A high affinity HSF-1 binding site in the 5′-untranslated region of the murine tumor necrosis factor-α gene is a transcriptional repressor
A high affinity HSF-1 binding site in the 5′-untranslated region of the murine tumor necrosis factor-α gene is a transcriptional repressor
复制标题
DOI：
10.1074/jbc.m108154200
复制
发表时间：
2002-02-15
影响因子：
4.8
通讯作者：
Hasday, JD
中科院分区：
文献类型：
--
作者：
Singh, IS;He, JR;Hasday, JD
肿瘤坏死因子-α(TNFpha)是宿主防御的关键早期介质，对感染后的生存至关重要。我们先前曾报道，巨噬细胞暴露在高温(FRT)(38.5-40摄氏度)下，通过导致转录的突然和过早停止，显著减弱TNFpha的表达。我们发现，FRT的这种抑制作用是由热休克因子1(HSF-1)的交替激活的抑制形式介导的，并且包含最小85核苷酸(NT)近端启动子和138-NT 5‘-非翻译区(UTR)的TNFpha基因片段足以介导这一作用。在本研究中，我们使用电泳迁移率改变分析(EMSA)来确定HSF-1在TNFpha基因5‘-UTR区的高亲和力结合位点，并使用染色体免疫沉淀试验证明HSF-1与内源性TNFpha基因的这一区域结合。该位点的突变失活阻断了过表达的hsf-1对RAW 264.7小鼠巨噬细胞中最小的肿瘤坏死因子α启动子(-85/+138)活性的抑制作用，从而确定该位点为hsf-1依赖的抑制子。然而，相同的突变未能阻止通过HSF-1过表达构建的全长(-1080/+138)TNFpha启动子的抑制，并且HSF-1与EMSA中-1080/-845、-533/-196和-326/-39nt区域的上游序列结合，表明在最小的-85-nt启动子的上游存在额外的依赖于HSF-1的抑制元件。此外，尽管最小TNFpha启动子结构中HSF-1结合位点的突变取消了HSF-1介导的抑制，但相同的突变并不能通过高水平的HSF-1过表达或暴露在39.5℃下来消除该结构的抑制。这表明HSF-1可能通过冗余机制抑制TNFpha转录，其中一些机制可能不需要HSF-1的高亲和力结合。
Tumor necrosis factor-alpha (TNFalpha) is a pivotal early mediator of host defenses that is essential for survival in infections. We previously reported that exposing macrophages to febrile range temperatures (FRT) (38.5-40 degreesC) markedly attenuates TNFalpha expression by causing abrupt and premature cessation of transcription. We showed that this inhibitory effect of FRT is mediated by an alternatively activated repressor form of heat shock factor 1 (HSF-1) and that a fragment of the TNFalpha gene comprising a minimal 85-nucleotide (nt) proximal promoter and the 138-nt 5'-untranslated region (UTR) was sufficient for mediating this effect. In the present study we have used an electrophoretic mobility shift assay (EMSA) to identify a high affinity binding site for HSF-1 in the 5'-UTR of the TNFalpha gene and have used a chromosome immunoprecipitation assay to show that HSF-1 binds to this region of the endogenous TNFalpha gene. Mutational inactivation of this site blocks the inhibitory effect of overexpressed HSF-1 on activity of the minimal TNFalpha promoter (-85/+138) in Raw 264.7 murine macrophages, identifying this site as an HSF-1-dependent repressor. However, the same mutation fails to block repression of a full-length (-1080/+138) TNFalpha promoter construct by HSF-1 overexpression, and HSF-1 binds to upstream sequences in the regions -1080/-845, -533/-196, and -326/-39 nt in EMSA, suggesting that additional HSF-1-dependent repressor elements are present upstream of the minimal -85-nt promoter. Furthermore, although mutation of the HSF-1 binding site in the minimal TNFalpha promoter construct abrogates HSF-1-mediated repression, the same mutation fails to abrogate repression of this construct by high levels of HSF-1 overexpression or exposure to 39.5 degreesC. This suggests that HSF-1 might repress TNFalpha transcription through redundant mechanisms, some of which might not require high affinity binding of HSF-1.