Identification of Tooth-Specific Enhancer Element of Human Dentin Matrix Protein 1 (DMP1) Gene by Transgenic Assays
Date Issued
2009
Date
2009
Author(s)
Chen, Wen-Yu
Abstract
Bones and teeth are two major mineralized tissues in vertebrate. In the process of osteogenesis and dentinogenesis, osteoblasts and odontoblasts secrete non-collagenous proteins to form extracellular matrix (ECM). One group of the non-collagenous proteins is SIBLING family (SIBLING family; Small-Integrin-Binding Ligand, N-linked Glyco- protein). DMP-1 (Dentin Matrix Protein 1; DMP-1) is a member of the family. The members of SIBLING family share many common characteristics. For example, they have sialic acids, phosphates, and RGD sequence. Such acidic phosphorylated proteins will secrete to ECM to incorporate with hydroxyapatite, helping nucleation of hydroxyapatite crystal growth and mineralization.reliminarily, upstream 2,783 kb fragment of human DMP-1 promoter is functional by the expression of the reporter gene (EGFP, enhanced green fluorescent protein) in transgenic zabrafish and mice (Yu, 2004). The results suggest that location of the DMP-1 is not restricted to mineralized tissues and may be present in non-mineralized tissue (Lin, 2007). However, the reporter gene EGFP is not expressed in odontoblasts of teeth.ccording to this finding, I attempt to indentify tooth-specific enhancer elements of human Dentin Matrix Protein 1 (DMP1) gene. By analyzing sequences which are located in upstream and downstream of DMP-1 gene between different species from UCSC bio-informatics, I found out seven highly conserved DNA sequences. cloned the DNA fragments of these regions from HeLa cell genome, as candidate enhancers, using βB1-Crystallin 1.3 kb (Cr1.3) enhancer elements (-1290 bp ~ -62 bp), which confer tissue-specificity in lens, as a selection marker in transient transgenic experiments. Then Cr1.3 and the seven DNA fragments of the highly conserved regions were ligated with 2.783 kb human DMP-1 promoter with EGFP as a reporter gene. They were analyzed in zebrafish and mouse by transgenic assay. The constructed plasmid including E2 (Enhancer 2) was injected into both zebrafish and mouse fertilized eggs. The results demonstrated that the reporter is expressed in lens and regions of pharyngeal teeth in zebrafish embryo with E2 enhancer-construct. In addition, the reporter is expressed in lens and odontoblasts in mouse by the same construct. Taken together, the evolutionarily conserved DNA sequence E2 might provide a cis-element for rendering tooth-specificity to human DMP-1 gene.
Subjects
DMP-1
odontoblast
enhancer
zebrafish
pharyngeal teeth
EGFP
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