|Budget Amount *help
¥2,500,000 (Direct Cost : ¥2,500,000)
Fiscal Year 1998 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1997 : ¥1,400,000 (Direct Cost : ¥1,400,000)
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
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.
wtB配列は既にc-Myc複合体が結合する配列であることを以前に報告した.そこで,まず,wtB配列に結合するタンパク質cDNAを酵母One=hybrid法でスクリーニングし,得られたpositive clonesを更に,c-Mycをbaitとした酵母Two=hybrid法でスクリーニングした。得られたクローンはCCAAT結合タンパク質の一つであるNF-YC/CBF-Cであった.wtBは以前報告したMYC-B配列とCCAAT配列が一部重複している.そこで,NF-Y/CBFとc-Mycとの相互関係を検討したところ,,両者はin vivo ,in vitroで結合し,この両者はともにwtBのエンハンサー活性に必須であることが明らかとなった.更に,両者は細胞周期の移行に伴う発現量の差によって転写活性が左右されることが明らかとなった.則ち,c-Myc発現量の少ないG1期においては,NF-Yとc-Mycは安定な複合体形成をおこない,転写を活性化するが,S期に入りc-Myc量の増加に伴い,過剰なc-MycはNF-Y/CBF複合体よりそのサブユニットであるNF-YC/CBF-Cと選択的に結合し,NF-Y/CBF複合体を解離させ,転写活性と抑制することが判明した.c-Mycによるhsp70遺伝子の転写調節機構に大きく前進したと考えられる. Less