Penix L, Weaver W M, Pang Y, Young H A, Wilson C B
Department of Pediatrics, University of Washington, Seattle 98195.
J Exp Med. 1993 Nov 1;178(5):1483-96. doi: 10.1084/jem.178.5.1483.
Like interleukin 2 (IL-2), interferon gamma (IFN-gamma) is an early response gene in T cells and both are prototypical T helper cell type 1 (Th-1) lymphokines. Yet IL-2 and IFN-gamma production are independently regulated, as demonstrated by their differential expression in certain T cell subsets, suggesting that the regulatory elements in these two genes must differ. To explore this possibility, the 5' flank of the human IFN-gamma gene was analyzed. Expression of IFN-gamma promoter-driven beta-galactosidase reporter constructs containing 538 bp of 5' flank was similar to that by constructs driven by the IL-2 promoter in activated Jurkat T cells; expression nearly as great was observed with the construct containing only 108 bp of IFN-gamma 5' flank. These IFN-gamma promoter constructs faithfully mirrored expression of the endogenous gene, in that expression required activation both with ionomycin and PMA, was inhibited by cyclosporin A, and was not observed in U937 or THP-1 cells. The region between -108 and -40 bp in the IFN-gamma promoter was required for promoter function and contained two elements that are conserved across species. Deletion of 10 bp within either element reduced promoter function by 70%, whereas deletions in nonconserved portions of this region had little effect on promoter function. The distal conserved element (-96 to -80 bp) contained a consensus GATA motif and a potential regulatory motif found in the promoter regions of the GM-CSF and macrophage inflammatory protein (MIP) genes. Factors binding to this element, including GATA-3, were found in Jurkat nuclear extracts by electromobility shift assays and two of the three complexes observed were altered in response to activation. One or both of these motifs are present in the 5' flank of multiple, other lymphokine genes, including IL-3, IL-4, IL-5, and GM-CSF, but neither is present in the promoter of the IL-2 gene. The proximal conserved element (-73 to -48 bp) shares homology with the NFIL-2A element in the IL-2 promoter; these elements compete for binding of factors in Jurkat nuclear extracts, although the NFIL-2A element but not the IFN-gamma element binds Oct-1. Factors binding to this element in the IFN-gamma gene were present in extracts from resting and activated Jurkat T cells. However, by in vivo footprinting of intact cells, this element was protected from methylation only with activation.(ABSTRACT TRUNCATED AT 400 WORDS)
与白细胞介素2（IL-2）一样，干扰素γ（IFN-γ）是T细胞中的早期反应基因，二者均为典型的1型辅助性T细胞（Th-1）淋巴因子。然而，IL-2和IFN-γ的产生是独立调节的，这在某些T细胞亚群中的差异表达中得到了证明，表明这两个基因中的调节元件必定不同。为了探究这种可能性，对人IFN-γ基因的5'侧翼进行了分析。在活化的Jurkat T细胞中，含有538 bp 5'侧翼的IFN-γ启动子驱动的β-半乳糖苷酶报告基因构建体的表达与IL-2启动子驱动的构建体相似；仅含有108 bp IFN-γ 5'侧翼的构建体也观察到了几乎同样高的表达。这些IFN-γ启动子构建体如实地反映了内源性基因的表达，即表达需要离子霉素和佛波酯（PMA）共同激活，会被环孢素A抑制，并且在U937或THP-1细胞中未观察到表达。IFN-γ启动子中-108至-40 bp之间的区域对于启动子功能是必需的，并且包含两个在物种间保守的元件。任一元件内10 bp的缺失使启动子功能降低70%，而该区域非保守部分的缺失对启动子功能影响很小。远端保守元件（-96至-80 bp）包含一个共有GATA基序和一个在GM-CSF及巨噬细胞炎性蛋白（MIP）基因启动子区域中发现的潜在调节基序。通过电泳迁移率变动分析在Jurkat细胞核提取物中发现了与该元件结合的因子，包括GATA-3，并且观察到的三种复合物中的两种会因激活而改变。这些基序中的一个或两个存在于多种其他淋巴因子基因的5'侧翼，包括IL-3、IL-4、IL-5和GM-CSF，但在IL-2基因的启动子中均不存在。近端保守元件（-73至-48 bp）与IL-2启动子中的NFIL-2A元件具有同源性；这些元件竞争Jurkat细胞核提取物中因子的结合，尽管NFIL-2A元件而非IFN-γ元件结合Oct-1。在静止和活化的Jurkat T细胞提取物中均存在与IFN-γ基因中该元件结合的因子。然而，通过完整细胞的体内足迹分析，该元件仅在激活时才免受甲基化作用的影响。（摘要截短至400字）
