PIG3 (p53-inducible gene 3), originally identified as one of a set of genes induced by p53 before the onset of apoptosis, was assumed to contribute to early cellular response to DNA damage. Here, we studied the relati...PIG3 (p53-inducible gene 3), originally identified as one of a set of genes induced by p53 before the onset of apoptosis, was assumed to contribute to early cellular response to DNA damage. Here, we studied the relation between p53 status and the increased expression of PIG3 by ionizing radiation (IR), and the related clues regarding the involvement of PIG3 in the cellular response to IR-induced DNA damage signaling. We demonstrated that the pentanucleotide microsatellite sequence was responsible for the p53-dependent induction of PIG3 transcription after irradiation, while sequence upstream of PIG3 promoter could maintain the basal level of expression which was not inducible by irradiation. The interaction of PIG3 and the KRAB-ZFP-associated protein 1 (KAP1), a DNA damage response protein, was revealed. PIG3 nucleus foci were formed 15 min after γ-ray irradiation, and which were found to partially colocalize with the phospho-KAP-1 foci as well as γ-H2AX foci. Although the lac operator tagged EGFP based reporter system revealed that PIG3 does not remodel chromatin in large scale in the cells under normal growing condition, it indeed prompted the chromatin relaxation in the cellular response to DNA damage signaling. All these data suggest that PIG3 is involved in IR-induced DNA damage response, and which maybe partially attribute to its interaction with KAP1.展开更多
Of ICE protease family, CPP32 (apopain or Yama) plays a central role in different apoptotic pathways. To study the molecules regulating CPP32, yeast two_hybrid system was used to identify proteins (peptides) that inte...Of ICE protease family, CPP32 (apopain or Yama) plays a central role in different apoptotic pathways. To study the molecules regulating CPP32, yeast two_hybrid system was used to identify proteins (peptides) that interact with CPP32. First, the CPP32 gene was cloned into plasmid vector pGBT9. The resulting recombinant plasmid was designated as pGBT9/CPP. The pGBT9 /CPP plasmid was transformed into the yeast strain HF7C, then the leukemia library was introduced. The transformation mixture was plated on medium lacking Trp, Leu and His in the initial screen. Colonies growing on the selection medium were further assayed for β galactosidase activity. Within 42 Trp +Leu +His + colonies only 5 turned blue in the presence of X_Gal. Plasmid DNA from 5 positive yeast colonies was prepared respectively and used to transform HB101 by electroporation. The transformation mixture was plated on medium lacking Leu to be selected for the library plasmid. Finally, only one library plasmid, designated as pY1, was determined to be truly positive by retransformation of pGBT9/CPP and the library plasmid into HF7C. The inserted cDNA of pY1 encodes a peptide of 15 amino acids, suggesting that it may be the domain interacting with CPP32.展开更多
基金supported by the National Basic Research Program of China (2007CB914603)the National Natural Science Foundation of China (30970677)the Outstanding Youth Scientist Foundation of National Natural Science Foundation of China (30825011)
文摘PIG3 (p53-inducible gene 3), originally identified as one of a set of genes induced by p53 before the onset of apoptosis, was assumed to contribute to early cellular response to DNA damage. Here, we studied the relation between p53 status and the increased expression of PIG3 by ionizing radiation (IR), and the related clues regarding the involvement of PIG3 in the cellular response to IR-induced DNA damage signaling. We demonstrated that the pentanucleotide microsatellite sequence was responsible for the p53-dependent induction of PIG3 transcription after irradiation, while sequence upstream of PIG3 promoter could maintain the basal level of expression which was not inducible by irradiation. The interaction of PIG3 and the KRAB-ZFP-associated protein 1 (KAP1), a DNA damage response protein, was revealed. PIG3 nucleus foci were formed 15 min after γ-ray irradiation, and which were found to partially colocalize with the phospho-KAP-1 foci as well as γ-H2AX foci. Although the lac operator tagged EGFP based reporter system revealed that PIG3 does not remodel chromatin in large scale in the cells under normal growing condition, it indeed prompted the chromatin relaxation in the cellular response to DNA damage signaling. All these data suggest that PIG3 is involved in IR-induced DNA damage response, and which maybe partially attribute to its interaction with KAP1.
文摘Of ICE protease family, CPP32 (apopain or Yama) plays a central role in different apoptotic pathways. To study the molecules regulating CPP32, yeast two_hybrid system was used to identify proteins (peptides) that interact with CPP32. First, the CPP32 gene was cloned into plasmid vector pGBT9. The resulting recombinant plasmid was designated as pGBT9/CPP. The pGBT9 /CPP plasmid was transformed into the yeast strain HF7C, then the leukemia library was introduced. The transformation mixture was plated on medium lacking Trp, Leu and His in the initial screen. Colonies growing on the selection medium were further assayed for β galactosidase activity. Within 42 Trp +Leu +His + colonies only 5 turned blue in the presence of X_Gal. Plasmid DNA from 5 positive yeast colonies was prepared respectively and used to transform HB101 by electroporation. The transformation mixture was plated on medium lacking Leu to be selected for the library plasmid. Finally, only one library plasmid, designated as pY1, was determined to be truly positive by retransformation of pGBT9/CPP and the library plasmid into HF7C. The inserted cDNA of pY1 encodes a peptide of 15 amino acids, suggesting that it may be the domain interacting with CPP32.
