DIFFERENTIATION-INDUCED GENE-EXPRESSION IN 3T3-L1 PREADIPOCYTES - CCAAT ENHANCER BINDING-PROTEIN INTERACTS WITH AND ACTIVATES THE PROMOTERS OF 2 ADIPOCYTE-SPECIFIC GENES
DIFFERENTIATION-INDUCED GENE-EXPRESSION IN 3T3-L1 PREADIPOCYTES - CCAAT ENHANCER BINDING-PROTEIN INTERACTS WITH AND ACTIVATES THE PROMOTERS OF 2 ADIPOCYTE-SPECIFIC GENES
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DOI：
10.1101/gad.3.9.1323
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发表时间：
1989-09-01
影响因子：
10.5
通讯作者：
LANE, MD
中科院分区：
文献类型：
--
作者：
CHRISTY, RJ;YANG, VW;LANE, MD
先前的研究表明，3 T3-L1前脂肪细胞的分化导致一组脂肪特异性基因的转录激活。作为一种方法来定义负责激活这些基因的表达的机制，我们研究了核因子的结合的两个分化诱导的基因，422（aP 2）和硬脂酰辅酶A去饱和酶1（SCD 1）基因的启动子。DNA酶1足迹法和凝胶阻滞分析确定了两个结合区域内的每个基因的启动子，与核因子存在于分化的3 T3-L1脂肪细胞。一个分化诱导的核因子与每个基因启动子中的单个结合位点特异性相互作用。竞争实验表明，该核因子与SCD 1启动子的相互作用被合成的寡核苷酸特异性地阻止，该寡核苷酸对应于422（aP 2）启动子中的足迹位点。几条证据表明，分化诱导的核因子是CCAAT/增强子结合蛋白（C/EBP），一种首先从大鼠肝脏中分离的DNA结合蛋白。细菌表达的重组C/EBP结合到分化特异性核因子在每个基因的启动子内相互作用的相同位点。北方分析表明，C/EBP mRNA的丰度在分化过程中显着增加。使用C/EBP表达载体的瞬时共转染研究表明，C/EBP可以作为422（aP 2）和SCD 1基因启动子的反式激活因子。
Previous studies have shown that differentiation of 3T3-L1 preadipocytes leads to the transcriptional activation of a group of adipose-specific genes. As an approach to defining the mechanism responsible for activating the expression of these genes, we investigated the binding of nuclear factors to the promoters of two differentiation-induced genes, the 422(aP2) and stearoyl-CoA desaturase 1 (SCD 1) genes. DNase 1 footprinting and gel retardation analysis identified two binding regions within the promoters of each gene that interact with nuclear factors present in differentiated 3T3-L1 adipocytes. One differentiation-induced nuclear factor interacts specifically with a single binding site in the promoter of each gene. Competition experiments showed that the interaction of this nuclear factor with the SCD1 promoter was prevented specifically by a synthetic oligonucleotide corresponding to the site footprinted in the 422(aP2) promoter. Several lines of evidence indicate that the differentiation-induced nuclear factor is CCAAT/enhancer binding protein (C/EBP), a DNA-binding protein first isolated from rat liver. Bacterially expressed recombinant C/EBP binds to the same site at which the differentiation-specific nuclear factor interacts within the promoter of each gene. Northern analysis with RNA from 3T3-L1 cells shows that C/EBP mRNA abundance increases markedly during differentiation. Transient cotransfection studies using a C/EBP expression vector demonstrate that C/EBP can function as a trans-activator of both the 422(aP2) and SCD1 gene promoters.