Molecular cloning, structure, expression, and chromosomal localization of the human Osterix (SP7) gene
Introduction
The process of cell differentiation is fundamental to organ development, maintenance, repair, and regeneration. Multipotential mesenchymal stem cells contribute to the development of several tissues and organs including cartilage, fat, muscle, and bone. The differentiation of these multipotential cells into discrete lineages, such as fibroblasts, chondroblasts, adipoblasts, myoblasts, and osteoblasts, is under strict molecular control. The discovery of several “master” regulators in pluripotent mesenchymal cells has shown that cell differentiation is largely controlled at the level of gene transcription. Thus, muscle differentiation is controlled by the helix-loop-helix transcription factors myoD, myf5, and myogenin (Perry and Rudnicki, 2000), cartilage differentiation by the high-mobility-group (HMG)-box containing transcription factors Sox9, Sox5, and Sox6 Akiyama et al., 2002, de Crombrugghe et al., 2001, and bone development is regulated by the runt-domain factor Runx2 (Komori et al., 1997). However, recent studies have demonstrated that Runx2 has a dual role in the control of both osteoblast and chondrocyte differentiation Enomoto et al., 2000, Kim et al., 1999. Recently, the zinc finger transcription factor Osterix (Osx) has been isolated in mice and shown to be a specific regulator of osteoblast differentiation acting downstream of Runx2 (Nakashima et al., 2002). During mouse embryonic development, Osx is expressed specifically in osteoblasts. Consistent with its localized expression, Osx-deficient mice show a complete lack of osteoblast differentiation and therefore no signs of either endochondral or intramembranous bone formation (Nakashima et al., 2002). Mouse Osx is a 428-amino acid protein that contains three C2H2-type zinc fingers near the C terminus. This 85 amino acid zinc finger motif has a high degree of identity with the motifs present in the murine SP family of transcription factors (Sp1 to Sp6). The gene locus for Osx has therefore been assigned the symbol Sp7.
The C2H2 zinc finger gene family is the largest class of transcriptional regulators in the mammalian genome. The number and complexity of zinc finger genes have increased with the complexity of organisms during evolution, and the human genome contains several hundred individual members of this family Bellefroid et al., 1989, Tupler et al., 2001. Zinc finger genes have been recognized as critical regulators of many fundamental biological processes (Matise and Joyner, 1999), but until recently, little was known about the role of zinc finger transcription factors in the regulation of gene expression during skeletal development and the specification of the osteoblast phenotype. We have previously identified several members of the zinc finger gene family that are involved in skeletal and mineralized tissue development Ganss et al., 2002, Ganss and Kobayashi, 2002, Jheon et al., 2001b. Due to our interest in the role of zinc finger transcription factors in skeletal and mineralized connective tissue development, we have focused our attention on the human Osterix gene, which has not been characterized. The goal of this study, therefore, was to identify and characterize a human SP7 complementary DNA (cDNA) and gene structure, to examine the expression of SP7 messenger RNA (mRNA) in human cell lines and adult and embryonic tissues, and to determine the chromosomal localization of the SP7 gene.
Section snippets
Materials and methods
All experiments were performed under approved human ethics protocols in accordance with institutional and federal guidelines.
Structure of the SP7 gene
Two positive genomic DNA restriction fragments from BAC clone RP11-680A11 (PshAI; ∼15 kb and XbaI; ∼8 kb) were identified by Southern blot hybridization. Additional restriction sites were identified by endonuclease mapping (Fig. 1). The ∼15-kb PshAI fragment was found to contain the entire SP7 open reading frame (ORF), interrupted by one intron of approximately 6.2 kb, as well as ∼5.8 kb upstream of the translation start codon ATG and approximately 450 bp of sequence downstream of the
Discussion
We have isolated a 15-kb genomic DNA fragment of the human Osterix/SP7 gene, a zinc finger transcription factor that was previously identified as a critical regulator of bone formation and osteoblast differentiation in mice (Nakashima et al., 2002). Sequencing of the genomic DNA indicated that the presence of two exons in the SP7 gene is highly conserved between mouse and human. The organization of the SP7 coding sequence into two exons is distinct from other members of the SP gene family (SP1
Acknowledgements
This project was partially supported by a research grant from the Faculty of Dentistry at the University of Toronto. The assistance from the CIHR Genome Resource Facility at the Hospital for Sick Children (Toronto) in DNA sequencing, BAC library screening, and FISH analyses is gratefully acknowledged. We also thank Dr. Jaro Sodek and Dr. Sela Cheifetz for their support and helpful comments on the manuscript.
References (27)
- et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction
Anal. Biochem
(1987) - et al.
Physical basis of a protein-DNA recognition code
Curr. Oin. Struct. Biol
(1997) - et al.
Cbfa1 is a positive regulatory factor in chondrocyte maturation
J. Biol. Chem
(2000) - et al.
Characterization of a novel KRAB/C2H2 zinc finger transcription factor involved in bone development
J. Biol. Chem
(2001) - et al.
Regulation of chondrocyte differentiation by Cbfa1
Mech. Dev
(1999) - et al.
Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts
Cell
(1997) - et al.
The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation
Cell
(2002) - et al.
The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6
Genes Dev
(2002) - et al.
The human genome contains hundreds of genes coding for finger proteins of the Kruppel type
DNA
(1989) - et al.
The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences
Electrophoresis
(1993)