Identification Of ARTS Gene Promoter And The Effect Of Sp1 On Its Promoter Activity

Objective To identify the promoter of human ARTS gene and analyze the effect of Sp1 on its promoter activity.Methods1. In order to identify putative promoter region and transcription factor binding sites, the 3kb upstream sequence of the ARTS gene closed to translation initiation sites was analyzed using bioinformatics analysis. The luciferase reporter plasmids were generated and designated based on their variable 5’ end, including full length promoter, series of deletion promoter and mutation of the transcription factor binding sites constructs.2. The luciferase activity of these plasmids were measured using the Dual-Luciferase Reporter Assay system according to the manufacturer’s instructions. The proximal promoter and Sp1 binding sites were found by comparing the expression activity of different deletion constructs. To determine the contribution of the Sp1 binding sites to the gene regulation, ChIP assays and point mutation were performed.Results1. With software analysis, we found that the putative promoter region of ARTS gene is located at the-2045 bp to +263bp of the translational start site. Using the programs of Matinspector and TFSEARCH, we failed to find any canonical TATA box or CAAT box. However, four putative Sp1 transcription factor binding sites were identified within the promoter region. Using luciferase reporter gene, we tested the transcriptional activity of this fragment in LX-2 and HEK293 T cells, and found this region has promoter activity.2. In order to determine the region required for basal activity of the promoter, a series of deletion constructs were generated and designated as p GL3-A2, pGL3-A3, pGL3-A4, p GL3-A5 based on their variable 5’ end. These plasmids were tested for their ability to drive the luciferase reporter gene in transiently transfected LX-2 and HEK293 T cells. Comparing the expression activity of different deletion constructs, the basal promoter has been located at-824 bp and-5bp.3. In ChIP analysis using anti-Sp1 antibodies, DNA fragments from-735 bp to-718 bp and-173 bp to-157 bp can be detected from co-immunoprecipitated chromatin complex. This result confirmed this region can specificly bind to Sp1.4. To determine the contribution of the Sp1 sites to the promoter activity, constructs with either single mutant reporters were generated. The luciferase activity assay showed mutation of each Sp1 binding site led to a significant decrease in ARTS promoter activity.Conclusions These results indicate that the proximal promoter of human ARTS gene has been located at-824 bp and-5bp. Both Sp1 binding sites at-735 bp to-718 bp and-173 bp to-157 bp of human ARTS promoter contribute to the basal activity of human ARTS gene transcription.