Secretory interleukin-1 receptor antagonist gene expression requires both a PU.1 and a novel composite NF-κB/PU.1/GA-binding protein binding site
Secretory interleukin-1 receptor antagonist gene expression requires both a PU.1 and a novel composite NF-κB/PU.1/GA-binding protein binding site
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DOI：
10.1074/jbc.273.37.24272
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发表时间：
1998-09-11
影响因子：
4.8
通讯作者：
Fenton, MJ
中科院分区：
文献类型：
--
作者：
Smith, MF;Carl, VS;Fenton, MJ
人分泌型白细胞介素-1受体拮抗剂（分泌型IL-1 Ra）基因通过三个脂多糖（LPS）响应性启动子元件控制，其中一个被鉴定为NF-κ B结合位点。分泌型IL-1 Ra启动子的序列分析鉴定了位于与NF-κ B位点重叠的互补链上位置-80和-90之间的潜在PU. 1结合位点。凝胶位移分析使用这个潜在的结合位点与RAW 264.7巨噬细胞的核提取物证明了三个复合物的形成，一个LPS诱导型和两个组成型。诱导因子被鉴定为NF-κ B，组成因子被鉴定为PU. 1和GA结合蛋白。对-93至-79启动子区域的定点突变证明，单独的NF-κ B 5 '-半位点或PU. 1/GA结合蛋白半位点的突变不会显著降低LPS反应性。然而，破坏所有三种因子结合的突变导致LPS反应性降低50%。通过凝胶位移和超位移测定鉴定了以-230为中心的第二个PU. 1结合位点。核心GGAA区域的突变导致LPS应答启动子活性降低50%。远端和近端LPS反应元件的突变导致反应性几乎完全丧失。因此，这些数据表明，IL-1 Ra基因表达的调节是一个复杂的事件，涉及三种不同的转录因子与一个单一的顺式作用元件的相互作用，两个PU.1结合位点是LPS诱导的IL-1 Ra基因表达的主要反应元件。
The human secretory interleukin-1 receptor antagonist (secretory IL-1Ra) gene is controlled through three lipopolysaccharide (LPS)-responsive promoter elements, one of which was identified as an NF-kappa B binding site. Sequence analysis of the secretory IL-1Ra promoter identified a potential PU.1 binding site located between positions -80 and -90 on the complementary strand overlapping the NF-kappa B site. Gel shift analysis using this potential binding site with nuclear extracts from RAW 264.7 macrophages demonstrated the formation of three complexes, one LPS-inducible and two constitutive. The inducible factor was identified as NF-KB, and the constitutive factors were identified as PU.1 and GA-binding protein. Site-directed mutagenesis of the -93 to -79 promoter region demonstrated that mutation of either the NF-kappa B 5'-half site or the PU.1/GA-binding protein half-site alone did not significantly decrease LPS responsiveness. However, a mutation that disrupted the binding of all three factors resulted in a 50% decrease in LPS responsiveness. A second PU.1 binding site centered at -230 was identified by gel shift and supershift assays. Mutation of the core GGAA region resulted ill a 50% decrease in LPS-responsive promoter activity. Mutation of both the distal and proximal LPS response elements led to an almost complete loss of responsiveness. These data therefore suggest that the regulation of IL-1Ra gene expression is a complex event involving the interactions of three different transcription factors with a single cis-acting element and that the two PU.1 binding sites are the major response elements for LPS-induced IL-1Ra gene expression.