DNA damage-induced histone H1 ubiquitylation is mediated by HUWE1 and stimulates the RNF8-RNF168 pathway

I K Mandemaker,W Vermeulen,K Bezstarosti,J A Marteijn,R C Janssens,H Lans,L Van Cuijk,J A Demmers,J H Hoeijmakers

doi:10.1038/s41598-017-15194-y

I K Mandemaker, W Vermeulen + Show 7 more

Open Access

https://doi.org/10.1038/s41598-017-15194-y

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Journal: Scientific Reports	Publication Date: Nov 10, 2017
Citations: 56	License type: open-access

Affiliation: Oncode Institute, Erasmus University Rotterdam

Abstract

The DNA damage response (DDR), comprising distinct repair and signalling pathways, safeguards genomic integrity. Protein ubiquitylation is an important regulatory mechanism of the DDR. To study its role in the UV-induced DDR, we characterized changes in protein ubiquitylation following DNA damage using quantitative di-Gly proteomics. Interestingly, we identified multiple sites of histone H1 that are ubiquitylated upon UV-damage. We show that UV-dependent histone H1 ubiquitylation at multiple lysines is mediated by the E3-ligase HUWE1. Recently, it was shown that poly-ubiquitylated histone H1 is an important signalling intermediate in the double strand break response. This poly-ubiquitylation is dependent on RNF8 and Ubc13 which extend pre-existing ubiquitin modifications to K63-linked chains. Here we demonstrate that HUWE1 depleted cells showed reduced recruitment of RNF168 and 53BP1 to sites of DNA damage, two factors downstream of RNF8 mediated histone H1 poly-ubiquitylation, while recruitment of MDC1, which act upstream of histone H1 ubiquitylation, was not affected. Our data show that histone H1 is a prominent target for ubiquitylation after UV-induced DNA damage. Our data are in line with a model in which HUWE1 primes histone H1 with ubiquitin to allow ubiquitin chain elongation by RNF8, thereby stimulating the RNF8-RNF168 mediated DDR.

Highlights

Ubiquitylation[17,18], SUMOylation[19,20,21] and PARylation[22,23], likely to allow swift and reversible regulation of the UV-DNA damage response (DDR)
We identified several peptides from proteins that were previously described to be ubiquitylated in response to DNA damage, including XPC19,24, DDB234, and FANCD235 (Supplemental Fig. S1c), providing the proof of principle of this approach
In line with previously reported data[36,37], we observed a 1.5 fold UV-induced increase in di-Gly modified ubiquitin peptides at lysine 6 (K6), while the abundance of all other di-Gly modified ubiquitin peptides remained largely unaffected (Supplemental Fig. S1d). This suggests that the overall amount of endogenous K6-linked ubiquitin chains is increased after UV-induced DNA damage, indicative for a role of this atypical ubiquitin chain[38] in the UV-DDR

Summary

Introduction

Ubiquitylation[17,18], SUMOylation[19,20,21] and PARylation[22,23], likely to allow swift and reversible regulation of the UV-DDR. Ubiquitylated peptides can be enriched by immunopurification using antibodies that recognize this di-Gly remnant, which will increase the identification efficiency of ubiquitylation sites on proteins by MS29–31. We propose a model in which HUWE1 primes histone H1 by ubiquitylation to provide substrates on which RNF8 can generate poly-ubiquitin chains. In line with this model, depletion of HUWE1 results in a reduced damage signalling, as shown by the decreased recruitment of RNF168 and 53BP1 to sites of DNA-damage, while MDC1 and RNF8 remained unaffected, indicating that HUWE1 affects the RNF8-RNF168 pathway downstream of RNF8

Methods

Results

Conclusion