A distal enhancer in the interferon-γ (IFN-γ) locus revealed by genome sequence comparison
A distal enhancer in the interferon-γ (IFN-γ) locus revealed by genome sequence comparison
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DOI：
10.1074/jbc.m307904200
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发表时间：
2004-02-06
影响因子：
4.8
通讯作者：
Rao, A
中科院分区：
文献类型：
--
作者：
Lee, DU;Avni, O;Rao, A
大规模跨物种DNA序列比较已成为鉴定基因保守顺式调控模块的有力工具。然而，生物信息学分析本身无法揭示进化上保守的区域如何调节基因表达：它是否作为增强子，沉默子或绝缘子发挥作用;它的功能是否受到细胞类型的限制;以及生物相关的转录因子是否与该元件结合。在这里，我们将联合收割机生物信息学与湿实验室技术相结合，阐述了识别基因功能保守调节区的通用且系统的方法。我们将这种方法应用于干扰素-γ（IFN-γ）基因。人和小鼠IFN-γ的比较揭示了一个高度保守的非编码序列，位于转录起始位点的5'端。该区域与产生IFN-γ的Th 1细胞中存在的组成型和诱导型DNA酶I超敏位点相一致，但不产生IFN-γ的Th 2细胞中不存在。组蛋白5'端保守非编码序列的甲基化表明，与Th 2细胞相比，Th 1细胞中的染色质结构更易接近。该元件结合两种已知对IFN-γ表达必不可少的转录因子：活化T细胞的核因子（一种诱导型转录因子）和T细胞中表达的T-box蛋白（一种细胞谱系限制性转录因子）。总之，这些发现确定了一个高度保守的远端增强子在IFN-γ细胞因子基因座，并验证我们的方法作为一个成功的方法来检测顺式调节元件。
Large-scale cross-species DNA sequence comparison has become a powerful tool to identify conserved cis-regulatory modules of genes. However, bioinformatic analysis alone cannot reveal how an evolutionarily conserved region regulates gene expression: whether it functions as an enhancer, silencer, or insulator; whether its function is cell-type restricted; and whether biologically relevant transcription factors bind to the element. Here we combine bioinformatics with wet-lab techniques to illustrate a general and systematic method of identifying functional conserved regulatory regions of genes. We applied this approach to the interferon-gamma (IFN-gamma) gene. Comparison of human and mouse IFN-gamma reveals a highly conserved non-coding sequence located similar to5 kb 5' of the transcription start site. This region coincides with constitutive and inducible DNase I hypersensitivity sites present in IFN-gamma-producing Th1 cells but not in Th2 cells that do not produce IFN-gamma. Histone methylation at the 5' conserved non-coding sequences indicates a more accessible chromatin structure in Th1 cells compared with Th2 cells. This element binds two transcription factors known to be essential for IFN-gamma expression: nuclear factor of activated T cells, an inducible transcription factor, and T-box protein expressed in T cells, a cell lineage-restricted transcription factor. Together, these findings identify a highly conserved distal enhancer in the IFN-gamma cytokine locus and validate our approach as a successful method to detect cis-regulatory elements.