Fig 1.
Identification of the putative ARTS promoter.
(A) Schematic representation of the human Sept4 gene features. The distinct transcript variants of human Sept4 gene were obtained and analyzed using NCBI Epigenomics. The structure of the putative ARTS promoter region (from -2045 bp to +263 bp), containing a CpG island. (B) Schematic representation of the ARTS promoter deletion constructs generated in the pGL3-basic vector. (C) The relative luciferase activity of pGL3-A1 in HEK-393T and LX-2 cell lines was detected by luciferase reporter assay. *P<0.05, as compared with the relative luciferase activity of pGL3-basic vector. (D) The endogenous expression of ARTS (32 kDa) in HEK-393T and LX-2 cell lines was detected by western blot. (E) The relative luciferase activities of the deletion constructs in both HEK-393T and LX-2 cell lines were observed by luciferase reporter assay. *P<0.05, as compared with the relative luciferase activity of pGL3-A1 in each cell line. #P<0.05, as compared with the relative luciferase activity of pGL3-A4 in each cell line.
Fig 2.
Putative transcription factor binding sites in ARTS promoter.
The binding sites in the region from -824 to -5 bp of the promoter were screened using TFSEARCH and MatInspector. Boxed sequences indicate the predicted sites.
Fig 3.
Sp1 protein binds to Sp1 binding sites in ARTS promoter.
(A) The schematic representation of the position of four putative Sp1 binding sites in the human ARTS promoter region (-824 bp to -5 bp). (B) The specific binding of Sp1 protein to the putative Sp1 binding sites in the ARTS promoter region was analyzed by ChIP. Protein/DNA complexes from the sonicated lysates of HEK-293T cells were immunoprecipitated with anti-Sp1 antibody with rabbit IgG used as a control. The results showed that the expression of Sp1 could be detected at the putative binding sites of -735/-718 and -173/-157, but not -434/-418 and -23/-6.
Fig 4.
Sp1 binding sites are required for ARTS promoter activation.
(A) The schematic representation of the mutation project. The constructs were then transiently transfected into HEK293 cells (B) and LX-2 cells (C) and promoter activities were determined by luciferase assay. The activities for the promoter of M1 and M2 plasmids were reduced in both cell lines, compared with the activity of the wild-type plasmid (*P<0.05).