Localization of 5' distal enhancer-like region and identification of trans-acting factors in human toll like receptor 4 gene / 解放军医学杂志

ABSTRACT

Objective To further localize 5' distal enhancer-like region of the Toll-like receptor 4 (TLR4) gene, and identify the trans-acting factors involved in its transcriptional regulation. Methods The molecular cloning technique and reporter gene analysis were used to analyze the effects of different regions of TLR4 gene 5' flank on its transcriptional activity. The pGL-3 vectors with TLR4 gene 5' flank -1337 to -683bp from transcriptional start site and their deletants were constructed and transferred into K562 cell line by liposomal transfection method. The instant luciferase expression of different clones was detected and the effects of the deletants on the transcriptional activity of TLR4 gene were evaluated. The electrophoretic mobility shift analysis and transcription factor database search were used to locate and identify the trans-acting factors. Results A 245 bp enhancer-like region was further identified in the range of -923 to -679bp from the TLR4 gene transcriptional start site, and -923 to -742 and -721 to -679 regions were refined as two functional units. The activator protein 1 (AP1) was confirmed as a trans-acting factor in the -721 to -679 functional units, and lymphoid enhancer-binding factor (LEF-1) may be another one. Conclusion AP1 acts as a trans-acting factor interacting with -923 to -679 region of TLR4 5' flank, and plays an enhancer-like role together with LEF-1.