Transcriptional mutagenesis dramatically alters genome-wide p53 transactivation landscape

Shuo Liang,Monika Ezerskyte,Kristian Dreij,Vicent Pelechano,Jingwen Wang

doi:10.1038/s41598-020-70412-4

Shuo Liang, Monika Ezerskyte + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-70412-4

Copy DOI

Abstract

The transcriptional error rate can be significantly increased by the presence of DNA lesions that instruct mis-insertion during transcription; a process referred to as transcriptional mutagenesis (TM) that can result in altered protein function. Herein, we determined the effect of O6-methylguanine (O6-meG) on transcription and subsequent transactivation activity of p53 in human lung H1299 cells. Levels of TM and effects on transactivation were determined genome wide by RNA-seq. Results showed that 47% of all p53 transcripts contained an uridine misincorporation opposite the lesion at 6 h post transfection, which was decreased to 18% at 24 h. TM at these levels reduced DNA binding activity of p53 to 21% and 80% compared to wild type p53, respectively. Gene expression data were analysed to identify differentially expressed genes due to TM of p53. We show a temporal repression of transactivation of > 100 high confidence p53 target genes including regulators of the cell cycle, DNA damage response and apoptosis. In addition, TM repressed the transcriptional downregulation by p53 of several negative regulators of proliferation and differentiation. Our work demonstrates that TM, even when restricting its effect to an individual transcription factor, has the potential to alter gene expression programs and diversify cellular phenotypes.

Highlights

Various DNA lesions from plethora of agents present in our environment pose a constant threat to cells’ genomic integrity, regardless of their proliferation state
We recently showed that O6-meG-induced transcriptional mutagenesis (TM) at codon 248 reduced the p53 tumour-suppressor function through deregulation of cell cycle control and impaired activation of apoptosis in human c ells[10]
We have recently shown that expression of mutant R248W p53 due to TM resulted in repressed transactivation of p53 target genes with downstream impairment of G1/S cell-cycle checkpoint and activation of apoptosis in H1299 cells[10]

Summary

Introduction

Various DNA lesions from plethora of agents present in our environment pose a constant threat to cells’ genomic integrity, regardless of their proliferation state. The p53 protein primarily functions as a transcription factor and exerts its tumour suppressive function mainly through transcriptional activation of a large number of target genes, which in turn mediate multiple important cellular processes including regulation of cell cycle, apoptosis, DNA repair, metabolism, autophagy, translational control, and feedback m echanisms[14]. This is managed by a differential time-dependent transactivation of target genes with cell-cycle genes generally being induced early upon p53 activation while induction of most apoptotic genes is a relatively later e vent[15,16]. The timedependent impact of TM at codon 248 on the transcriptional regulation of p53 target genes was assessed on a genome-wide level in human cells by RNA-seq

Methods

Results

Conclusion