CELL-CYCLE DEPENDENT REGULATION OF HSP70 GENE EXPRESSION BY C-MYC

我们之前已经在人类hsp70启动子转录起始位点-150处发现了HSP-MYC-B元件，该元件被c-Myc蛋白复合物识别，并有助于该基因的早期G1特异性表达。为了确定在HSP-MYC-B上形成的c-Myc复合物参与G1特异性表达的蛋白，首先利用人脑cDNA文库对HSP-MYC-B序列进行酵母单杂交筛选，并以c-Myc为诱饵进行双杂交筛选。克隆的两个cdna中的一个编码CCAAT盒结合蛋白亚基CBF-C/NF-YC。体外结合实验表明，c-Myc与CBF-C/NF-YC直接结合，在体外培养细胞中也观察到两者之间的关联。用小鼠Balb3T3细胞核提取物对wtB探针进行带移实验，观察到两种不同的含有c-Myc的dna -蛋白复合物，复合物I和II，复合物I除了含有c-Myc外还含有CBF。当纯化的CBF亚基与wtB探针孵育时，即使存在纯化的c-Myc，蛋白质也只识别wtB序列中的CCAAT，而具有核提取物的复合物I需要HSP-MYC-B元素以及wtB中的CCAAT。同样，HSP-MYC-B元件和CCAAT序列都是wtB序列转录活性所必需的。在小鼠Balb3T3细胞的瞬时转染实验中，根据引入c-Myc的剂量，c-Myc先刺激然后抑制来自wtB-hsp70 TATA的转录。高剂量c-Myc抑制的转录被CBF/NF-Y以剂量依赖的方式消除。使用类似转染细胞的核提取物进行带移实验显示，复合物I的形成效率与wtB的转录活性相似。此外，具有wtB结合活性的CBF/NF-Y-c-Myc复合物在细胞周期的早期Gi期c-Myc表达不高时出现，在c-Myc表达水平达到最大值后逐渐消失。结果表明，hsp70基因表达的细胞周期依赖性上调和下调是由CBF/NF-Y和c-Myc之间的复合物形成状态决定的，受细胞内c-Myc数量的控制。少

We have previously identified at about -150 from the transcriptional initiation site in the human hsp70 promoter the HSP-MYC-B element which is recognized by a c-Myc protein complex and contributes to the early G1 specific expression of the gene. To identify the proteins involved in the c-Myc complex formed on the HSP-MYC-B and contributes the G1 specific expression, the one-hybrid screening in yeast targetin the HSP-MYC-B sequence was first carried out using a human brain cDNA library, and positive clones were further screened by the two-hybrid assays on the c-Myc as a bait. One of the two cDNAs thus cloned encoded the CCAAT box binding protein subunit, CBF-C/NF-YC.In vitro binding assays showed that c-Myc directly bound to CBF-C/NF-YC, and the association between the two proteins was also observed in vivo in cultured cells. In bandshift assays on the wtB probe with the nuclear extract prepared from mouse Balb3T3 cells, two distinct DNA-protein complexes containing c-Myc, the complexe … More s I and II, were observed, and the complex I contained CBF in addition to c-Myc. When the purified CBF subunits were incubated with the wtB probe, the proteins recognized only CCAAT in the wtB sequence even in the presence of purified c-Myc, while the complex I with the nuclear extract required the HSP-MYC-B element as well as CCAAT in the wtB.Similarly, both the HSP-MYC-B element and the CCAAT sequence were required for the transcriptional activity of the wtB sequence. In the transient transfection experiments in mouse Balb3T3 cells, c-Myc first stimulated and then repressed the transcription derived from wtB-hsp70 TATA, according to the doses of c-Myc introduced. The repressed transcription by a high dose of c-Myc was abrogated by the introduction of CBF/NF-Y in a dose-dependent manner. Bandshift assays using the nuclear extract of the cells similarly transfected revealed that the efficiency of the complex I formation w a parallel to the transcriptional activity of wtB.Furthermore, CBF/NF-Y-c-Myc complex possessing the wtB binding activity appeared in the early Gi phase of the cell cycle when the c-Myc expression was not high, and gradually disappeared after the expression level of c-Myc reached maximal. The results indicate that the cell cycle-dependent up- and down- regulation of the hsp70 gene expression is determined by the complex formation states between CBF/NF-Y and c-Myc, controlled by the intracellular amount of c-Myc. Less