INTERLEUKIN-2 TRANSCRIPTION IS REGULATED IN-VIVO AT THE LEVEL OF COORDINATED BINDING OF BOTH CONSTITUTIVE AND REGULATED FACTORS
INTERLEUKIN-2 TRANSCRIPTION IS REGULATED IN-VIVO AT THE LEVEL OF COORDINATED BINDING OF BOTH CONSTITUTIVE AND REGULATED FACTORS
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DOI：
10.1128/mcb.14.3.2159
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发表时间：
1994-03-01
影响因子：
5.3
通讯作者：
WOLD, BJ
中科院分区：
文献类型：
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作者：
GARRITY, PA;CHEN, D;WOLD, BJ
白细胞介素-2（IL-2）转录在发育上限于T细胞，并且在生理上依赖于特异性刺激，例如抗原识别。先前的研究表明，这种严格的两级调控是通过转录启动子/增强子DNA片段介导的，该片段由不同的识别元件组成。与这些元件中的一些结合的因子组成性地存在于许多细胞类型中，而其他因子是信号依赖性的、T细胞特异性的或两者兼有。这就提出了几个关于IL-2表达调节的分子机制的问题。在信号依赖性诱导之前，可用因子与IL-2增强子之间的稳定相互作用是否反映了T细胞的发育承诺？在何种水平上，与DNA结合的因子或一旦结合的因子活性，天然增强子内的单个调节元件受到调节？考虑到许多相对非特异性的因素的参与，是什么机制促使了发育和生理特异性的产生？为了回答这些问题，我们使用体内足迹法来确定和比较细胞环境中天然IL-2位点的蛋白质-DNA相互作用模式，包括ELA T淋巴瘤细胞和32 D克隆5肥大细胞，它们表达不同的IL-2 DNA结合因子亚群。我们还使用免疫抑制剂环孢菌素A作为药理学试剂，以进一步剖析环孢菌素A敏感因子在蛋白质-DNA复合物的组装和维持中所起的作用。所有位点类型的占据仅在T细胞中观察到，然后仅在信号转导途径激发时观察到。这是真的，即使部分重叠的子集的IL-2结合活性显示存在于32 D克隆5肥大细胞。这一观察结果在32 D细胞中特别引人注目，因为在信号刺激下，它们动员了大量的IL-2 DNA结合活性，如通过使用核提取物的体外测定所测量的。我们的结论是，所有类别的结合活性不能稳定地占据其同源位点的IL-2，除了以下活化的T细胞，和IL-2转录的特异性是在染色体占有率的水平，这似乎是一个全有或全无的现象。
Interleukin-2 (IL-2) transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. Prior studies have shown that this stringent two-tiered regulation is mediated through a transcriptional promoter/enhancer DNA segment which is composed of diverse recognition elements. Factors binding to some of these elements are present constitutively in many cell types, while others are signal dependent, T cell specific, or both. This raises several questions about the molecular mechanism by which IL-2 expression is regulated. Is the developmental commitment of T cells reflected molecularly by stable interaction between available factors and the IL-2 enhancer prior to signal-dependent induction? At which level, factor binding to DNA or factor activity once bound, are individual regulatory elements within the native enhancer regulated? By,what mechanism is developmental and physiological specificity enforced, given the participation of many relatively nonspecific elements? To answer these questions, we have used in vivo footprinting to determine and compare patterns of protein-DNA interactions at the native IL-2 locus in cell environments, including ELA T-lymphoma cells and 32D clone 5 premast cells, which express differing subsets of IL-2 DNA-binding factors. We also used the immunosuppressant cyclosporin A as a pharmacological agent to further dissect the roles played by cyclosporin A-sensitive factors in the assembly and maintenance of protein-DNA complexes. Occupancy of all site types was observed exclusively in T cells and then only upon excitation of signal transduction pathways. This was true even though partially overlapping subsets of IL-2-binding activities were shown to be present in 32D clone 5 premast cells. This observation was especially striking in 32D cells because, upon signal stimulation, they mobilized a substantial set of IL-2 DNA-binding activities, as measured by in vitro assays using nuclear extracts. We conclude that binding activities of all classes fail to stably occupy their cognate sites in IL-2, except following activation of T cells, and that specificity of IL-2 transcription is enforced at the level of chromosomal occupancy, which appears to be an all-or-nothing phenomenon.