Sterol regulation of human fatty acid synthase promoter I requires nuclear factor-Y- and Sp-1-binding sites.
Sterol regulation of human fatty acid synthase promoter I requires nuclear factor-Y- and Sp-1-binding sites.
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DOI：
10.1073/pnas.040574197
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发表时间：
2000-04
影响因子：
11.1
通讯作者：
S. Xiong;S. Chirala;S. Wakil
中科院分区：
文献类型：
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作者：
S. Xiong;S. Chirala;S. Wakil
为了了解胆固醇介导的人脂肪酸合成酶启动子I的调节，我们在HepG 2细胞中测试了启动子I-荧光素酶报告基因构建体的各种5 '缺失构建体。仅包含Sp-1结合位点（核苷酸-82至-74）和两个串联甾醇调节元件（SRE;核苷酸-63至-46）的报告基因构建体对胆固醇没有反应。只有含有核因子-Y（NF-Y）序列、CCAAT序列（核苷酸-90至-86）、Sp-1序列和两个串联SRE的报告基因结构对胆固醇有反应。因此，NF-Y结合位点对于胆固醇反应是必不可少的。突变SRE或NF-Y位点并在Sp-1-和NF-Y-结合位点之间插入4 bp均导致报告基因的最小胆固醇应答。使用抗SRE结合蛋白（SREBP）和抗NF-Ya抗体的电泳迁移率变化测定证实，这些SRE和NF-Y位点结合各自的因子。我们还鉴定了位于核苷酸-40和-30之间的第二个Sp-1位点，其可以取代位于核苷酸-82和-74之间的突变的Sp-1位点。当SREBP 1a [SREBP的N-末端结构域（氨基酸1-520）]组成型过表达时，野生型启动子和Sp-1位点（核苷酸-82至-74）突变启动子的报告基因表达相似，表明Sp-1将SREBP募集至SRE。在相同条件下，NF-Y位点突变导致报告基因表达的显著损失，表明NF-Y是激活胆固醇反应所必需的。
To understand cholesterol-mediated regulation of human fatty acid synthase promoter I, we tested various 5'-deletion constructs of promoter I-luciferase reporter gene constructs in HepG2 cells. The reporter gene constructs that contained only the Sp-1-binding site (nucleotides -82 to -74) and the two tandem sterol regulatory elements (SREs; nucleotides -63 to -46) did not respond to cholesterol. Only the reporter gene constructs containing a nuclear factor-Y (NF-Y) sequence, the CCAAT sequence (nucleotides -90 to -86), an Sp-1 sequence, and the two tandem SREs responded to cholesterol. The NF-Y-binding site, therefore, is essential for cholesterol response. Mutating the SREs or the NF-Y site and inserting 4 bp between the Sp-1- and NF-Y-binding sites both resulted in a minimal cholesterol response of the reporter genes. Electrophoretic mobility-shift assays using anti-SRE-binding protein (SREBP) and anti-NF-Ya antibodies confirmed that these SREs and the NF-Y site bind the respective factors. We also identified a second Sp-1 site located between nucleotides -40 and -30 that can substitute for the mutated Sp-1 site located between nucleotides -82 and -74. The reporter gene expression of the wild-type promoter and the Sp-1 site (nucleotides -82 to -74) mutant promoter was similar when SREBP1a [the N-terminal domain of SREBP (amino acids 1-520)] was constitutively overexpressed, suggesting that Sp-1 recruits SREBP to the SREs. Under the same conditions, an NF-Y site mutation resulted in significant loss of reporter gene expression, suggesting that NF-Y is required to activate the cholesterol response.