Multiple transcriptional elements in the avian type X collagen gene. Identification of Sp1 family proteins as regulators for high level expression in hypertrophic chondrocytes.
During the cartilage-to-bone transition, participating chondrocytes eventually undergo hypertrophy and are replaced by bone and marrow. Type X collagen is synthesized by chondrocytes specifically when they become hypertrophic, and this specificity is primarily regulated at the level of transcription. Previously, we demonstrated that a proximal promoter region from nucleotide -562 to +86 contained cis-acting elements that directed high level expression of a reporter gene in a cell-specific manner (Long, F., and Linsenmayer, T. F. (1995) J. Biol. Chem. 270, 31310-31314). In the present study, we have further dissected this region by generating a series of constructs and examining their expression in hypertrophic versus nonhypertrophic chondrocytes. Several positive and negative elements have been delineated within the proximal promoter region to mediate the regulation of transcription in hypertrophic chondrocytes. Most notably, a sequence from nucleotide -139 to +5 was sufficient to direct high level expression in this cell type. Electrophoresis mobility shift assay and supershift experiments identified within this sequence two 10-base pair noncanonical binding sites for Sp1 proteins. Mutations within the Sp1 binding sites either diminished or abolished the expression driven by the sequence from -139 to +5. These results indicate that the Sp1 proteins mediate the cell-specific expression of type X collagen.
Long
F
F
Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Sonenshein
G E
GE
Linsenmayer
T F
TF
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002356:Cartilage / Q000166:cytology / Q000196:embryology*; D002642:Chick Embryo; D019902:Chondrocytes / Q000502:physiology*; D003094:Collagen / Q000235:genetics*; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D005091:Exons; D018507:Gene Expression Regulation, Developmental; D006984:Hypertrophy / Q000235:genetics; D007438:Introns; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D009687:Nuclear Proteins; D016329:Sp1 Transcription Factor; D003094:Collagen