding gene expression, but PBX1 was absent from promoters during the transcription of bone-related genes, including osterix (Osx). Further, PBX1 complexes were associated with histone deacetylases normally linked with chromatin inactivation. Loss of PBX1 but not of HOXA10 from the Osx promoter was associated with increases in the recruitment of histone acetylases (p300), as well as decreased H3K9 methylation, reflecting transcriptional activation. We propose PBX1 plays a central role in attenuating the activity of HOXA10 as an activator of osteoblast-related genes and functions to establish the proper timing of gene expression during osteogenesis, resulting in proper matrix maturation and mineral deposition in differentiated osteoblasts.

Fig. 1.

Depletion of PBX1 by shRNA results in increased expression of osteoblast-related genes in mesenchymal cells. a–c BMSC cells were infected with ∼100 plaque-forming units/cell of shRNA-encoding recombinant lentivirus (NS or Pbx1-specific target). a Relative mRNA and protein levels of PBX1 in treated BMSCs. b BMSCs treated with Pbx1-shRNA demonstrate increased alkaline phosphatase activity and mineral deposition. Scale bar = 500 μm. c Relative expression of osteoblast-related genes, monitored by RT-qPCR, was significantly increased in Pbx1-shRNA-infected BMSCs. Statistical significance was determined by 1-way ANOVA followed by a Bonferroni post hoc test. Data are presented as the means of 3 experiments ± SEM.

Fig. 2.

PBX1 displays a pattern of gene-repressive functionality on the osterix promoter. a Diagram of the osterix promoter displaying the relative binding sites and primer sites used for ChIP. BMSCs were isolated from the long bones of 6-week-old mice and collected during the proliferative stage (day 4) or cultured in differentiating conditions and collected during the increase in Osx expression (day 12). b BMSCs were analyzed at the indicated differentiation stages by RT-qPCR to determine the levels of Osx gene expression. c–e ChIP analysis was performed on cleared lysates from BMSCs using ∼5 μg of the indicated antibody. The recovered DNA was then quantified by qPCR using primers specific for the proximal promoter region of the osterix gene to determine the relative occupancy of transcriptional activators (c), activation markers (d), or repressive markers (e). BMSCs (as above) were infected with Pbx1-shRNA or NS-shRNA (NS)-lentiviral constructs (f) and the relative expression of Osx was determined by RT-qPCR. g–i ChIP analysis was performed on cleared lysates from treated BMSCs (as above) to determine the relative occupancy of chromatin modifiers. Statistical significance was determined by Student's t test (∗ p < 0.05 vs. matched control). ChIP experiments were repeated at least 2 times with similar results, and 1 representative experiment is presented ± SD.