Cloning of human LUNX gene enhancers and analysis of transcriptional regulation / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To identify the enhancers of human lung specific X protein (LUNX) and their regulation at the transcription level in vitro.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>LUNX gene sequences from +3770 to +3959 bp and +14553 to +14652 bp possess the capacity to enhance gene transcription.</p>