Pbx1 Represses Osteoblastogenesis by Blocking Hoxa10-Mediated Recruitment of Chromatin Remodeling Factors

Abstract

Abdominal-class homeodomain-containing (Hox) factors form multimeric complexes with TALE-class homeodomain proteins (Pbx, Meis) to regulate tissue morphogenesis and skeletal development. Here we have established that Pbx1 negatively regulates Hoxa10-mediated gene transcription in mesenchymal cells and identified components of a Pbx1 complex associated with genes in osteoblasts. Expression of Pbx1 impaired osteogenic commitment of C3H10T1/2 multipotent cells and differentiation of MC3T3-E1 preosteoblasts. Conversely, targeted depletion of Pbx1 by short hairpin RNA (shRNA) increased expression of osteoblast-related genes. Studies using wild-type and mutated osteocalcin and Bsp promoters revealed that Pbx1 acts through a Pbx-binding site that is required to attenuate gene activation by Hoxa10. Chromatin-associated Pbx1 and Hoxa10 were present at osteoblast-related gene promoters preceding gene expression, but only Hoxa10 was associated with these promoters during transcription. Our results show that Pbx1 is associated with histone deacetylases normally linked with chromatin inactivation. Loss of Pbx1 from osteoblast promoters in differentiated osteoblasts was associated with increased histone acetylation and CBP/p300 recruitment, as well as decreased H3K9 methylation. We propose that Pbx1 plays a central role in attenuating the ability of Hoxa10 to activate osteoblast-related genes in order to establish temporal regulation of gene expression during osteogenesis.

We are grateful to A. Imbalzano for advice during these studies. We also thank Judy Rask for editorial assistance, members of the laboratory for helpful discussions, and Sadiq Hussain for technical support.

The studies reported here were supported in part by grants from the National Institutes of Health (DE12528, AR39588, and AR48818). Core resources supported by Diabetes Endocrinology Research Center (DERC) grant DK32520 from the National Institute of Diabetes and Digestive and Kidney Diseases were used; Jane Lian and Gary Stein are members of the University of Massachusetts DERC (DK32520).

The contents of this work are solely our responsibility and do not necessarily represent the official views of the National Institutes of Health.