Deciphering the Nature of Trp73 Isoforms in Mouse Embryonic Stem Cell Models: Generation of Isoform-Specific Deficient Cell Lines Using the CRISPR/Cas9 Gene Editing System

SIMPLE SUMMARY: The Trp73 gene is involved in the regulation of multiple biological processes such as response to stress, differentiation and tissue architecture. This gene gives rise to structurally different N and C-terminal isoforms which lead to differences in its biological activity in a cell type dependent manner. However, there is a current lack of physiological models to study these isoforms. The aim of this study was to generate specific p73-isoform-deficient mouse embryonic stem cell lines using the CRISPR/Cas9 system. Their special features, self-renewal and pluripotency, make embryonic stem cells a useful research tool that allows the generation of cells from any of the three germ layers carrying specific inactivation of p73-isoforms. Characterization of the generated cell lines indicates that while the individual elimination of TA- or DN-p73 isoform is compatible with pluripotency, it results in alterations of the transcriptional profiles and the pluripotent state of the embryonic stem cells in an isoform-specific manner. ABSTRACT: The p53 family has been widely studied for its role in various physiological and pathological processes. Imbalance of p53 family proteins may contribute to developmental abnormalities and pathologies in humans. This family exerts its functions through a profusion of isoforms that are generated by different promoter usage and alternative splicing in a cell type dependent manner. In particular, the Trp73 gene gives rise to TA and DN-p73 isoforms that confer p73 a dual nature. The biological relevance of p73 does not only rely on its tumor suppression effects, but on its pivotal role in several developmental processes. Therefore, the generation of cellular models that allow the study of the individual isoforms in a physiological context is of great biomedical relevance. We generated specific TA and DN-p73-deficient mouse embryonic stem cell lines using the CRISPR/Cas9 gene editing system and validated them as physiological bona fide p73-isoform knockout models. Global gene expression analysis revealed isoform-specific alterations of distinctive transcriptional networks. Elimination of TA or DN-p73 is compatible with pluripotency but prompts naïve pluripotent stem cell transition into the primed state, compromising adequate lineage differentiation, thus suggesting that differential expression of p73 isoforms acts as a rheostat during early cell fate determination.