|
Download
article
155kb
pdf? |
|
PIASy Regulation of STAT5 Activity: Different Reporter Vectors, Different Responses
Hassan Mziaut1, Mirko Trajkovski1,
Anke Altkrüger1, and Michele Solimena1,2
1Experimental Diabetology and Department of Medicine III
2School of Medicine, Dresden University of Technology,
Dresden 01307, Germany
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.
|
