FIGURE 3:

Cloning and analysis of TIS11b promoter. (A) The sequence of the human TIS11b promoter region from −1670 nucleotides upstream of the transcription start site (+1) to the translation initiation ATG was determined using Ensembl Genome Browser (www.ensembl.org). The CRE is shown in red, and the TATAA box (bold) is underlined. (B) The CRE in TIS11b promoter is conserved among species. (C) Schematic representations of TIS11b promoter region constructs. The WT construct contains 1088 base pairs of the promoter region. The mutCRE construct results from the substitution of four nucleotides within the CREB consensus sequence as described in the Supplemental Material. (D) WT and mutCRE constructs were inserted in pGL3-luciferase reporter plasmid and transfected in COS7 cells as described in the Supplemental Material. Promoter-driven luciferase activity was measured after cell stimulation with 25 μM forskolin in the presence or absence of RpcAMP (10 nM) for 24 h. Results are represented as a percentage of luciferase activity in control nonstimulated cells. Transfections were performed in triplicate, and values are means ± SD from three independent experiments. *, significantly different from pWT luciferase activity in control nonstimulated cells, with p < 0.05; #, significantly different from pWT luciferase activity in forskolin-treated cells, with p < 0.05.