TA*p63 and GTAp63 achieve tighter transcriptional regulation in quality control by converting an inhibitory element into an additional transactivation domain

Susanne Pitzius,Birgit Schäfer,Christian Münch,Jakob Gebel,Christian Osterburg,Volker Dötsch,Huiqing Zhou,Georg Tascher

doi:10.1038/s41419-019-1936-z

Abstract

The p53 homolog p63 plays important roles in development of epithelial tissues and quality control in germ cells. These two functions are executed by two distinct isoforms of p63. They are created by different promotors resulting in isoforms having either an N-terminal transactivation domain (TAp63) or a truncated form (ΔNp63). In addition to these two N-terminal isoforms a third one with an even longer N-terminus, named TA*p63, has been found. A fourth N-terminal isoform, GTAp63, that closely resembles TA*p63 was discovered in male germ cells where it is involved in genetic quality control. Here, we characterize TA*p63α and GTAp63α and show that their N-terminal extensions stabilize the closed and only dimeric conformation adopted by the shorter TAp63α protein. Both proteins can be activated by the two kinases Chk2 and CK1 resulting in the open tetrameric state. In this conformation, the N-terminal extension acts as an additional transactivation domain enhancing transcriptional activity. Through this mechanism, the difference in transcriptional activity between the repressed and the active state of the protein gets enhanced relative to TAp63α. Finally, we show by mass spectrometry that TA*p63α is expressed in the breast cancer cell line Sum159 at the protein level together with mutant p53. Upon doxorubicin treatment, TA*p63α gets activated, providing a potential new tool to fight cancer.

Highlights

Sequencing of the human genome has identified~23,000 open reading frames, which is significantly lower than the number of genes initially estimated and lower than the number found in other, more primitive organisms, e.g., Daphnia pulex, which has ~31,000 genes[1]
GTAp63 specific N-termini can each be divided into two subdomains: While the C-terminal 18 aa of both peptides are identical the Nterminal sequences diverge
Size exclusion chromatography (SEC) analysis confirmed this finding (Supplementary Fig. S1). These results suggest that the transcriptional activity of TA*p63α and GTAp63α is regulated via their oligomeric state to TAp63α

Summary

Introduction

Sequencing of the human genome has identified~23,000 open reading frames, which is significantly lower than the number of genes initially estimated and lower than the number found in other, more primitive organisms, e.g., Daphnia pulex, which has ~31,000 genes[1]. The second p63 isoform for which a specific cellular function has been identified is TAp63α, which includes the full length N-terminal transactivation domain (TAD) This isoform is highly expressed in oocytes where it serves as a genetic quality control factor[6,7]. Upon detection of DNA double strand breaks it gets activated adopting an active, open and tetrameric conformation that triggers oocyte death via the expression of the two BH3-only proteins NOXA and PUMA10,11. This activation requires the consecutive action of the priming kinase Chk[212], phosphorylating a single serine in TAp63α and the executioner kinase CK1 which phosphorylates four more residues[13]

Methods

Results

Conclusion