Figure 4

Dual luciferase reporter assays examining promoter and enhancer activities for DNA fragments containing HS site. The pGL4.10 vector, which contains the promoterless synthetic firefly luc2, was used in the promoter assay (left). The pGL4.23 vector, which contains the synthetic firefly luc2 driven by a minimal promoter, was used in the enhancer assay (right). The histograms show a summary of the ratio of luciferase activity (adjusted by dividing the firefly luciferase with the control Renilla luciferase) for each insert (from the HS sites shown in lower panel) relative to the luciferase activity for pGL4.10 or pGL4.23. Each fragment was tested in triplicate and experiments were carried out three times independently. Error bars represent the standard deviations of three trials. While fragments upstream of the XIST promoter containing HS sites showed background luciferase activity, fragment -65 displayed five fold and seven fold increases in promoter activity and ten fold and six fold increases in enhancer activity in the XIST and antisense orientation, respectively.