Browsing by Subject "Tip110"
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Item CHARACTERIZATION OF THE REGULATORY MECHANISMS OF TIP110 IN EARLY EMBRYONIC DEVELOPMENT AND BEYOND(2013-04-12) Whitmill, AmandaPurpose: 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.Item Tip110 is required for embryonic stem cell survival and embryonic development(2016-08-01) Whitmill, Amanda J.; Johnny J. He; Geoffrey Guttmann; Khalid TimaniHIV-1 Tat-interacting protein of 110 kDa, Tip110, has roles in tumor antigen presentation, pre-mRNA splicing, transcription of viral and host genes, and protein degradation. Tip110 is also known to be up-regulated in a variety of cancers and to regulate and/or interact with a variety of transcription factors, oncogenes, and pluripotency factors. As such, Tip110 has been shown to effect pluripotency, proliferation, apoptosis, and the cell cycle when knocked down in vitro. However, the function of Tip110 in embryonic development remains largely uncharacterized. One early study has shown that loss of a Tip110 ortholog leads to embryonic lethality in zebrafish. Our studies have shown that transgenic mouse embryos lacking expression of a functional Tip110 protein die several days post-implantation in vivo. In the present study, we determined how Tip110 knockout affects mouse embryonic development and investigated the underlying molecular mechanisms. We found that Tip110 loss did not impair embryo growth from the zygote to the blastocyst stage nor did it impair the blastocysts ability to implant into the uterine lining in vivo. Extended culture of blastocysts in vitro revealed that Tip110 loss impaired both blastocyst outgrowth formation and derivation of mouse embryonic stem cells from blastocysts. In vivo embryos could survive until the post-implantation stage where they eventually perished. The premature death of these embryos was characterized by a clear retardation in embryonic development resulting in underdeveloped or more commonly, completely resorbed mouse embryos around 8.5 or 9.5 days post coitum. Microarray analysis of Tip110-/- cells derived from mouse blastocysts revealed that Tip110 loss favored differentiation but not self-renewal, pluripotency, or cell cycling through a complex regulatory network of stem cell factors. Tip110-/- cells also had perturbations in many other signaling and cellular processes including mRNA processing and proteasome degradation. Taken together, these findings document for the first time the lethal effects of complete loss of Tip110 on mammalian embryonic development and suggest that Tip110 is an important regulator of not only embryonic development but also stem cell factors.