Evidence for a factor required for transcriptional stimulation by the chimeric acidic activator GAL-VP16 in HeLa cell extracts.
We provide biochemical evidence for the existence of a transcriptional intermediary factor (TIF) in HeLa whole-cell extracts (WCE) that is distinct from the basic transcription factors and that is required for transcriptional stimulation by the chimeric acidic activator GAL-VP16. We have fractionated HeLa WCE by heparin-agarose chromatography. Of transcriptionally active fractions eluting in a step between 0.24 and 0.6 M KCl, the initial fractions are refractory to GAL-VP16 stimulation, whereas subsequent fractions are strongly stimulated by the activator. Aliquots of GAL-VP16-responsive fractions efficiently complement refractory fractions for transcriptional stimulation. Aliquots of responsive fractions are also far more efficient than those of refractory fractions in overcoming transcriptional inhibition that is brought about by high concentrations of GAL-VP16. Experiments performed with heat-treated WCE support the idea that HeLa cells contain a TIF that is essential for GAL-VP16 stimulation, but that is not required for basal transcription. Addition of recombinant yeast or human transcription factor TFIID (rTFIIDY and rTFIIDH, respectively) to a WCE heated at 48 degrees C for 15 min restores basal transcription, but in neither case is the reconstituted system activated by GAL-VP16. However, a 45 degrees C heat-treated WCE reconstituted with either rTFIIDH or rTFIIDY is stimulated by GAL-VP16, suggesting that a HeLa TIF can be selectively inactivated by heating at 48 degrees C, but not at 45 degrees C. Interestingly, a TFIID fraction partially purified from HeLa cell extracts, but not rTFIIDH, efficiently relieves transcriptional inhibition by GAL-VP16, suggesting that there may be an association between TIF(s) and TFIID and, moreover, that TIF(s) may be the direct target of the acidic domain of GAL-VP16. In summary, our results support the existence of a TIF that is not essential for basal transcription but that is required to mediate the stimulatory activity of the acidic activator GAL-VP16.
White
J H
JH
Laboratoire de Génétique Moléculaire des Eucaryotes du Centre National de la Recherche Scientifique, Unité de Génie Génétique et de Biologie Moléculaire, Faculté de Médecine, Strasbourg, France.
Brou
C
C
Wu
J
J
Burton
N
N
Egly
J M
JM
Chambon
P
P
D001685:Biological Factors / Q000302:isolation & purification / Q000378:metabolism*; D002678:Chimera; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D005656:Fungal Proteins / Q000378:metabolism*; D006367:HeLa Cells / Q000502:physiology; D006801:Humans; D011994:Recombinant Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D001685:Biological Factors; D005656:Fungal Proteins; C070633:Gal-VP16; D011994:Recombinant Proteins; D015534:Trans-Activators; D035362:Transcription Factor TFIID; D014157:Transcription Factors