Investigation of the interaction of p63 with p300 and iASPP in oocytes.

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p63 belongs to the family of the tumor suppressor p53. Despite high sequence homology between both proteins, p63 is not a typical tumor suppressor. So far two different functions have been identified for p63: 1) It is highly expressed in the basal compartment of stratified epithelial tissues where it regulates the proliferative potential of these cells and 2) it serves as a quality control factor in germ cells. We have focused on characterizing how the longest isoform, TAp63a, is involved in the quality control of oocytes. In mammals oocytes enter the dictyate arrest phase after completion of homologous recombination. In this stage oocytes are kept for extended periods of time, lasting in humans several decades, until being recruited for ovulation. During the dictyate arrest oocytes express high levels of TAp63a. We have shown that TAp63a in resting oocytes is kept in an inactive, closed and only dimeric conformation. Detection of DNA double strand breaks leads to phosphorylation of TAp63a, resulting in the formation of an open, active and tetrameric state. Based on mutational analysis, SAXS measurements and structure determination of individual domains we have created a structural model of the closed state and shown that this state is a kinetically trapped state. Activation follows a spring loaded mechanism that creates the thermodynamically more stable tetrameric form. We have demonstrated that the DNA binding affinity of the closed and dimeric form is 20 fold less than that of the open tetramer. However, this reduction of the DNA binding affinity is only part of the inhibitory mechanism. In addition, our preliminary data show that the interaction with the transcriptional machinery is inhibited. We want to study the interaction between p63 and the domains of p300 that interact with transactivation domains and develop a quantitative model of the entire inhibitory mechanism. In addition, we have demonstrated that the activation of TAp63a after irradiation with a low dose of irradiation is significantly slower than the activation after irradiation with a higher dose. Since most oocytes survive a low irradiation dose cells must have a mechanism to inactivate activated TAp63a. In addition to ubiquitination and proteasomal degradation, inhibition by interaction with iASPP is a likely mechanism. In C. elegans only one member of the p53 family (CEP-1) and one member of the ASPP family (Ce-iASPP) are expressed in germ cells. Depletion of Ce-iASPP by RNAi enhances germ cell apoptosis, suggesting that Ce-iASPP is a negative regulator of Cep-1 and that this inhibitory interaction between both proteins plays an important role in maintaining a viable germ cell population. In this application we propose to study the regulatory function of iASPP by interaction and structural analysis as well as functional studies in mouse oocytes in collaboration with the laboratory of Xin Lu, Oxford, that has created an iASPP knock out mouse.