Defective transcription of ATF3 responsive genes, a marker for Cockayne Syndrome

Alexey Epanchintsev,Frederic Coin,Nadege Calmels,Vincent Laugel,Federico Costanzo,Alain Sarasin,Marc-Alexander Rauschendorf,Cathy Obringer,Jean-Marc Egly

doi:10.1038/s41598-020-57999-4

Abstract

Cockayne syndrome (CS) is a rare genetic disorder caused by mutations (dysfunction) in CSA and CSB. CS patients exhibit mild photosensitivity and severe neurological problems. Currently, CS diagnosis is based on the inefficiency of CS cells to recover RNA synthesis upon genotoxic (UV) stress. Indeed, upon genotoxic stress, ATF3, an immediate early gene is activated to repress up to 5000 genes encompassing its responsive element for a short period of time. On the contrary in CS cells, CSA and CSB dysfunction impairs the degradation of the chromatin-bound ATF3, leading to a permanent transcriptional arrest as observed by immunofluorescence and ChIP followed by RT-PCR. We analysed ChIP-seq of Pol II and ATF3 promoter occupation analysis and RNA sequencing-based gene expression profiling in CS cells, as well as performed immunofluorescence study of ATF3 protein stability and quantitative RT-PCR screening in 64 patient cell lines. We show that the analysis of few amount (as for example CDK5RAP2, NIPBL and NRG1) of ATF3 dependent genes, could serve as prominent molecular markers to discriminate between CS and non-CS patient’s cells. Such assay can significantly simplify the timing and the complexity of the CS diagnostic procedure in comparison to the currently available methods.

Highlights

The human genome is constantly exposed to genotoxic attack, which might lead, in absence of proper DNA repair, to genomic instability resulting in rare disorders such as xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS)
The present study proposes an easy and straightforward molecular assay for identifying CS patients based on the inability of CSA and CSB to perform their ubiquitin/proteasome degradation function
In CS deficient cells, the ATF3 is maintained on its target site, abrogating the expression of amount of ATF3 responsive genes leading to subsequent cellular defects as exemplified in our blind assay (Fig. 3a and Supplementary Fig. S1d)

Summary

Introduction

The human genome is constantly exposed to genotoxic attack, which might lead, in absence of proper DNA repair, to genomic instability resulting in rare disorders such as xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS). CS can be caused by mutations in ERCC6 (CSB, [MIM: 609413]) or ERCC8 (CSA, [MIM: 609412]) while certain mutations in XPB (MIM: 610651), XPD (MIM: 278730) and XPG (MIM: 278780) genes develop some CS clinical features[4,5,6,7]. We later have shown that genotoxic stress triggered the overexpression of immediate early genes such as the Activating Transcription Factor 3 (ATF3, MIM: 603148)[16]. On the contrary in CS cells, CSA and CSB dysfunction impaired ATF3 degradation In these cells, ATF3 remained bound to the CRE/ATF3 response element, thereby preventing the comeback of Pol II and the restart of transcription (Fig. 1 and Supplementary Fig. S1a). We hypothesized that ATF3 direct downstream transcriptional targets may be responsible for mediating CS clinical outcomes

Methods

Results

Conclusion