Resources to Study the Isoforms of the p53 Family of Transcription Factors

The p53 family of transcription factors regulates diverse gene regulatory networks such as those involved in DNA damage repair, epidermal commitment, and neuronal development. As a result, dysfunction in these pathways lead to numerous human health conditions, including multiple types of cancer and craniofacial developmental disorders. Each family member regulates a distinct gene network leading to varied cellular and organismal phenotypes, but all are capable of regulating programmed cell death, likely due to considerable similarity between protein structure and binding sites in DNA. Analysis of these shared and distinct functions is complicated by each p53 family member gene (TP53, TP63, and TP73) generating multiple N- and C-terminal isoforms with potential differences in activity. Here, we develop a novel p53 family expression library for comprehensively and uniquely interrogating their shared and distinct biological activities. The first collection allows for in vitro expression and transient expression of all 35 uniquely codon-shifted isoforms in a range of mammalian models. We used this library to demonstrate its utility in distinguishing isoforms based on their unique sequences enabling their unequivocal identification in complex biological settings. We also showcase the advantage of utilizing this collection for knockdown – rescue style experiments that are challenging due to the sequence homology present within this family. The second collection allows for stable integration and inducible expression of all 35 isoforms coupled with unique molecular identifiers (UMIs) suitable for use in pooled screens. We used this library to perform pooled drop-out screens to compare anti-proliferative activity between isoforms of this transcription factor family. We identified both known and novel activities that would have been difficult with conventional loss-of-function (LOF) approaches. This newly constructed tool provides the means to answer previously intractable questions about the p53 family and has the potential to significantly impact evolutionary biology, developmental biology, and cancer biology.