Dlx3 Transcriptional Regulation of Osteoblast Differentiation: Temporal Recruitment of Msx2, Dlx3, and Dlx5 Homeodomain Proteins to Chromatin of the Osteocalcin Gene

Abstract

Genetic studies show that Msx2 and Dlx5 homeodomain (HD) proteins support skeletal development, but null mutation of the closely related Dlx3 gene results in early embryonic lethality. Here we find that expression of Dlx3 in the mouse embryo is associated with new bone formation and regulation of osteoblast differentiation. Dlx3 is expressed in osteoblasts, and overexpression of Dlx3 in osteoprogenitor cells promotes, while specific knock-down of Dlx3 by RNA interference inhibits, induction of osteogenic markers. We characterized gene regulation by Dlx3 in relation to that of Msx2 and Dlx5 during osteoblast differentiation. Chromatin immunoprecipitation assays revealed a molecular switch in HD protein association with the bone-specific osteocalcin (OC) gene. The transcriptionally repressed OC gene was occupied by Msx2 in proliferating osteoblasts, while Dlx3, Dlx5, and Runx2 were recruited postproliferatively to initiate transcription. Dlx5 occupancy increased over Dlx3 in mature osteoblasts at the mineralization stage of differentiation, coincident with increased RNA polymerase II occupancy. Dlx3 protein-DNA interactions stimulated OC promoter activity, while Dlx3-Runx2 protein-protein interaction reduced Runx2-mediated transcription. Deletion analysis showed that the Dlx3 interacting domain of Runx2 is from amino acids 376 to 432, which also include the transcriptionally active subnuclear targeting sequence (376 to 432). Thus, we provide cellular and molecular evidence for Dlx3 in regulating osteoprogenitor cell differentiation and for both positive and negative regulation of gene transcription. We propose that multiple HD proteins in osteoblasts constitute a regulatory network that mediates development of the bone phenotype through the sequential association of distinct HD proteins with promoter regulatory elements.

Cationic Nanogel-mediated Runx2 and Osterix siRNA Delivery Decreases Mineralization in MC3T3 Cells

“…Mid-and late-stage osteogenic markers including OPN, bone sialoprotein (BSP), and OCN are temporal successors to RUNX2 and OSX expression after rhBMP-2 stimulation [63]. The complex temporal expression profiles of OPN, BSP, and OCN have been elucidated [15,16,23,25,26,30,53,59,66]; therefore, a coordinated, temporal RNAi treatment cycle may enhance the duration and intensity of gene silencing and prevent HA deposition in vitro. Consequently, the evolution of RNAi therapeutics must match RNAi targets to their expression profiles.…”

Expression of the dlx gene family during formation of the cranial bones in the zebrafish (Danio rerio): Differential involvement in the visceral skeleton and braincase

“…Krauss and Lufkin (1999) have shown Dlx5 to be expressed in the perichondral region of all the developing fetal skeletal elements starting as early as cartilage initiation and continuing through the period of mineralization. In mice, Dlx5 occupancy increased over Dlx3 in mature osteoblasts at the mineralization stage of differentiation (Hassan et al, 2004).…”

“…Dlx3 is expressed in the osteoblasts, and overexpression of this gene in osteoprogenitor cells promotes, whereas specific knockdown of Dlx3 by RNA interference inhibits, induction of osteogenic markers (Hassan et al, 2004). In accordance with this result, we find strong expression of dlx3b during the different stages of ossification in almost all bones investigated: condensation of mesenchymal cells (the osteoprogenitors)/differentiation of osteoprogenitors into osteoblasts, synthesis and secretion of extracellular matrix by the osteoblasts, and matrix mineralization (Table 2A,B).…”

A 4 bp deletion mutation in DLX3 enhances osteoblastic differentiation and bone formation in vitro

“…[9][10][11][12] DLX3 is expressed in osteoblasts, and over-expression of DLX3 in osteoprogenitor cells promotes the induction of osteoblastic differentiation markers such as type 1 collagen, bone sialoprotein, osteocalcin, and alkaline phosphatase. [13] Chromatin immunoprecipitation assays have revealed a DLX3 binding element in the proximal promoter region of the osteocalcin (OC) gene. Transcriptional repression of the OC gene is controlled by MSX2 in proliferating osteoblasts.…”

“…DLX3, DLX5, and Runx2 are recruited in the differentiated osteoblast to initiate transcription of the OC gene, demonstrating that in addition to DLX5 and Runx2, DLX3 is also important in osteoblast proliferation and differentiation. [13] A 4 bp deletion mutation in the DLX3 gene is associated with Tricho-Dento-Osseous syndrome (TDO), [14][15][16] an autosomal dominant condition characterized by variable clinical expression of kinky/curly hair, taurodontism, thin enamel and enhanced bone thickness. Increased density and thickness of cranial bone, distal radius/ulna, femoral neck, and lumbar spine in TDO [17][18][19] suggest that DLX3 is important in remodeling and homeostasis of skeletal bone and that this DLX3 gene mutation affects both endochondral and intramembranous bone development.…”