Figure 6

Role of Sp1 in ACVR1 gene promoter regulation. A) Nucleotide sequence of the basal promoter of the ACVR1 gene lacks TATA boxes and has high GC-nucleotide content. The putative Sp1 binding sites are indicated by boxes and nucleotides subjected to site-directed mutagenesis are indicated with red circles. The arrow indicates the TSS (+1). B) In silico analysis of the region as it appears in the HMR Conserved Transcription Factor Binding Sites (TFBS conserved) track in the UCSC Genome Browser revealed a well-conserved putative recognition site for the Sp1 transcription factor. Custom tracks indicating the overlapping GC-boxes (ACVR1_Basal_Promoter_GC-Boxes) and the position of the mutagenized nucleotides (GC-boxes_mutagenesis) were added to the UCSC window and are shown. C) Both the wild-type (WT) and mutated Pr-0.072 constructs containing the 72 bp promoter region were transiently transfected into U2OS, HeLa, ATDC5, and C2C12 cell lines. Luciferase activities obtained with mutated constructs are expressed as a percentage of the activity of the wild-type (100%) in each cell line. D) The effect of Sp1 on basal promoter activity was tested by co-transfecting the Sp1 expression plasmid with the wild-type (WT) and mutated Pr-0.072 reporter constructs in the indicated cell lines. Luciferase activity is expressed as relative to the transcriptional activity of the Pr-0.072WT in cells transfected with the empty vector that was used to subclone the Sp1 cDNA (ev). The data represent the mean ± SD (error bars) of independent experiments (n=5 for C, n=3 for D) carried out in triplicate with p< 0.05^*, p< 0.01^**, or p< 0.001***.