CBP is required for sterol-regulated and sterol regulatory element-binding protein regulated transcription
CBP is required for sterol-regulated and sterol regulatory element-binding protein regulated transcription
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DOI：
10.1074/jbc.273.28.17865
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发表时间：
1998-07-10
影响因子：
4.8
通讯作者：
Edwards, PA
中科院分区：
文献类型：
--
作者：
Ericsson, J;Edwards, PA
在源自法呢基二磷酸 (FPP) 合酶、3-羟基-3-甲基戊二酰辅酶 A (HMG-CoA) 合酶、HMG-CoA 还原酶或低密度脂蛋白受体基因的启动子的控制下，用荧光素酶报告基因转染细胞。当细胞在甾醇耗尽的培养基中孵育或与编码甾醇调节元件结合蛋白（SREBP）-1a的cDNA共转染时，荧光素酶活性的增加被野生型E1A或Gal4-CBP（1-451）融合蛋白的共表达所阻止。 E1A 的抑制作用可以通过 CBP 的共表达来克服。当用 cDNA 共转染细胞时，上述报告基因活性的增加不受影响，所述 cDNA 编码不能与转录激活剂 CBP 相互作用的突变体 E1A 或编码 CBP 单独片段的 Gal4-CBP 融合蛋白，其跨越 CBP 分子的其余部分。预先形成的 SREBP-1a:[P-32]DNA 复合物与含有 CBP 氨基末端部分的膜固定化 GST-CBP 融合蛋白特异性结合，为了研究 CBP 在内源基因调节中的作用，我们分离了响应添加的强力霉素而表达 Ga14-CBP(1-451) 的稳定转化体。当细胞表达 Ga14-CBP(1-451) 时，会阻止内源性 FPP 合酶和 HMG-CoA 合酶 mRNA 的诱导，以响应细胞胆固醇消耗。我们得出的结论是，当细胞在没有甾醇的情况下孵育时，HMG-CoA 合酶、HMG-CoA 还原酶、FPP 合酶和低密度脂蛋白受体基因的转录激活取决于 SREBP 之间的特定相互作用，该相互作用与启动子 DNA 和 CBP 的氨基末端结构域（氨基酸 1-451）。
Cells were transfected with luciferase reporter genes, under the control of promoters derived from either the farnesyl diphosphate (FPP) synthase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, or low density lipoprotein receptor genes. The increase in luciferase activity that occurred when cells were either incubated in sterol-depleted medium or cotransfected with a cDNA encoding sterol regulatory element-binding protein (SREBP)-1a was prevented by coexpression of wild type E1A or a Gal4-CBP (1-451) fusion protein. The inhibitory effect of E1A was overcome by coexpression of CBP. The increase in reporter gene activity noted above was not affected when the cells were cotransfected with cDNAs that encoded either a mutant E1A that is unable to interact with the transcriptional activator CBP or Gal4-CBP fusion proteins encoding separate fragments of CBP, which span the remainder of the CBP molecule, A preformed SREBP-1a:[P-32]DNA complex bound specifically to membrane-immobilized GST-CBP fusion proteins that contained amino terminal portions of CBP,In order to investigate the role of CBP in the regulation of endogenous genes, we isolated stable transformants that express Ga14-CBP(1-451) in response to added doxycycline. Induction of endogenous FPP synthase and HMG-CoA synthase mRNAs, in response to cellular cholesterol depletion, was prevented when cells expressed Ga14-CBP(1-451),We conclude that when cells are incubated in the absence of sterols, the transcriptional activation of the HMG-CoA synthase, HMG-CoA reductase, FPP synthase, and low density lipoprotein receptor genes is dependent on a specific interaction between SREBP, which is bound to the promoter DNA, and the amino terminal domain (amino acids 1-451) of CBP.