A functional screening assay for the isolation of transcription factors.
Transcription factors (TFs) have been difficult to identify by bioinformatics, due to the heterogenous nature of the domains they are composed of. Therefore, we have developed a simple and generally applicable screening system for the identification of transcriptional activators, based on the presence of a functional transactivation domain (TAD). The system utilizes a retroviral vector to express a cDNA library as fusion genes with the yeast gal4 DNA-binding domain. This retroviral library is transduced into a murine NIH3T3-based reporter cell line carrying a stable integrated gal4 promoter-green fluorescent protein reporter gene. cDNA inserts encoding a functional TAD reconstitute a chimeric TF that activates the reporter gene. After fluorescence activated cell sorting (FACS) and expansion of GFP-positive cells, the responsible cDNA inserts are retrieved. From a cDNA library of cytokine-stimulated human umbilical vein endothelial cells (HUVEC), a number of known as well as potentially novel TFs were isolated, demonstrating the suitability of the system. The identification of other factors that are currently not associated with transcriptional regulation suggest additional functions for these proteins. Moreover, our results have focused attention on signaling pathways that have not been recognized previously in the context of endothelial cell biology.
Wiesner
Christoph
C
Department of Vascular Biology and Thrombosis Research, University of Vienna, and BMT-Biomolecular Therapeutics GmbH, Brunnerstrasse 59, A-1235 Vienna, Austria.
Hoeth
Martina
M
Binder
Bernd R
BR
de Martin
Rainer
R
D016475:3T3 Cells; D020816:Amino Acid Motifs; D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D004727:Endothelium / Q000166:cytology / Q000378:metabolism; D005189:False Positive Reactions; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015533:Transcriptional Activation; D014161:Transduction, Genetic; D014471:Umbilical Veins / Q000378:metabolism
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA