Objective: To identify the enhancers of human lung specific X protein (LUNX) and their regulation at the transcription level in vitro.
Methods: Three enhancer fragments (E1:+3770~+3959bp; E2: +6454~+6555bp; E3: +14553~+14652 bp) predicted by bioinformatics software were isolated from the human genomic DNA by PCR amplification. Luciferase assay was performed to detect the activities of the enhancers in transcriptional regulation.
Results: PCR products were confirmed by DNA sequencing. The amplified enhancers digested by Kpn I/Xho I and BamH I/Sal I, to generate the sticky-end fragments were inserted into PGL3-promoter in a reporter vector, and 6 luciferase expression vectors were obtained. All the reporter plasmids and pGL3-promoter were transiently transfected into HEK293 cells with an internal control of pSV-β-Galactosidase reporter vector. The enhancer activity of each construct was evaluated by luciferase assay of the cell extracts after transfection for 48 h. The results showed that the 3 fragments, when located upstream, did not increase transcription of reporter gene, but when at the downstream, E1 and E3 increased the transcription by 2.83 and 1.59 folds of that of pGL3-promoter, respectively.
Conclusion: LUNX gene sequences from +3770 to +3959 bp and +14553 to +14652 bp possess the capacity to enhance gene transcription.