Cloning, sequencing and activity analysis of human Dmp1 promoter

Objectives: To clone and sequence human dentin matrix protein 1 (Dmp1) promoter and to investigate luciferase activity of different segments of this promoter in human dental pulp stem cells (HDPSCs). Methods: The desired upstream DNA segment about 2.2 kb was obtained from human genomic DNA by PCR method. The positive clone was sequenced after it was inserted into PKey-TA vector and its correct sequence analyzed by biological information methods. The amplified promoter segments with different lengths were constructed into luciferase report gene vector pGL3-Basic, the correct orientation of those inserts was verified by cutting with two different restrict enzymes. The luciferase activities were observed after different pGL3-PDmp1 vectors were transfected transiently into HDPSCs. Results: The sequence we cloned was as long as 2195bp and it was consistent with sequence location of proper human genome that displayed on Pubmed. Promoter prediction and Matinspector software analytic results showed that there were many active regions and varied nuclear factors such as Smads, Tcf/Lef-1, Cbfá1, Meis1, Msx, NFs and so on in this obtained promoter region. The recombinants of pGL3-Basic of promoter segments with different lengths were successfully constructed and identified. They had different luciferase activities when they were transfected transiently into HDPSCs. The luciferase activities of pGL3-P-505~+86 was strongest (the mean relative fluorescence 164.23) and the difference of pGL3-P-193~+86 and pGL3-P-505~+86 was most distinguished, so the region of -505~-193 bp could be regarded as the core promoter of Dmp1 promoter. Conclusions: We had obtained the correct clone of upstream promoter sequence of human Dmp1, and promoter segments with different lengths in report gene had different luciferase activities in HDPSCs. These results will make an important basis for studying transcriptional regulation mechanisms of Dmp1 during dentinogenesis.