Identification of cooperative monomeric Brachyury sites conferring T-bet responsiveness to the proximal IFN-γ promoter
Identification of cooperative monomeric Brachyury sites conferring T-bet responsiveness to the proximal IFN-γ promoter
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DOI：
10.1093/intimm/dxg113
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发表时间：
2003-10-01
影响因子：
4.4
通讯作者：
Murphy, K
中科院分区：
文献类型：
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作者：
Cho, JY;Grigura, V;Murphy, K
据报道，T-box转录因子T-bet可增强IFN-γ报告构建体的活性，并且是CD 4（+）T细胞产生IFN-γ所需的，而不是CD 8（+）T细胞产生IFN-γ所需的。尽管有这些观察结果，T-bet在IFN-γ基因座内的精确序列靶标尚未被鉴定，并且T-bet在选择性增加CD 4（+）T细胞中IFN-γ产生中的作用的性质尚未阐明。作为该过程的初始步骤，我们检查了IFN-γ报告构建体的T-bet依赖性扩增的基础，以鉴定IFN-γ基因座内该因子的特异性靶点。删除先前提出的TDB和TRU元件，使T-bet诱导的IFN-γ报告活性不变，表明存在额外的T-bet响应元件。我们在近端IFN-γ启动子内鉴定了几个额外的单体Brachyury共有元件，它们协同作用以增加组成型和刺激型启动子活性。这些Brachyury元件的最近端在介导T-bet依赖性启动子增强方面在定量上是最显著的。用任何其他Brachyury元件突变该元件导致T-bet依赖性启动子激活的几乎根除。IFN-γ基因座内这些单体Brachyury结合位点的鉴定应有助于在IFN-γ基因调控中T-bet的谱系和背景依赖性需求中T-bet的功能的体内分析。
The T-box transcription factor T-bet has been reported to augment the activity of IFN-gamma reporter constructs and to be required for CD4(+), but not CD8(+), T cell production for IFN-gamma. Despite these observations, the precise sequence targets of T-bet within the IFN-gamma locus have not been identified and the nature of T-bet's role in selectively augmenting IFN-gamma production in CD4(+) T cells has not been elucidated. As an initial step in this process, we examined the basis of T-bet-dependent augmentation of IFN-gamma reporter constructs to identify specific targets of this factor within the IFN-gamma locus. Deletion of previously proposed TDB and TRU elements left T-bet-induced IFN-gamma reporter activity unchanged, suggesting the existence of additional T-bet-responsive elements. We identified several additional monomeric Brachyury consensus elements within the proximal IFN-gamma promoter that operate cooperatively to increase both constitutive and stimulated promoter activity. The most proximal of these Brachyury elements is most significant quantitatively in mediating T-bet-dependent promoter augmentation. Mutation of this with any of the other Brachyury elements leads to a near eradication of T-bet-dependent promoter activation. The identification of these individual monomeric Brachyury-binding sites within the IFN-gamma locus should facilitate the in vivo analysis of the function of T-bet in the lineage- and background-dependent requirement for T-bet in IFN-gamma gene regulation.