Waterman M L, Jones K A
Salk Institute, La Jolla, CA 92037.
New Biol. 1990 Jul;2(7):621-36.
The differentiation of T cells into functionally diverse subpopulations is controlled in part, by transcriptional activation and silencing; however, little is known in detail about the proteins that influence this developmental process. We have purified a new T-cell-specific factor, TCF-1 alpha, that is implicated in the activation of genes encoding a major component of the human T-cell receptor (TCR). TCF-1 alpha, originally identified and purified through its binding sites on the HIV-1 promoter, was found to bind to the TCR alpha enhancer and to promoters for several genes expressed at significantly earlier stages of T-cell development than the TCR alpha gene (e.g., p56lck and CD3 delta). Sequences related to the TCF-1 alpha binding motif (5'-GGCACCCTTTGA-3') are also found in the human TCR delta (and possibly TCR beta) enhancers. Southwestern and gel renaturation experiments with the use of purified protein fractions revealed that TCF-1 alpha activity is derived from a family of 57- to 53-kD proteins that are abundantly expressed in mature and immature T-cell lines (Jurkat, CCRF-CEM) and not in mature B cells (JY, Namalwa) or nonlymphoid (HeLa) cell lines. A small 95-bp fragment of the TCR alpha control region that contains the TCF-1 alpha binding site juxtaposed between a cAMP-response element (the CRE or T alpha 1 motif) and the binding site for a distinct lymphoid-specific protein (TCF-2 alpha) behaved as a potent T-cell-specific enhancer in vivo. Tandem copies of this enhancer functioned synergistically in mature (Jurkat) T-cell lines as well as resting and activated immature (CCRF-CEM) T-cell lines. Mutation of the TCF-1 alpha binding site diminished enhancer activity and disrupted the synergism observed in vivo between tandem enhancer repeats. The TCF-1 alpha binding site was also required for TCR alpha enhancer activity in transcriptionally active extracts from Jurkat but not HeLa cells, confirming that TCF-1 alpha is a T-cell-specific transcription factor. Curiously, the TCF-1 alpha binding element was inactive in vivo when removed from its neighboring elements on the TCR alpha enhancer and positioned in one or more copies upstream of a heterologous promoter. Thus, the transcriptional activity of TCF-1 alpha appears to depend on the TCF-2 alpha and T alpha 1 (CREB) transcription factors and the context of its binding site within the TCR alpha enhancer.
T细胞分化为功能多样的亚群部分受转录激活和沉默的控制；然而，对于影响这一发育过程的蛋白质，我们知之甚少。我们纯化了一种新的T细胞特异性因子TCF-1α，它与编码人类T细胞受体（TCR）主要成分的基因激活有关。最初通过其在HIV-1启动子上的结合位点鉴定和纯化的TCF-1α，被发现可与TCRα增强子以及在T细胞发育的比TCRα基因更早阶段显著表达的几个基因的启动子结合（例如p56lck和CD3δ）。在人类TCRδ（可能还有TCRβ）增强子中也发现了与TCF-1α结合基序（5'-GGCACCCTTTGA-3'）相关的序列。使用纯化的蛋白质组分进行的蛋白质印迹和凝胶复性实验表明，TCF-1α活性源自一族57至53kD的蛋白质，它们在成熟和未成熟T细胞系（Jurkat、CCRF-CEM）中大量表达，而在成熟B细胞（JY、Namalwa）或非淋巴细胞（HeLa）细胞系中不表达。TCRα控制区的一个95bp小片段，包含并列于cAMP反应元件（CRE或Tα1基序）和一个不同的淋巴样特异性蛋白质（TCF-2α）结合位点之间的TCF-1α结合位点，在体内表现为一个有效的T细胞特异性增强子。该增强子的串联拷贝在成熟（Jurkat）T细胞系以及静止和活化的未成熟（CCRF-CEM）T细胞系中协同发挥作用。TCF-1α结合位点的突变降低了增强子活性，并破坏了体内串联增强子重复序列之间观察到的协同作用。在Jurkat细胞而非HeLa细胞的转录活性提取物中，TCRα增强子活性也需要TCF-1α结合位点，这证实了TCF-1α是一种T细胞特异性转录因子。奇怪的是，当TCF-1α结合元件从TCRα增强子上的相邻元件中移除并置于异源启动子上游的一个或多个拷贝中时，它在体内是无活性的。因此，TCF-1α的转录活性似乎依赖于TCF-2α和Tα1（CREB）转录因子以及其在TCRα增强子内结合位点的背景。
