CELL-FREE TRANSCRIPTION DIRECTED BY THE 422 ADIPOSE P2 GENE PROMOTER - ACTIVATION BY THE CCAAT ENHANCER BINDING-PROTEIN
CELL-FREE TRANSCRIPTION DIRECTED BY THE 422 ADIPOSE P2 GENE PROMOTER - ACTIVATION BY THE CCAAT ENHANCER BINDING-PROTEIN
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DOI：
10.1073/pnas.88.19.8465
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发表时间：
1991-10-01
影响因子：
11.1
通讯作者：
LANE, MD
中科院分区：
文献类型：
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作者：
CHENEVAL, D;CHRISTY, RJ;LANE, MD
先前的研究表明，CCAAT/增强子结合蛋白（C/EBP）可以作为几种脂肪细胞特异性基因启动子的反式激活因子，即，3 T3-L1小鼠前脂肪细胞中的422脂肪P2（422/aP 2）、硬脂酰辅酶A去饱和酶1（SCD 1）和葡萄糖转运蛋白4（GLUT 4）基因。 我们现在描述一种无细胞系统，该系统由3 T3-L1细胞的细胞核制备，其进行由这些启动子指导的转录。 为了测量转录物的形成，我们采用了聚合酶链反应辅助分析。 来自表达C/EBP的3 T3-L1脂肪细胞的核提取物比来自缺乏C/EBP的前脂肪细胞的核提取物支持嵌合422（aP 2）启动子-氯霉素乙酰转移酶（CAT）报告基因构建体的更高的转录速率。 含有C/EBP结合位点序列的竞争寡核苷酸和针对C/EBP产生的抗体抑制由422（aP 2）启动子指导的转录。 前脂肪细胞核提取物的转录限制因子似乎是C/EBP，因为重组C/EBP（rC/EBP）显著激活前脂肪细胞提取物而非脂肪细胞提取物的422（aP 2）启动子-CAT基因的转录。 rC/EBP还激活SCD 1启动子-CAT和GLUT 4启动子-CAT嵌合基因的无细胞转录。 422（aP 2）启动子中C/EBP结合位点内的点突变显著降低rC/EBP激活的转录。 与完整前脂肪细胞中cAMP激活422（aP 2）启动子一致，cAMP依赖性蛋白激酶通过无细胞系统的启动子激活转录，这种作用不依赖于C/EBP。 因此，在无细胞系统中由422（aP 2）启动子指导的转录调节类似于在完整的3 T3-L1细胞中发生的。
Previous investigations have shown that CCAAT/enhancer binding protein (C/EBP) can function as a trans-activator of the promoters of several adipocyte-specific genes-i.e., the 422 adipose P2 (422/aP2), stearoyl-CoA desaturase 1 (SCD1), and glucose transporter 4 (GLUT4) genes, in 3T3-L1 mouse preadipocytes. We now describe a cell-free system prepared from nuclei of 3T3-L1 cells that carries out transcription directed by these promoters. To measure transcript formation, we employed a polymerase chain reaction-assisted analysis. Nuclear extract from 3T3-L1 adipocytes that express C/EBP supports a higher rate of transcription of chimeric 422(aP2) promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs than nuclear extract from preadipocytes that lack C/EBP. A competitor oligonucleotide containing the C/EBP binding site sequence and antibodies raised against C/EBP inhibit transcription directed by the 422(aP2) promoter. The factor limiting transcription by nuclear extract from preadipocytes appears to be C/EBP, since recombinant C/EBP (rC/EBP) markedly activates transcription of the 422(aP2) promoter-CAT gene with preadipocyte extract but not with adipocyte extract. rC/EBP also activates cell-free transcription of SCD1 promoter-CAT and GLUT4 promoter-CAT chimeric genes. Point mutations within the C/EBP binding site in the 422(aP2) promoter markedly decrease transcription activated by rC/EBP. Consistent with activation by cAMP of the 422(aP2) promoter in intact preadipocytes, cAMP-dependent protein kinase activates transcription through this promoter with the cell-free system, this effect being independent of C/EBP. Thus, regulation of transcription directed by the 422(aP2) promoter in the cell-free system resembles that which occurs in intact 3T3-L1 cells.