Single cell assay of a transcription factor reveals a threshold in transcription activated by signals emanating from the T-cell antigen receptor.
Stimulation of T lymphocytes through their antigen receptor leads to the appearance of several transcription factors, including NF-AT and NF-kappa B, which are involved in regulating genes required for immunologic activation. To investigate the activity of a single transcription factor in individual viable cells, we have applied an assay that uses the fluorescence-activated cell sorter to quantitate beta-galactosidase (beta-gal). We have analyzed the distribution of NF-AT transcriptional activity among T cells undergoing activation by using a construct in which three tandem copies of the NF-AT-binding site directs transcription of the lacZ gene. Unexpectedly, stimulation of cloned stably transfected Jurkat T cells leads to a bimodal pattern of beta-gal expression in which some cells express no beta-gal and others express high levels. This expression pattern cannot be accounted for by cell-cycle position or heritable variation. Further results, in which beta-gal activity is correlated with NF-AT-binding activity, indicate that the concentration of NF-AT must exceed a critical threshold before transcription initiates. This threshold likely reflects the NF-AT concentration-dependent assembly of transcription complexes at the promoter. Similar constructs controlled by NF-kappa B or the entire interleukin-2 enhancer show bimodal expression patterns during induction, suggesting that thresholds set by the concentration of transcription factors may be a common property of inducible genes.
Fiering
S
S
Department of Genetics, Stanford University, California 94305.
Northrop
J P
JP
Nolan
G P
GP
Mattila
P S
PS
Crabtree
G R
GR
Herzenberg
L A
LA
D002453:Cell Cycle / Q000502:physiology; D002478:Cells, Cultured; D002934:Cinnamates; D004268:DNA-Binding Proteins / Q000502:physiology; D004351:Drug Resistance / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D012260:Ribonucleases / Q000037:antagonists & inhibitors; D015398:Signal Transduction / Q000235:genetics*; D013601:T-Lymphocytes / Q000378:metabolism*; D014157:Transcription Factors / Q000032:analysis* / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology*; D014162:Transfection / Q000235:genetics; D001616:beta-Galactosidase / Q000032:analysis
D002934:Cinnamates; D004268:DNA-Binding Proteins; D011948:Receptors, Antigen, T-Cell; D014157:Transcription Factors; D006921:Hygromycin B; C026273:hygromycin A; D012260:Ribonucleases; D001616:beta-Galactosidase