SINGLE CELL ASSAY OF A TRANSCRIPTION FACTOR REVEALS A THRESHOLD IN TRANSCRIPTION ACTIVATED BY SIGNALS EMANATING FROM THE T-CELL ANTIGEN RECEPTOR
SINGLE CELL ASSAY OF A TRANSCRIPTION FACTOR REVEALS A THRESHOLD IN TRANSCRIPTION ACTIVATED BY SIGNALS EMANATING FROM THE T-CELL ANTIGEN RECEPTOR
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DOI：
10.1101/gad.4.10.1823
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发表时间：
1990-10-01
影响因子：
10.5
通讯作者：
HERZENBERG, LA
中科院分区：
文献类型：
--
作者：
FIERING, S;NORTHROP, JP;HERZENBERG, LA
T淋巴细胞通过其抗原受体刺激产生多种转录因子，包括核因子-AT和核因子-kappa.B，它们参与调节免疫激活所需的基因。为了研究单个活细胞中单个转录因子的活性，我们应用了一种使用荧光激活细胞分选器来定量β-半乳糖苷酶(β-GAL)的方法。我们使用了一个由三个串联的NF-AT结合位点来指导LacZ基因转录的结构，分析了核因子-AT转录活性在经历激活的T细胞中的分布。出乎意料的是，对克隆的稳定转染的Jurkat T细胞的刺激导致了β-Gal表达的生物模式，其中一些细胞表达NO-β-Gal，另一些细胞表达高水平。这种表达模式不能用细胞周期位置或遗传变异来解释。进一步的结果表明，β-半乳糖活性与核因子-AT结合活性相关，表明在转录启动之前，核因子-AT的浓度必须超过一个临界阈值。这一阈值可能反映了核因子-AT浓度依赖的转录复合体在启动子上的组装。在诱导过程中，由NF-kappa.B或整个IL-2增强子控制的类似结构显示出生物模式的表达模式，这表明由转录因子浓度设定的阈值可能是可诱导基因的共同属性。
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 requires for immunologic activation. To investigate the activity of a single transcription factor in individual viable cells, we have applied an assay that used 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 biomodal 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 biomodal expression patterns during induction, suggesting that thresholds set by the concentration of transcription factors may be a common property of inducible genes.