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Deciphering the Nature of Trp73 Isoforms in Mouse Embryonic Stem Cell Models: Generation of Isoform-Specific
Lorena López-Ferreras1,2, Nicole Martínez-García1,3, Laura Maeso-Alonso1,2
1Instituto de Biomedicina (IBIOMED), Universidad de León, 24071 León, Spain.
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.
Insights
The p53 family protein p73 has distinct isoforms (TA and DN-p73) crucial for development. Creating knockout models revealed these isoforms regulate stem cell fate and differentiation.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- The p53 protein family plays critical roles in cell physiology and pathology.
- Isoforms of p53 family proteins, generated through alternative splicing and promoter usage, contribute to diverse cellular functions.
- The Trp73 gene specifically produces Tumor Apoptosis (TA) and Dominant Negative (DN) p73 isoforms, influencing p73's dual biological roles.
Purpose of the Study:
- To generate and validate cellular models for studying individual p73 isoforms in a physiological context.
- To investigate the specific functions of TA-p73 and DN-p73 isoforms in stem cell biology.
- To understand the role of p73 isoforms in early cell fate determination and differentiation.
Main Methods:
- Utilized CRISPR/Cas9 gene editing technology to create specific TA-p73 and DN-p73 deficient mouse embryonic stem cell lines.
- Validated these cell lines as bona fide knockout models for studying p73 isoform function.
- Performed global gene expression analysis to identify isoform-specific transcriptional network alterations.
Main Results:
- Generated validated mouse embryonic stem cell lines lacking either TA-p73 or DN-p73.
- Demonstrated that p73 isoform deficiency is compatible with pluripotency but induces a transition from the naive to the primed pluripotent state.
- Observed compromised lineage differentiation in p73 isoform-deficient cells, indicating a role in developmental processes.
Conclusions:
- Differential expression of p73 isoforms acts as a critical regulator, or rheostat, in early cell fate determination.
- p73 isoforms are essential for maintaining the naive pluripotent state and ensuring proper stem cell differentiation.
- The generated knockout models provide a valuable tool for further research into p73 isoform functions in development and disease.
