Molecular cloning, structure, expression, and chromosomal localization of the human Osterix (SP7) gene

Abstract

We report the isolation of the human orthologue of the mouse Osterix (Osx/Sp7) gene, a C2H2 zinc finger transcription factor of the SP gene family and putative “master” regulator of bone cell differentiation. The human SP7 cDNA encodes a putative 431 amino acid protein that contains three consecutive C2H2 zinc finger repeats. The SP7 protein is highly conserved between mice and humans with an overall sequence identity of 95%. The expression of a SP7 mRNA transcript of approximately 3.2 kb is restricted to bone-derived cell lines in vitro but undetectable in any adult tissues including mandibular bone by Northern blot hybridization. The specific expression of SP7 mRNA in osteoblasts in vivo was further confirmed by in situ hybridization on human embryonic tissues. The highly restricted expression pattern and the divergence of the sequence outside of the zinc finger region distinguish SP7 as a unique member of the SP family. The SP7 gene consists of two exons, with exon 2 containing most of the protein coding sequence. The gene locus was mapped to chromosome 12q13.13 by fluorescent in situ hybridization (FISH). The identification and initial characterization of the SP7 gene will facilitate the study of the molecular regulation of osteoblast differentiation in humans.

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.