PIASy Regulation of STAT5 Activity: Different Reporter Vectors Different Responses

Luciferase reporter vectors are widely used to study regulation of gene expression because they are sensitive, easy to quantify and detectable over a broad dynamic range. However, presence of cryptic cis-acting regulatory sequences in some vectors can induce anomalous responses when activated by undesired transcription factors. Here we used dual-reporter assays with firefly and Renilla luciferases to investigate whether PIASy regulates the transcriptional activity of STAT5 in insulinoma INS-1 cells. Unexpectedly, we found that the expression of Renilla luciferase encoded by the phRL-null Vector was highly regulated by PIASy, an E3-SUMO ligase that inhibits STATs. Analysis of the pRL-null Vector backbone revealed the presence of three previously undetected regulatory elements for STAT5. The pGL4.70[hRluc] Vector, from which these elements and those for many other transcription factors have been removed, was found to be a more reliable means for assessing PIASy function. These data highlight the limitation of the phRL Vector backbone and the enhanced performance of the pGL4 Vector backbone. The results emphasize the need to perform appropriate controls to determine whether differences in the expression of reporter genes result from variability in transfection efficiency or from diversity in the response of the promoter and promoterless vectors.

Cell Notes 17, 6–8.