The Screening And Functional Study Of P53 Regulators Among The KRAB-type Zinc-finger Protein Family

The p53 tumor suppressor is a transcriptional factor which lies at the center of a protein network that controls cell cycle progression and commitment to apoptosis. In response to various types of stress including DNA damage and abnormal proliferative signals, p53 is activated and selectively regulats the expression of its target genes that are involved in cell cycle arrest, apoptosis and DNA repair. The regulation of p53 target genes is partly achieved through interaction between p53 and its regulators, such as Hzf, CAS/CSE1L, ASPP1 and ASPP2 . Tight regulation of p53 is essential for maintaining normal cell growth and preventing tumorigenesis.The zinc finger proteins containing the KRAB (Kruppel-associated box) domain (KZNF) are the single largest class of transcription factors in mammals. They repress transcription in conjuction with the co-repressor, KAP-1 (KRAB box-associated protein-1)and other transcriptional factors. We recently identified and characterized a novel KZNF member, named Apak(ATM and p53 associated KZNF protein) as a negative regulator of p53. In unstressed cells, Apak interacts with p53 and KAP-1 through its zinc-fingers and KRAB domain, respectively. KAP-1 then recruits ATM and HDAC1 to regulate the acetylation of p53, thereby repressing p53 activity and selectively turning off the expression of pro-apoptotic genes. We also revealed that Apak displays this repression activity dependent of ATM; whereas in response to DNA damage signals, Apak is phosphorylated by ATM and dissociates from p53, resulting in p53 acivation, which are also dependent of ATM. Thus we identified the dual role of ATM on p53 regulation. Considering that the criticial domains of Apak are highly conserved among about 400 members of the KZNF superfamily, these homologues may also be pivotal to p53 regulation. We are interested with whether other memebers among the KZNF family could negatively regulate p53 like Apak and what the correlation is between these members.In this regard, we found 48 members among 423 ones within the whole KZNF family contain the potential ATM phosphorylaation site SQ/TQ motif and the phylogenetic tree of these 48 members was estabilished. Then we successfully constructed the expression plasmids of 21 members and confirmed their expression in mammalian cells. We detected that 5 members(ZNF8 , ZNF195 , ZNF248, ZNF566, ZNF568)could strongly downregulate the transcriptional activity of p53. According to the types of KRAB domain and the regulatory effects on p53, twelve and four members were selected to examine their interactions with p53 and ATM, respectively. These analyses showed that 8 members of the twelve could interact with p53 and 2 members of the four could interact with ATM. KZNF protein which could bind to both ATM and p53 could display significant repression activity on p53.Taken together, this thesis explored the possible role of the KZNF family in p53 regulation. We totally obtained 48 potential ATM substrates among KZNF family and constructed the expression palsmids of 21 members. Several members were shown to repress p53 activity significantly, which function seems to be related to their interaction with p53 and ATM. These findings might deepen our understandings of the physiological role of KZNF family and contribute to elucidate the spatio-temporal complexity of p53 regulation.