CHARACTERIZATION OF THE REGULATORY MECHANISMS OF TIP110 IN EARLY EMBRYONIC DEVELOPMENT AND BEYOND

Purpose: HIV Tat-interacting protein of 110 kDa (Tip110) plays important roles in various cellular processes including pre-mRNA splicing, antigen presentation, regulation of transcription, and even embryonic development. Previous studies pinpointing Tip110 knockout in the perinatal death of zebrafish led to the current study concerning the effect of Tip110 knockout in a transgenic mouse model. Taking from the zebrafish study we predicted that Tip110 knockout might primarily affect endoderm differentiation and thus mainly disrupt thymic development, lung development, and the functions of the exocrine pancreas. Thus far our data indicate that embryonic lethality of Tip110-/- mice occurs early after uterine implantation, suggesting a key role for this protein in mammalian development. We have also began investigating differences in the human and mouse versions of this protein as it has been discovered that human Tip110 is processed at the cellular level in a different fashion than mouse Tip110. Methods: A floxed/cre transgenic mouse model was used to determine the time point at which embryonic death occurs. Tip110 floxed mice were bred with eIIa-Cre transgenic mice, the embryos were dissected at various time points post coitum to estimate time of death. In the future tissue fixation and immunohistochemistry, RT-PCR, and in vitro culture of embryos will be performed. To characterize the differences between human and mouse tip110 we have developed recombinant constructs and expressed them in both human and mouse cell lines followed by Western blot analysis, immunoprecipitation, and use of various enzymes and inhibitors to determine their differences. Results: Tip110-/- mice do not survive long enough after uterine implantation for key developmental milestones such as establishment of the placenta, organogenesis, and limb formation to occur. Human and mouse Tip110 have different protein expression patterns depending on whether vector expression occurs in a human or mouse cell line. They also differ in the degree at which they are (de)ubiquitinated and degraded by the 26S proteasomal system. Conclusions: Tip110-/- must disturb a crucial step of early embryogenesis. Future studies with other tissue-specific conditional knockouts may provide insight into the effects of Tip110-/-. There is some subtle change occurring in the human and/or mouse Tip110 protein that will likely explain some of the differences we have noted in terms of the size, protein expression, and rate of degradation in Tip110.