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DNA Damage Triggers SAF-A and RNA Biogenesis Published online 16 July 2014 Nucleic Acids Research, 2014, Vol. 42, No. 14 9047–9062 doi: 10.1093/nar/gku601 DNA damage triggers SAF-A and RNA biogenesis factors exclusion from chromatin coupled to R-loops removal Sebastien´ Britton1,2,3,†, Emma Dernoncourt1,2,3,†, Christine Delteil1,2,3, Carine Froment1,2, Odile Schiltz1,2, Bernard Salles1,2, Philippe Frit1,2,3,* and Patrick Calsou1,2,3,* 1CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), BP 64182, 205 route de Narbonne, F-31077 Toulouse, Cedex 4, France, 2Universite´ de Toulouse, UPS, IPBS, F-31077 Toulouse, France and 3Equipe Labellisee´ Ligue Nationale Contre le Cancer Received March 11, 2014; Revised June 01, 2014; Accepted June 23, 2014 ABSTRACT INTRODUCTION We previously identified the heterogeneous ribonu- Deoxyribonucleic acid (DNA) double-strand break (DSB) cleoprotein SAF-A/hnRNP U as a substrate for DNA- is the most toxic type of DNA damage. If improperly re- PK, a protein kinase involved in DNA damage re- paired, DSBs can cause cell death or mutations and gross sponse (DDR). Using laser micro-irradiation in hu- chromosomal rearrangements promoting cancer develop- man cells, we report here that SAF-A exhibits a two- ment (1–4). In mammalian cells, DSBs initiate a global DNA damage response (DDR) to overcome their toxicity phase dynamics at sites of DNA damage, with a and maintain genome stability. DDR includes lesions detec- rapid and transient recruitment followed by a pro- tion, checkpoint activation, modulation of gene expression longed exclusion. SAF-A recruitment corresponds to and DNA repair (5–9). DDR defects manifest as a variety its binding to Poly(ADP-ribose) while its exclusion of human diseases, including neurodegenerative disorders, is dependent on the activity of ATM, ATR and DNA- immunodeficiency, infertility and cancer (5). PK and reflects the dissociation from chromatin of Another component of the DDR is local transcription SAF-A associated with ongoing transcription. Hav- arrest triggered by DNA breaks (10–13). More generally, ing established that SAF-A RNA-binding domain re- an expanding aspect of the DDR is its connection with ri- capitulates SAF-A dynamics, we show that this do- bonucleic acid (RNA) metabolism. Indeed, the DNA dam- main is part of a complex comprising several mRNA age activated kinases ATM or ATR phosphorylate numer- biogenesis proteins of which at least two, FUS/TLS ous proteins involved in RNA metabolism (14,15) and links with the DDR have been established for several members of and TAFII68/TAF15, exhibit similar biphasic dynam- the heterogeneous ribonucleoprotein (hnRNP) family (16), ics at sites of damage. Using an original reporter for RNA-binding proteins (RBPs) (17–25) or pre-RNA pro- live imaging of DNA:RNA hybrids (R-loops), we show cessing factors (26,27). Moreover, RNA-processing factors a transient transcription-dependent accumulation of are major mediators of genome stability, some of them by R-loops at sites of DNA damage that is prolonged preventing interactions between the nascent RNA and tem- upon inhibition of RNA biogenesis factors exclusion. plate DNA (R-loops) (28–33) which are relevant source of We propose that a new component of the DDR is DNA breaks (33,34). an active anti-R-loop mechanism operating at dam- We and another group have identified SAF-A/hnRNP U aged transcribed sites which includes the exclusion (hereinafter referred to as SAF-A), as a substrate for DNA- of mRNA biogenesis factors such as SAF-A, FUS and PK, a key protein kinase involved in DSB repair by non- TAF15. homologous end-joining (NHEJ) (35,36). In NHEJ, DNA- PK operates together with the DSBs sensor Ku70/80 het- erodimer and the XRCC4/DNA ligase IV ligation com- plex (37). SAF-A is an abundant nuclear protein found in *To whom correspondence should be addressed. Tel: +33 561 175 970; Fax:+33 561 175 933; Email: [email protected] Correspondence may also be addressed to Philippe Frit. Tel: +33 561 175 937; Fax:+33 561 175 933; Email: [email protected] †The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors. Present address: Bernard Salles, TOXALIM (Research Centre in Food Toxicology), UMR 1331 INRA/INP/UPS, 180 chemin de Tournefeuille, F-31027 Toulouse Cedex 3, France. C The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 9048 Nucleic Acids Research, 2014, Vol. 42, No. 14 hnRNP particles and contains both DNA-binding domain RNase HI including a 5 start codon in a strong kozak se- (DBD) and RNA-binding domain (RBD) (38,39)(Figure quence and a 3 in frame nuclear localization signal from 1A). The HNRNPU gene coding for SAF-A is essential SV40 large T antigen (NLS) were generated by gene syn- for cell viability (40) and the protein participates in chro- thesis (GeneArt, LIfe Technologies). The RNase HI-NLS matin organization and transcription repression in special- sequences were recovered by HindIII and AgeI digestion ized territories (41,42). SAF-A is implicated in several as- and cloned together with AgeI and NotI-digested mCherry pects of RNA metabolism, including transcription elonga- from pmCherry-C1 (Clontech) between HindIII and NotI tion through interaction with nuclear actin and RNA poly- restriction sites of pICE, a new synthetic plasmid allow- merase II (43,44), RNA stability control (45) and alternative ing doxycline-inducible expression and conferring to hu- splicing through regulation of U2 snRNP maturation (46). man cells resistance to puromycin (47). A control plas- Here, we investigate further the involvement of SAF-A in mid expressing NLS-mCherry was generated by replac- the DDR. We document the local post-damage exclusion ing RNase HI cDNA by annealed NLS-S and NLS-AS of an RBP complex including SAF-A and at least two of its oligonucleotides cloned between HindIII and AgeI. partners FUS/TLS and TAFII68/TAF15, which is uncou- pled from their initial poly(ADP-ribose) (PAR)-dependent PAR-binding assay recruitment at these sites. In addition, we present several results supporting that RBP exclusion is part of an anti- For experiments carried in HEK293T, 140-mm dishes were R-loop mechanism operating at DNA damage sites. There- seeded with 5 million cells 2 days before transfection with fore, the present data further substantiate the links between lipofectamine 2000 (Thermo scientific) according to man- RBPs, the DDR and genome stability. ufacturer’s instructions using 20 ␮g of plasmid DNA cod- ing for each FLAG-GFP tagged constructs. Two days af- MATERIALS AND METHODS ter transfection, cells were collected, washed in phosphate- buffered saline (PBS) and lysed 15 min on ice plus 5 min Cell culture at room temperature in 300 ␮l of lysis buffer [10-mM Human HT1080 fibrosarcoma cells, human U2OS os- Tris-HCl pH 7.8, 150-mM NaCl, 1-mM ethylenediaminete- teosarcoma cells (ECCAC, Salisbury, UK) and human traacetic acid (EDTA), 0.5% NP-40] containing 0.2-mg/ml HEK293T were grown in Dulbecco’s modified Eagle’s RNase A and protease and phosphatase inhibitors (HALT, Thermo Scientific). Extracts were then clarified by 4-min medium medium (Fisher Scientific, Illkirch, France) sup- ◦ plemented with 10% foetal calf serum (Lonza, Basel, centrifugation at 14 000 rpm at 4 C. Supernatant diluted Switzerland), 2-mM glutamine, 125-U/ml penicillin and with 400-␮l dilution buffer (20-mM Tris-HCl pH 7.8, 150- 125-␮g/ml streptomycin. All cells were grown in a humidi- mM NaCl, 1-mM EDTA, 0.05% NP-40 containing pro- ◦ tease and phosphatase inhibitors (HALT, Thermo scien- fied atmosphere, at 37 C with 5% CO2. tific) was incubated 4 h at 4◦C on gentle shaking with 50 ␮l of GFP-trap magnetic beads (Chromotek). Beads Plasmids and DNA manipulations were washed three times with 500-␮l stringent washing A list of primers used in the study is provided in Sup- buffer (20-mM Tris-HCl pH 7.8, 500-mM NaCl, 1-mM plementary Table S1. All DNA constructs were confirmed EDTA, 0.05% NP-40 buffer containing protease and phos- mutation free as tested by DNA sequencing. pEGFP- phatase inhibitors (HALT) and once in dilution buffer. N1-FLAG plasmid containing a FLAG tag with a start For experiments carried in HT1080, two 140-mm dishes codon and a strong kozak sequence between BamHI and were seeded with 2.5-million cells GFP-sorted for stable ex- MluI was generated by insertion of annealed FLAG-S pression of FLAG-GFP,SAF-A-WT-FLAG-GFP,SAF-A- and FLAG-AS oligonucleotides between BamHI and AgeI dRBD-FLAG-GFP and SAF-A-RBD-FLAG-GFP. After restriction sites of pEGFP-N1 (Clontech). For expres- two days, cells were collected by scrapping and sonicated (24 sion of SAF-A-FLAG-GFP, SAFA was sub-cloned into pulses of 2 s and 30 of intensity, Vibra-cell sonicator) in 1 ml pEGFP-N1-FLAG between BamHI and MluI after am- of GFP-trap lysis buffer [20-mM Tris-HCl pH 7.8, 1-mM plification by polymerase chain reaction (PCR) with SAF- EDTA, 150-mM NaCl, 0.1% NP-40 with HALT protease A-F and SAF-A-R primers and using pcDNA3-SAF-A- and phosphatase inhibitors Cocktail (Thermo scientific) FLAG (36) as a matrix.
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