Regulation of the Cell Type-specific dentin sialophosphoprotein gene expression in mouse odontoblasts by a novel transcription repressor and an activator CCAAT-binding factor.
Dentin sialophosphoprotein (DSPP) is an extracellular matrix protein that is cleaved into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) with a highly restricted expression pattern in tooth and bone. Mutations of the DSPP gene are associated with dentin genetic diseases. Regulation of tissue-specific DSPP expression has not been described. To define the molecular basis of this cell-specific expression, we characterized the promoter responsible for the cell-specific expression of the DSPP gene in odontoblasts. Within this region, DNase I footprinting and electrophoretic mobility shift assays delineated one element that contains an inverted CCAAT-binding factor site and a protein-DNA binding site using nuclear extracts from odontoblasts. A series of competitive electrophoretic mobility shift assay analyses showed that the protein-DNA binding core sequence, ACCCCCA, is a novel site sufficient for protein binding. These two protein-DNA binding sequences are conserved at the same proximal position in the mouse, rat, and human DSPP gene promoters and are ubiquitously present in the promoters of other tooth/bone genes. Mutations of the CCAAT-binding factor binding site resulted in a 5-fold decrease in promoter activity, whereas abolishment of the novel protein-DNA binding site increased promoter activity by about 4.6-fold. In contrast to DSPP, expression levels of the novel protein were significantly reduced during odontoblastic differentiation and dentin mineralization. The novel protein was shown to have a molecular mass of 72 kDa. This study shows that expression of the cell type-specific DSPP gene is mediated by the combination of inhibitory and activating mechanisms.
Department of Pediatric Dentistry, Dental School, UNiversity of Texas, San Antonio, TX 78229, USA.
Shuo
S
Chen
Aaron
A
Unterbrink
Sheela
S
Kadapakkam
Juan
J
Dong
Ting Ting
TT
Gu
Julie
J
Dickson
Hui-Hsiu
HH
Chuang
Mary
M
MacDougall
D000818:Animals; D001665:Binding Sites; D023081:CCAAT-Binding Factor / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D017124:Conserved Sequence; D003804:Dentin / Q000378:metabolism; D016326:Extracellular Matrix Proteins; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009804:Odontoblasts / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D010750:Phosphoproteins; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D012097:Repressor Proteins / Q000502:physiology*; D012795:Sialoglycoproteins; D014158:Transcription, Genetic; D014162:Transfection; D000099311:Dentin Sialophosphoprotein
D023081:CCAAT-Binding Factor; D016326:Extracellular Matrix Proteins; D010750:Phosphoproteins; D011498:Protein Precursors; D012097:Repressor Proteins; D012795:Sialoglycoproteins; D000099311:Dentin Sialophosphoprotein