oehgn l ale e ,4,20 oehgnN Denmark. N, Copenhagen 2200 4., of 5, University Vej Centre, Maaløes Innovation Ole & Copenhagen, Research Biotech address: *Present 310-8512, Ibaraki Mito, 2-1-1, Bunkyo Japan. University, Ibaraki Science, Biological oy erpltnUiest,Hcij,Tko1209,Japan. 192-0397, Tokyo Hachioji, University, Metropolitan Tokyo ohd-ooco ay-u yt 0-51 Japan. University, 606-8501, Kyoto Kyoto Center, Sakyo-ku, Biology Yoshida-konoecho, Radiation Network, Regulatory nvriy ohd-ooco ay-u yt 0-51 Japan. 606-8501, Kyoto Sakyo-ku, Yoshida-konoecho, University, oga .Oliveira V. RNF20 Douglas with interaction through by remodeling recombination chromatin homologous regulates FACT Histone ARTICLE RESEARCH ß eevd2 a 03 cetd9Nvme 2013 November 9 Accepted 2013; May 29 Received { radiomimetic (IR), radiation ionizing to 1 sources, of exposure plethora a by as caused be form can such they deleterious and most damage, the DNA are of (DSBs) breaks double-strand DNA INTRODUCTION SUPT16H RNF20, ubiquitylation, recombination, H2B Homologous remodeling, Chromatin WORDS: KEY Indeed, that pathway. indicating RNF20 RNF20-mediated cells, to bound for RNF20-deficient directly to essential SUPT16H similar is fashion SUPT16H mitomycin- a and a (IR) in radiation SUPT16H, ionizing C This to activity. of sensitivity HRR enhanced Depletion decreased to consequently, led HRR. and, accumulations in of repair defects of pronounced steps caused FACT, early of chromatin key component the a of as FACT presence in chaperone how the histone in the in unresolved identified DNA we DNA remains Here, the the structure. it approaches access itself However, can RNF20 repair. homologous structure early proteins during that chromatin (HRR) eukaryotes so regulates repair H2B, histone RNF20 recombination of ligase ubiquitylation ubiquitin through E3 The ABSTRACT cuuain swl sta fRD1adBC1 tstsof sites and at BRCA1, interaction and RAD51 its of abolished that as RNF20 well as in accumulation, domain RING-finger niaeapiayrl fFC nRF0rcutetadthe and recruitment RNF20 results HRR. in by of FACT initiation our for of counteracted remodeling together, chromatin role resulting effectively Taken primary defective a relaxation. the were indicate observation, SUPT16H this enforced of with remodeling Consistent chromatin phenotypes the SNF2h. of factor accumulation SUPT16H impaired of ubiquitylation thereby, H2B depletion and RNF20-mediated RNF20 Furthermore, in defects and SUPT16H. pronounced FACT caused with of RNF20 interaction mediator DNA-damage-induced of a for been dispensable as has was of association, transcription which localization PAF1, in Interestingly, the implicated intact. whereas remained (DSBs), SUPT16H breaks double-strand DNA uy Kobayashi Junya uhrfrcrepnec ([email protected]) correspondence for Author iiino eoeRpi yais aito ilg etr Kyoto Center, Biology Radiation Dynamics, Repair Genome of Division 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,7372doi:10.1242/jcs.135855 763–772 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 1 ioiYanagihara Hiromi , 1 khr Kato Akihiro , nvivo in 1 ysk Nakamura Kyosuke , n uaina the at mutation and , 3 etrfrPirt Areas, Priority for Center 1 ucioSaito Yuichiro , 4 eatetof Department 2 iiinof Division 1 ioh Tauchi Hiroshi , 1, ,Tuoh Ikura Tsuyoshi *, ta. 02.Peiul,w n te rushv hw htthe that shown have groups other and we Previously, (Mallette 2012). DSBs recruit of al., to sites et functions to 53BP1, and as IR such components, to occurs subsequent which exposure 2004), following al., minutes et (Morrison within H2AX ubiquitylation variant and histone phosphorylation the of including (PTMs) 2011), modifications al., post-translational et by (Zhou led often dynamics Such are 2011). changes Crabtree, and and (Suganuma Hargreaves remodeling processes 2011; early chromatin Workman, key the the are and which accessibility (Ciccia al., in DNA and injuries 2010), et detrimental Murr, such 2010; Limoli circumvent the Elledge, in to exists 1988; order however, in (DDR), (Ward, cell response damage stress DNA The oxidative 2002). or compounds, eitn nietascaino ATwt N2 (Orphanides RNF20 thus with region, FACT promoter 2003; the of al., to association et RNF20 indirect recruits mediating (Ng and 2012), al., active et PAF1 in Piro 2009). FACT Roeder, with and Kim interacts 2006; physically al., by et elongation (Pavri H2B transcription PAF1 the regulator of by engaged ubiquitylation is which requires RNF20–RNF40, elongation process, 2008; and and this transcription In (Winkler 2013). breathing’ al., the al., et Hondele ‘nucleosome 2012; of Formosa, et 2011; configuration enhances Luger, open FACT (Heo the support that stabilizes studies template polymerase recent model RNA However, nucleosomal the 2003). transcribing al., the the et of Belotserkovskaya through passage the nucleosome II the for from been allow dimer has H2A–H2B FACT to one transcription, displace In to 1998). proposed al., has et SSRP1, Orphanides and Luger, 2011; and Spt16) (Winkler reorganization yeast nucleosome comprising in of implicated been ortholog complex (facilitates human heterodimeric (the FACT SUPT16H chaperones, a and histone transcription), (Winkler the chromatin machinery of One cellular 2011). for Luger, template an provide DNA to still accessible nucleosomes individual is reorganize site and architecture damage DNA the to RNF20 of elusive. recruitment responsible the is that for mechanism the However, DSBs. of rejoining during the remodeling chromatin in operates H2AX- that alternative pathway an independent is there the that ( indicating of accumulation, independently 53BP1 phosphorylation functions RNF20 H2AX It that 2011). canonical shown al., been et also (Nakamura RAD51 has by and followed DSBs, BRCA1 of of sites factor, accumulation to in SMARCA5), remodeling result as to chromatin known (also shown ATP-dependent SNF2h been an has PTM al., of histone the et recruitment this (Kao of DDR, elongation histone the transcription ortholog In of the 2004). regions an monoubiquitylates at is Lys123 and on Bre1, RNF20 H2B protein and 2010). yeast DSBs al., al., budding et of et Moyal sites 2011; Chernikova al., at 2011; et (Nakamura accumulates ubiquitylation H2B RNF20 induces ligase E3 ubiquitin itn hprnsaekont osnhg-re nucleosome high-order loosen to known are chaperones Histone 4 n esiKomatsu Kenshi and 2 aaioOkada Masahiro , c 2X n h resulting the and H2AX) 3 , 1, { 763

Journal of Cell Science rtisa h ie fDB Nkmr ta. 01 Moyal the 2011; that shown al., have et experiments (Nakamura yeast DSBs Similarly, of 2011). al., sites et the remodeling (HRR) at chromatin repair recombination proteins promote homologous of to accumulation the RNF20–RNF40 and by driven lacking is cells in 2006). defective Sims, and are (Reinberg and PAF1 ubiquitylation recruitment functional RNF20 H2B transcription, in resulting result, the a As 1998). al., et ARTICLE RESEARCH 764 against antibodies using and cells DNA immunostained SUPT16H to first response in we SUPT16H damage, of involvement to the response investigate in To RNF20 of recruitment damage the DNA for required is SUPT16H in RESULTS observed that on from depend mechanism not distinct RNF20 did transcription. a FACT for suggesting and Intriguingly, RNF20 PAF1, responsible HRR. between subsequent was interaction and such DNA chromatin which the during to RNF20, localization SUPT16H proteins DSBs, with of HRR generation upon interacted and that in show FACT RNF20 results of Our role repair. of the investigated MAT accumulation herein the we the observed regard, at as that of DSBs, changes In of 2005). recruitment H2B sites the histone for to RAD51, by prerequisite as such a proteins, HRR is displacement nucleosome ntal oaie otencel,cnitnl omdfoci, which formed SUPT16H, consistently that nucleoli, with the found colocalized partially to We which localizes exposure. initially IR following 2 HB10,wihi h bqiyaintre fRNF20, of target lysine ubiquitylation on the is ubiquitylation which H2B (H2BK120), Histone 120 RNF20. of downstream 1D). (Fig. sensitization further any both cause of not depletion concurrent did and factors IR, RNF20-depleted to sensitive or equally supplementary were SUPT16H- (supplementary cells Interestingly, 8; outcome S1E). immunofluor- similar Our and Fig. a 8). material and 7 showed whole-cell 7 experiments lanes lanes in panel, escence panel, right observed S1D, right Fig. decreased, were material dramatically 1C, changes (Fig. were significant extracts fraction no chromatin short RNF20- whereas the with RNF20 and in (shSUPT16H), cells SUPT16H control levels transfected against we (shRNA) both RNA when the in hairpin in However, SUPT16H observed cells. of presence was depleted the 4), fraction and 3 chromatin lanes panel, small in (left with shown 1C As transfected Fig. (siRNF20). cells RNF20 examined RNF20 against in (siRNA) we exposure of RNA RNF20, interfering IR on level upon test rely levels To similar would SUPT16H S1C). function a Fig. SUPT16H material by whether supplementary 1B; accompanied (Fig. was accumulation the sites results at SUPT16H the damage of in accumulation kinetics observed the recruitment Interestingly, colocalization their earlier. limited described in the difference for this account that S1B). might possible Fig. material is (supplementary it period later Thus, prolonged a a at that for occurred but formation focus time, revealed SUPT16H DSBs. whereas S1A), hour, analysis first of the Fig. immunostaining sites the time-lapse material c at a accumulates (supplementary However, SUPT16H tracks that confirming induced damage to the at us SUPT16H allowed endogenous DNA focus of which portion of micro-irradiation, distinct a focused in kinetics observe using DSBs different by localized cell spatially the the induced to we due Furthermore, formation. be could colocalization 2Xfc ekdsotyatrI xoue eraigwithin decreasing exposure, IR after shortly peaked foci H2AX nDArpi,i a lobe hw htHBubiquitylation H2B that shown been also has it repair, DNA In enx esndta UT6 ol fetevents affect would SUPT16H that reasoned next We c 2X elkonmre o DSBs, for marker well-known a H2AX, c 2X(i.1) hslimited This 1A). (Fig. H2AX a ou Tuuae al., et (Tsukuda locus ta. 01 oa ta. 01.Smlrrslswr observed were results previous Similar 2011). with (Nakamura al., inhibitors et consistent Moyal transcription is 2011; different al., observed of This et intact presence still 8). was the ubiquitylation H2B was and in IR-induced that 6 exposure showing evidence lanes IR 2B, although following RNF20, (Fig. and mg/ml) association SUPT16H Surprisingly, 0.2 between interaction and their DSBs. overall mg/ml the chromatin (0.03 affect of ActD did the an of in generation concentrations was increasing RNF20 the there and contrast, following SUPT16H By that the 6). between in and showed 5 association even lanes analysis 2A, inhibitor, (Fig. co- transcription damage However, with DNA a 4). treatment of did presence (ActD), and after PAF1 reduced 3 D dramatically lanes actinomycin conditions, was 2A, interaction unstressed (Fig. in their SUPT16H under our with Similarly, RNF20 that 2009). associate Roeder, by with and showed intermediated Kim 2006; and results associates al., indirect et (Pavri is complex PAF1 interaction FACT their transcription, the PAF1 Although from independently DDR, in and RNF20 with interacts SUPT16H uha A5 n RA,t h aaests Transient sites. damage in impairment the an to to led RNF20 BRCA1, or SUPT16H either and proteins, of knockdown RAD51 HRR of as recruitment of such the absence compromise the whether would speculated SUPT16H we agents, DNA-damaging to sites. at damage modification DNA histone for the subsequent crucial the is and SUPT16H Taken accumulation, that S1H). RNF20 Fig. indicate material observations those supplementary these than away 1E; together, pronounced further (Fig. less positions control were the in differences in whereas those the sites, sites at the DSB dimethylation from the H3K4 to and next endonuclease. reduction ubiquitylation region a I-SceI H2BK120 to led the the SUPT16H in of DSBs with abrogation the that transfection DR-GFP and revealed A Analysis cells, by 2010). HeLa generated al., into et were introduced (Niida was system (ChIP) cassette reporter DSB assay the H2B inducible immunoprecipitation at an chromatin accumulation were the their with a analyzed by dimethylation whether we sites DSBs, H3K4 damage the determine subsequent at them place to was the taking of (H3K4) and each 4 Moreover, a lysine ubiquitylation or on observed. Similarly, dimethylation siRNA S1F,G). H3 also of histone Fig. of a sets material reduction either different (supplementary with three downregulation separately RNF20 of after mixture decreased also was htbt rtisaecmoet facmo aha nthe in pathway indicating common HRR. a activity, through DSBs of HRR of components in rejoining are impairment proteins did further both proteins any that both to of lead in activity depletion not significantly HRR concurrent was 1F, cells, Importantly, Fig. RNF20-depleted reduced. in in as shown well as as and SUPT16H-, construct 1999), reporter al., SCneo-containing assessment HRR et an foci specific (Johnson used more that reduces we a activity, BRCA1 For reported HRR significantly 1999). of been al., and dysfunction et has (Moynahan BRCA1 activity RAD51 It and S1L). IR-induced RAD51 Fig. or both material show BRCA1 (supplementary not of did performed either also RNF20 we lacking and formation cells confirmation, that RNF20 observed and further detect RAD51, For to S1K). immunofluorescence when (supplementary Fig. observed foci was disturbance material BRCA1 cell Fig. no and material whereas (supplementary S1I,J), RAD51 control respective their IR-induced to compared of formation the wn otefc htSP1Hdpee el eesensitive were cells SUPT16H-depleted that the to Owing c H2AX ora fCl cec 21)17 6–7 doi:10.1242/jcs.135855 763–772 127, (2014) Science Cell of Journal

Journal of Cell Science EERHARTICLE RESEARCH rnfce el ihsRAaantPF (siPAF1) PAF1 against siRNA next we with repair, DNA in cells RNF20 and transfected SUPT16H of interaction interaction. although their transcription, induces DNA be still of might damage disturbance interaction DNA the possible RNF20 by is and for SUPT16H it accounted in Thus, decline S2A). some Fig. that material against (supplementary blotting and RNF20 anti-SUPT16H of immunoprecipitation upon anti- using immunostained later, hours 2 and Gy, 5 with with irradiated colocalized were partially 10 RNF20 which bar: Gy, Flag-tagged Scale 5 with antibodies. to transfected anti-Flag exposure cells and after U2OS SUPT16H hours damage. 2 DNA cells following damage. U2OS RNF20 DNA in to response foci in discrete RNF20 formed of recruitment SUPT16H-dependent 1. Fig. h fiinyo R.HL el ihteSnorpre eecntutwr umte oISe-nue laae n ek ae,G418-resitant later, weeks 2 and cleavage, r I-SceI-induced together, to both or submitted RNF20, were or construct SUPT16H either of reporter means and Depletion SCneo (F) ChIP are anti-monoubiquitylated-H2BK120 break. the site. Results the DNA with means damage counted. the by are cells of the were immunoprecipitated site Results HeLa at colonies was the exposure. HRR. H3K4 from chromatin IR of bp methylated Soluble following efficiency 180 and performed. days of the H2B was position 10 ubiquitylated a DSB assay of at I-SceI-induced colony antibodies, after accumulation of a anti-dimethylated-H3K4 hours the by sites 2 compromised determined the and SUPT16H as at siRNF20, of IR, analysis analysis. or Depletion to immunoblotting shSUPT16H (E) cells either with MCF7 experiments. with assayed of independent transfected was sensitivity were fraction the cells chromatin enhanced HeLa their SUPT16H cells. Gy, RNF20-depeted 5 in with unchanged irradiation remained SUPT16H of accumulation oeaiewehrPF ol ersosbefrthe for responsible be would PAF1 whether examine To 6 ...o he needn xeiet.** experiments. independent three of s.e.m. m .()Acmlto fRF0a h hoai a mardi UT6-eltdcls whereas cells, SUPT16H-depleted in impaired was chromatin the at RNF20 of Accumulation (C) m. i A1(ioe l,21) nspoto hsie,H3K4 idea, this drastically of was support transcription, In active 2012). for marker al., a et RNF20 methylation, (Piro and FACT 2D), PAF1 of is interaction (Fig. which via indirect DSBs decreased, the of slightly the and because was to probably interaction SUPT16H mobilization overall their between the not or although did interaction 2C), (Fig. PAF1 IR-induced RNF20 of the depletion abrogate Intriguingly, immunopreci- experiments. performed and pitation S2B) Fig. material (supplementary A muotiiguigat-UT6 nioyrvae htSUPT16H that revealed antibody anti-SUPT16H using Immunostaining (A) P c , tblnwsue salaigcnrl D elto of Depletion (D) control. loading a as used was -tubulin 0.01. ora fCl cec 21)17 6–7 doi:10.1242/jcs.135855 763–772 127, (2014) Science Cell of Journal c 2X cl a:10 bar: Scale H2AX. m .()SP1Hclclzdwith colocalized SUPT16H (B) m. 6 ...o three of s.e.m. educed 765

Journal of Cell Science EERHARTICLE RESEARCH 1) hs bevtossgetta h ehns of mechanism one different 766 the a be that might RNF20 homologous suggest and FACT which observations between Fig. in interaction material These did S/G2, supplementary 2F, of depletion S1K). (Fig. was place changes although IR takes minor recombination to cells, some sensitization siPAF-transfected significant induce to in further contrast significant no in observed knockdown a Also, activity, concomitant their 2E). display HRR or (Fig. depletion, of not RNF20 efficiency or SUPT16H did the in cells perturbation revealed assay siPAF1-transfected reporter DR-GFP that the using Fig. analysis material Further (supplementary S2C). cells siPAF1-transfected means in are Results reduced counted. were means cells are GFP-positive Results later, anti-SUPT16H exposure. days using IR immunostained 2 following later, and hours la days DSBs, 2 hours I-SceI-induced and IR- 2 ** Gy, to and formed experiments. 5 subjected Gy, independent RNF20 with were 5 and irradiated construct with were SUPT16H siPAF1 gene irradiated Both with reporter were 10 (D) transfected cells antibody. bar: cells Scale U2OS HeLa anti-RNF20 PAF1. antibodies. siPAF1-transfected against of anti-Flag cells. blotted absence irradiated the and Depleti in in (C) antibody foci antibody. RNF20 anti-SUPT16H induced anti-RNF20 was and with against damage SUPT16H performed blotted DNA between and was following antibody interaction co-immunoprecipitation RNF20 anti-SUPT16H the (30 with using abolish ActD performed SUPT16H not or was of DMSO did fraction Interaction with chromatin treated (B) the antibody. were of anti-PAF1 cells co-immunoprecipitation HeLa against transcription. blotted with and (30 associated antibody ActD and not or PAF1 anti-SUPT16H DMSO from with with performed independently treated were was DDR, cells in HeLa RNF20 treatment. ActD with following interacts SUPT16H 2. Fig. P , .1 F iA1tasetdclsddntso n infcn estzto oI xoue sdtrie yacln sa 10 assay colony a by determined as exposure, IR to sensitization significant any show not did cells siPAF1-transfected (F) 0.01. m .()Argto fPF i o infcnl fetteefcec fHR eaclssal rnfce ihteDR-GFP the with transfected stably cells HeLa HRR. of efficiency the affect significantly not did PAF1 of Abrogation (E) m. 6 ...o he needn experiments. independent three of s.e.m. m /l ro oepsr o5G,ad2husatrirdain co-immunoprecipitation irradiation, after hours 2 and Gy, 5 to exposure to prior g/ml) m 2) diinly 2Xdpeinb iN i not did micro-irradiation siRNA laser SUPT16H- after by both accumulation depletion Fig. in SUPT16H H2AX material foci affect (supplementary Additionally, of cells 53BP1 number S2E). RNF20-depleted of expected and recruitment the H2AX-dependent formed it Moreover, that of observed modifications IR-induced and S2D,E). SUPT16H affect through depleted not DSBs we did end, of that rejoining To H2AX. the in factors. SUPT16H two these essential of is association PAF1 indirect where the transcription, for during triggered that from /lad200 and g/ml c enx ogtt lcdt h ucinlrltosi of relationship functional the elucidate to sought next We H2AX. A h neato ewe UT6 n A1wsabrogated was PAF1 and SUPT16H between interaction The (A) ora fCl cec 21)17 6–7 doi:10.1242/jcs.135855 763–772 127, (2014) Science Cell of Journal m /l ro oepsr o5G,ad2husatrirradiation, after hours 2 and Gy, 5 to exposure to prior g/ml) c 2X(upeetr aeilFig. material (supplementary H2AX 6 ...o three of s.e.m. no PAF1 of on ter, and

Journal of Cell Science sabnigpafr o h ont 2lgs,i a been has Bre1 yeast it of ligase, N-terminus E2 the to cognate binds the Rad6 for yeast serve that platform to reported believed binding generally a is the ligases as Although E3 SUPT16H. of 2001; and domain RNF20 al., crucial RING-finger is between et domain interaction (Ito RING-finger the that ligase for showed ubiquitin 2007), al., of et activity Mailand the for responsible upre yimnsann fSP1HadRNF20. and SUPT16H of further immunostaining was RNF20 evidence Intriguingly, This by 2003). al., et supported Hwang 2009; al., et (Kim IGfne oan hc sesnilfrrcutetof recruitment repair. DNA for in the proteins essential HRR at downstream RNF20 is the and and which SUPT16H RNF20 of domain, interaction of RING-finger idea the enforce N2,mgtfnto neednl rmtecanonical the with from together independently SUPT16H, that function c suggest might al., results RNF20, et These (Nakamura our cells cells with 2011). RNF20-depleted consistent shSUPT16H-transfected in is observation in which previous S2G), period Fig. longer material (supplementary a Interestingly, for S2F). persisted Fig. material (supplementary ARTICLE RESEARCH 4) iia eut eeosre nclsperae iha with pretreated hypotonic cells and Fig. (TSA), in A trichostatin material observed inhibitor, and deacetylase were supplementary histone RAD51 results 4C; were Similar (Fig. as S4B). formation S4A), 7 4, foci Fig. chromatin 3, material BRCA1 lanes the supplementary 4B, (Fig. 8; SUPT16H- at chloroquine in and with RAD51 alleviated pretreated were with cells and ubiquitylation, depleted Consistent nucleosome H2B RNF20 4A). and a (Fig. of fraction, chloroquine, otherwise accumulation not with this, but pretreated recover agent, to were relaxation RNF20 seemed cells cells IR-induced SUPT16H-depleted when that in by observed reversed formation be focus We could SUPT16H DSB relaxation. of of lack impairment nucleosome the the machinery from whether examined resulting transcription we repair of the 2008), passage from al., histones the et core (Heo for sequester allow to to FACT nucleosome of function the Given chromatin by counteracted relaxation is phenotype SUPT16H-defective The RNF20 and a (RNF20 both using RNF20 analyses domain of Interestingly, 9). RING-finger more mutant and cells, C-terminal-located 4 in deletion lanes RNF20 its experiment, 3C, of this (Fig. C-terminus to with the Consistent specifically to al., 2013). et bound al., Evans 2011; SUPT16H et al., et Hondele (Myers deficient to findings was 1998; seems previous 2011), which with functionally 3B), al., line (Fig. in et RNF20 be Myers the with interaction 2013; its al., for of essential et acids (Hondele (amino 3A). most cluster latter (Fig. mutation the comprising that proteins revealed lacking C-terminus, 758–1047), RNF20 or mutants, N- SUPT16H and different the Flag-tagged either generated SUPT16H using analysis subsequently both Domain we of RNF20, mutants between association and the for SUPT16H responsible domain domain the finger determine RING To RNF20 to binds SUPT16H usiuina ytiepsto 2 (RNF20 922 position cysteine at substitution A5 n RA oiwsas eetv nRNF20 in defective of is also formation was accumulation foci IR-induced RNF20 BRCA1 Similarly, that and SUPT16H. supplementary RAD51 suggesting by 3D; (Fig. S3), mediated unaffected Fig. remained material SUPT16H full-length IR-induced formation of cases, both introduction in foci the However, normal 3D). after whereas (Fig. observed RNF20 cells, were RNF20-depleted foci in RNF20 foci RNF20 induced 2Xdie D pathway. DDR H2AX-driven C922S tasetdcls(i.3) hs observations These 3E). (Fig. cells -transfected D RING n RNF20 and C922S D RING aldt etr IR- restore to failed ,adaserine a and ), C922S c ,wihis which ), 2Xfoci H2AX D RING earmcieyt ie fDB nodrt aiiaeHRR. facilitate the to order result, in DSBs a the of of sites RNF20 as recruitment to the mediates machinery for and, important repair is FACT damage Taken that remodeling that DNA events. chromatin such indicate following for sites, results accumulation dispensable damage these again at together, accumulation was SNF2h PAF1 for whereas that crucial suggesting to sites, is irradiated the failed SUPT16H SNF2h at normally also cells, localize PAF1-depleted to SUPT16H micro- seemed in lacking Conversely, the SNF2h. laser cells cells accumulate at RNF20-depleted Importantly, in accumulation observations, 4D). reduced (Fig. SNF2h substantially was these al., that sites damage revealed et with analysis (Nakamura irradiation Consistent ubiquitylation regulated is H2B SNF2h 2011). Nakanishi that RNF20-dependent DNA-damaging shown 2012; previously al., to have by et We Smeenk sensitivity 2007). 2009; al., al., high et al., et et with (Yoshimura (Santos-Rosa cells agents complex confers remodeling 2003), chromatin ISWI the Fig. material (supplementary 1999) and S4C,D). Krajewski, (Bakkenist relaxation 2003; chromatin Kastan, promote also which buffer, ATi rti ope epnil o nucleosome for responsible complex protein a is FACT DISCUSSION rncito Pr ta. 02.Smlry thsbe reported been as has well it as Similarly, H2B 2012). PAF1, al., resulting functional et the on al., (Piro transcription and dependent et (Pavri recruitment are ubiquitylation ubiquitylation RNF20 binds H2B Therefore, then, induce 2006). PAF1, to interaction. RNF20–RNF40 subsequently physical and to elongation, through II PAF1, complex Pol is RNA FACT recruits of ligase whereby sites model at ubiquitin a recruited proposed first the al. et recruits Pavri RNF20–RNF40. directly FACT whether at repair. DNA RNF20 in recruits protein upstream FACT an as that sites suggesting target focus intact, SUPT16H the was although 3D,E), formation (Fig. this those BRCA1 also in and and RAD51 foci, Mutation of RNF20 of 3C). formation (Fig. the compromised domain domain we RING-finger through C-terminus RNF20 where with its not interacted further physically experiments but SUPT16H was that co-immunoprecipitation SUPT16H, showed This of by S1E). absence an Fig. supported the be material in (supplementary to impaired otherwise seems was formation there RNF20 focus IR-induced of Furthermore, because FACT, pathway. for function upstream HRR in RNF20 together, and same Taken SUPT16H 1F). of the (Fig. participation two cells the the suggest these in results stress these activity DNA of HRR under nor depletion sensitization 1D) further concomitant (Fig. any cause and not did 1B), proteins (Fig. HRR those RNF20 of with colocalized reduction of DNA were a foci following SUPT16H by Interestingly, survival characterized activity. cell reduced, drastically abrogated, stress was SUPT16H phosphorylation. When H2AX and PAF1 of of domain independently remodeling RING-finger HRR chromatin showed the for the DNA We facilitating damage, 1997). with with DNA al., upon interacts MCM and RNF20 et SUPT16H and Wittmeyer 2009), that alpha 2006; al., here al., polymerase et et DNA and (Tan Kim as helicase II 2006; such factors, polymerase al., with replication interacts RNA et and directly (Pavri including it Wittmeyer PAF1 and factors, 2006; 2009), al., protein transcription al., et gene et further (Tan Kim site and 1997; target Formosa, transactions the to DNA recruitment during reorganization eetrprshv hw htdlto fSFh uui of subunit a SNF2h, of deletion that shown have reports Recent hr scnlcigltrtr ntasrpinsuisa to as studies transcription in literature conflicting is There exposure. IR following foci formed SUPT16H that found We ora fCl cec 21)17 6–7 doi:10.1242/jcs.135855 763–772 127, (2014) Science Cell of Journal 767

Journal of Cell Science EERHARTICLE RESEARCH 768 immunoprecipitat to used were RNF2 beads with M2 interacted Flag SUPT16H sequences. (B) recombinant C-terminus. Flag–SUPT16H SUPT16H SUPT16H and C, HA–RNF20 harboring mid-terminus; using SUPT16H Flag-constructs performed MID, was N-terminus; Analysis domain. SUPT16H C-terminus. RING-finger N, its study. RNF20 through this in the used with as interacts mutants respective physically SUPT16H 3. Fig. ihat-A5 n niBC1atbde.()rpeet rnfcinwt mt etr cl a:10 immun bar: irradiation, Scale after vector. hours anti-SUPT16H empty 2 and with and anti-Flag Gy, transfection with 5 represents to immunostained (-) exposure irradiation, to antibodies. after prior anti-BRCA1 hours siRNF20, and with 2 anti-RAD51 transfected and were with Gy, mutants 5 RNF20 Flag-tagged to siRNA-resistant exposure expressing to prior 10 bar: siRNF20, Scale with antibodies. transfected were mutants RNF20 RNF20 neatdwt UT6 hog t IGfne oan h hoai rcin fFa-agdRNF20 Flag-tagged of fractions chromatin The domain. RING-finger its through SUPT16H with interacted RNF20 D D RING 2 RNF20 , n RNF20 and D 3 RNF20 , C922S m .()TeRN-igrdmi fRF0i rca o rprfraino Ridcdfc otiigRD1adBC1 2Scells U2OS BRCA1. and RAD51 containing foci IR-induced of formation proper for crucial is RNF20 of domain RING-finger The (E) m. D RING tasetdcls lhuhI-nue UT6 oirmie nhne.UO el xrsigsRArssatFlag-tagged siRNA-resistant expressing cells U2OS unchanged. remained foci SUPT16H IR-induced although cells, -transfected n RNF20 and N SUPT16H , C922S MID el eec-muorcpttdwt lgM ed.()I-nue N2 oiwr opoie nboth in compromised were foci RNF20 IR-induced (D) beads. M2 Flag with co-immunoprecipitated were cells rSUPT16H or C uat n ute lte gis niH nioyfrRF0dtcin C RNF20 (C) detection. RNF20 for antibody anti-HA against blotted further and mutants A ceai ftedmi rhtcueo N2 n UT6 n their and SUPT16H and RNF20 of architecture domain the of Schematic (A) ora fCl cec 21)17 6–7 doi:10.1242/jcs.135855 763–772 127, (2014) Science Cell of Journal m FL m. fl egh,RNF20 length), (full D C RNF20 , D N RNF20 , ostained D 1 , e 0

Journal of Cell Science hra osgiiatdfeec a bevdi iA1tasetdcls 2Sclswr ie 0mntsatrlsrirdain n tie w stained and irradiation, laser after minutes 30 fixed were cells U2OS cells. siPAF1-transfected anti- in and observed GFP was difference significant no whereas EERHARTICLE RESEARCH A1(i.2.Ti sfrhrspotdb rvosstudies previous in intact by is supported ubiquitylation further H2B such RNF20-dependent is by that Surprisingly, This mediated showing 2011). nor 2). transcription, al., (Fig. domains on et PAF1 functional dependent Myers not its was 2013; of interaction al., most et (Kari encompasses (Hondele binds domain which C-terminal FACT activity its that 3B), through an DSBs (Fig. here transcription of demonstrated formation of upon decreased we RNF20 to However, because the 2011). al., possibly of et radiomimetics, accumulation effect SUPT16H with indirect affects treatment RNF40 of after knockdown the that means are results and counted were cells Foci-positive nuc antibody. their (20 anti-RAD51 later, chloroquine with hours with immunostained treated 2 were and and shSUPT16H IR, Gy, or of 5 * siRNF20 Gy with to experiments. transfected 5 exposure independent cells to to U2OS exposure prior cells. to SUPT16H-depleted chloroquine, in prior with foci treated RAD51 IR-induced were blotting. of shSUPT16H western formation with by transfected analyzed cells was HeLa fraction fraction. chromatin the in (20 10 chloroquine bar: Scale with relaxation. antibodies. treated SUPT16H chromatin were by cells recovered U2OS was cells. phenotype SUPT16H-depleted 4. Fig. c 2Xantibodies. H2AX P , .5 ** 0.05; c 2Xwsue saDAdmg edu.Saebr 10 bar: Scale readout. damage DNA a as used was H2AX m .()Clrqietetetsgiiatyrsudteipie cuuaino N2 n bqiyaino 2 (H2Bub) H2B of ubiquitylation and RNF20 of accumulation impaired the rescued significantly treatment Chloroquine (B) m. P , .1 D cuuaino N2 tlsridcddmg ie a mardi AT n N2-eltdcells, RNF20-depleted and FACT- in impaired was sites damage laser-induced at SNF2h of Accumulation (D) 0.01. c tblnwsue salaigcnrl C hooun ramn ed oasgiiatrcvr nteimpaired the in recovery significant a to leads treatment Chloroquine (C) control. loading a as used was -tubulin m /l,pirt xouet yo R n or ae,imnsandwt niRF0adanti- and anti-RNF20 with immunostained later, hours 2 and IR, of Gy 5 to exposure to prior g/ml), idnssgetta h ehns fitrcinbetween interaction of mechanism in the 2F), (Fig. that cells These suggest 2006). the al., of et findings (Game sensitivity observations previous radiation with the accordance affect 2B,C; PAF1 not of (Fig. abrogation did inhibition Moreover, S2C). transcription reduced Fig. and material slightly supplementary depletion were 4 PAF1 and not lysine both RNF20, on do by and dimethylation SUPT16H H3 results between resulting the such once the interaction 2007), of Importantly, al., level et 2011). (Fink overall previously al., observed those et contradict al., et Moyal (Nakamura inhibitors 2011; transcription different of presence the A hooun ece h oaiaino N2 nSUPT16H-depleted in RNF20 of localization the rescued Chloroquine (A) ora fCl cec 21)17 6–7 doi:10.1242/jcs.135855 763–772 127, (2014) Science Cell of Journal m m. 6 ...o three of s.e.m. t h anti- the ith m lear g/ml), 769

Journal of Cell Science htol hnPF osbemdao fteinteraction level, the transcription robust of a reconstituted observe mediator their they to did possible added assay, was a transcription their RNF20, and mediates PAF, SUPT16H shown exclusively when between have it studies only Previous that transcription. that in unlikely least with is at interact interaction, to it known also proteins, is during H2B both association triggered Although indirect factors. that the two from for these of essential one is different PAF1 a where transcription, is RNF20 and FACT ARTICLE RESEARCH 770 in the reorganization nucleosome phosphorylation-dependent with H2B compared of cells, H2AX the contribution in 2008). remodeling dominant chromatin to the al., the to to ubiquitylation et leading due be (Heo nucleosomes, might DNA difference The the nucleosomal of from accessibility FACT-mediated enhanced H2AX facilitated of phosphorylation dissociation H2AX that shown stability an chromatin However, on of 2007). effect al., replacement no et (Fink a had acid, by glutamic yeast, with 129 that in serine shown phosphorylation has Moyal H2A study another 2011; of this, inhibition al., with H2AX accordance et In from 2011). (Nakamura independent al., et repair is the DNA which in in is RNF20, phosphorylation, FACT with that pathway indicating evidence same with observa- consistent These are S2F). tions Fig. material (supplementary of damage generation after DNA accumulation FACT SUPT16H affect of not did depletion depletion H2AX similarly, of that, after and S2D,E), unchanged Fig. material remained (supplementary chromatin 53BP1 the of accumulation at resulting the and phosphorylation, the that H2AX indicate of results accumulation present The the proteins. to repair-related subsequent leading H2AX, of repair. phosphorylation DNA the in place takes mechanism needed this for is how sufficient investigation elucidate further not help Nonetheless, was to 2006). itself al., by et H2B (Pavri of it presence the that showing rmtdb yfnto fSuf,aHC-oanE3 HECT-domain a is Smurf2, tumorigenesis of that dysfunction shown have by this studies promoted with recent Consistent susceptibility. prediction, cancer could increase pathway SUPT16H conceivably dysfunctional (Cousineau the genomic 2007), mutations sufficient Belmaaza, trigger cancer-promoting H2B and to able accelerate In is to HRR RNF20-dependent activity. in instability defect HRR the compromises as through much depletion so its HRR and transcription. ubiquitylation, initiates in function well-established SUPT16H its outside SUPT16H 4D). (Fig. mediated SNF2h from then and is RNF20 which by independently FACT remodeling, chromatin that but facilitates and suggest activity thereby reorganization nucleosome DSBs, results its through these RNF20 more recruits of together, recruit to Taken generation able S4). transcription. be upon Fig. SUPT16H- might form material FACT the nucleosomal supplementary open suppressed the 4, Thus, (Fig. 1999), phenotype Krajewski, defective and (Bakkenist 2003; solution relaxing hypotonic chromatin Kastan, of and of TSA types form our chloroquine, different manner, agents, open-reorganized with similar treatment a after the In Spt16- 2011). results that al., the of et is mutation that (McCullough levels histone nucleosomes model showed a increased by This which suppressed promoted study, 2012). was genetic phenotype Formosa, defective a 2011; dimer by action Luger, FACT supported H2A–H2B of and byproduct hence, nonessential (Winkler a and, be could nucleosomes displacement open of the stabilizes FACT configuration that suggests which FACT, for proposed nDArpi,tecnnclsgaigptwyi ntae by initiated is pathway signaling canonical the repair, DNA In osqety h rsn bevtosdmntaearl for role a demonstrate observations present the Consequently, been has model accessibility/non-eviction global a Recently, nvitro in experiment. nvitro in td yaohrgophas group another by study hr-ari N lsi (1 plasmid RNA short-hairpin (5 GCUCAAGGAUCUUGGCUUGTT-3 GGAGAAAGATGAGGCAGAGGA-3 GGAUTT-3 3 asatrsRAsRAtaseto,tasetdclswr sdfor used were cells transfected 2000 transfection, 2 At experiments. Lipofectamine protocol. siRNA/shRNA following manufacturer’s using the after to cells according days into CA), Diego, transfected San (Invitrogen, were (B-Bridge) RNAs AAGGAUUATT-3 0 ea oiesrmsplmne ihatboisadicbtdat incubated and 5 antibiotics with with DMEM in supplemented 37 grown serum were bovine cells fetal 293E 10% and MCF7 U2OS, transfection HeLa, and Human constructs RNAi cultures, Cell METHODS AND MATERIALS el eetetdwt cioyi (30 D actinomycin with treated were Cells fraction chloroquine chromatin of and Preparation hours, IR 6 to analyses. exposure immunostaining for following solution and hours 7.4), blotting 2 hypotonic pH western harvested and further and PBS hours, for 2 mM) in for (0.2 mg/ml), solution were (20 (TSA) Cells salt A blotting. mM western (50 trichostatin for later with (30 hours D treated 2 actinomycin harvested with and treated irradiation, were cells agents chromatin-modifying HeLa and inhibitors with Treatment by cells plasmid into pCßASce enhancer/chicken site, SCneo, I hCMV transfected the I-Sce the a of at was from DSBs copy of one induction plasmid For with isolated. were transformants, SCneo stable al., et and analysis, the (Johnson electroporation, repair previously DSB Briefly, described DNA as site-specific 1999). cells for HeLa-SCneo used in was performed assay SCneo The assays DR-GFP sequencing. and SCneo DNA by sequence. confirmed the PCR, then within was by mutations Japan), DNA Ohtsu, respective insert generated The the Inc., were Bio containing primers (Takara constructs FLAG using polymerase (Invitrogen). DNA SUPT16H with backbone Pyrobest plasmid and tagged employing pcDNA3 RNF20 N-terminally a into were Mutant subcloned cDNAs and C-termini epitope N-, SUPT16H mutant and (Invitrogen). and backbone respective epitopes, MID- plasmid HA pcDNA3 its or a FLAG and into either subcloned with RNF20 tagged N-terminally full-length were cDNAs resistant and Wild-type manipulation DNA and Plasmid neato ewe ATadRNF20. and PAF1-independent FACT a between by interaction driven stability, thus of genomic the and repair, of underscores types DNA maintenance study during various our remodeling chromatin Therefore, in of by 2012). importance al., downregulated et and/or degradation with (Hahn tumors interaction mutated its RNF20 is CDC73, through Similarly, RNF20, ubiquitylation 2012). H2B induces al., regulates et which which (Blank machinery proteasomal ligase, ubiquitin pcfe)2husbfr raito,adte avse o further for harvested then and irradiation, before hours 2 specified) a using was assessed plasmid were (Becton were cells pCßASce software cells CellQuest and GFP-positive with Dickinson). selected. site, equipped cytometer I hours. flow 48 I-Sce was FACSCalibur the for at DR-GFP DSBs incubated of induction of for electroporated with copy clone a and G418 cells the single into al., of transfected et was plasmid (Pierce DR-GFP previously mg/ml The described DR-GFP 1.6 1999). as the assessed were containing with cassette cells hours reporter medium HRR HeLa 48 transfected for in stably incubated Similarly, then grown (Calbiochem). were subsequently cells The and electroporation. by cells the 9 9 m -GCUAAAAGAGUCAGAAAAATT-3 ˚ -CAACAAGAAGACGCGAAUCTT-3 .Amxueo N lgncetds(o niiino N2 o 1, no. RNF20 of inhibition (for oligonucleotides RNA of mixture A C. /l,frihbto fPF(o ,5 1, PAF1(no. of inhibition for g/ml), ora fCl cec 21)17 6–7 doi:10.1242/jcs.135855 763–772 127, (2014) Science Cell of Journal 9 o ,5 2, no. ; 9 o ,5 3, no. ; 9 GGCAGGAGGUT o ,5 3, no. -GGGACCAGGAGGAGGAGAUTT; 9 m -GAAGAAAUCUUGUGGGUAUTT-3 /l o niiino UT6 (5 SUPT16H of inhibition for g/ml) b atnpooe a nrdcdinto introduced was promoter -actin 9 9 ,adnntreigshort-interfering non-targeting and ), ,3 9 9 m o ,5 2, no. ; -rde uetn,C) a CA), Cupertino, B-Bridge, ; /lfrihbto fH2AX), of inhibition for g/ml 9 -UGAGAAGACUUUUGA- m /lo 200 or g/ml 9 m -UGGAAGAGAUU- /l orbefore hour 1 g/ml) m /l where g/ml, 9 9 9 - - ;

Journal of Cell Science S ctb -cI ihta nteasneo S (uncut). DSB of of absence presence the the in in that amounts with protein I-SceI) accumulated by (cut of DSB ratio the as defined was induction (5 h ua AD ou a mlfe sn hl eoi N with DNA genomic (5 whole using amplified amplified, GAPDH-F was fragments locus specific GAPDH the human of the normalization the for control internal 3 CGCTACAGC n c60R(5 AGGACCAT-3 Sce600-R and TCCGCCCTGAGCAAAGAC) ,0 pfo h S ie c10F(5 bp, Sce180-F 600 bp, site; 180 of DSB distances I- the at CGT-3 the sets from of different bp detection three 3,100 for were PCR used site Green real-time Primers break SYBR quantitative SceI Biosystems). Power then by using (Applied analyzed was system, Mix described and PRISM7500 Master DNA buffer ABI as precipitated the TE essentially with of The PCR ml performed 2011). 50 was in al., dissolved assay et (Nakamura ChIP previously The glycine. M EDTA, mM sheared 1 mM and (Roche), [3 centrifugation cocktail by buffer sonication. collected inhibitor by was extraction protease chromatin Insoluble in complete nuclei. DTT]. isolate EGTA, resuspended to X-100] mM then Triton 0.2 0.1% were and DTT Nuclei mM 1 (Roche), cocktail nlss hoai rcin eeioae codn otemto of method the to according isolated Me were fractions Chromatin analysis. ARTICLE RESEARCH el ncvrlp eewse ihPS ie n4 omlnand formalin 4% in fixed 4 PBS, at solution with detergent a washed (Nakamura with were analysis, previously incubated immunostaining coverslips For described on antibodies. as indicated cells the out using 2011) carried al., et was blotting analysis Western immunostaining SP5 and analysis. blotting TCS immunostaining Western for Leica used were a Cells (Nakamura into 2011). previously al., coupled described et as laser microscope, 405-nm laser-scanning confocal a 4 with with treated concentration microirradiated were (final SNF2h 33258 and NBS1 Hoechst GFP-tagged expressing stably Cells analysis Microirradiation HEPES-NaOH EDTA, mM mM 1 [20 glycerol, Na IP-buffer mM 25% 1 in NaCl, NaF, mM mM minutes 10 150 30 inhibitor, NP-40, protease for 0.2% 7.4), ice and (pH PBS cold on with washed were lysed cells assay, immunoprecipitation the For Immunoprecipitation 1% by with them treated to were introduced cells 37 was later at pCßASce hours formaldehyde 24 50 vector then, (BIO-RAD). expression and electroporation 1 I-SceI hour 10 enzyme, 1 the for with restriction of Calbiochem) pre-treated I-SceI inhibitor; were the (DNA-PKcs cells Nu7026 of HeLa-DRGFP means transfected by stably DSBs generate To assay ChIP umte o5G fI.Clswr hnwse n oiae nIP- in 20 sonicated with and hours washed 3 then for incubated were fraction Cells soluble IR. the of and later buffer, Gy hours tagged- 7.5 5 21 the and to with LTX, of submitted Lipofectamine case co-transfected using HA-RNF20 the were were and In SUPT16H cells The analysis. proteins sequences, and blotting antibodies. boiled, and western nucleotide buffer, for respective to loading buffer in submitted the buffer resuspended then IP and with IP washed with hours were with 2 precipitates this diluted centrifugation, for extracted was subsequent was pellet immunoprecipitated and and sonication The fraction buffer After minutes. chromatin ice. 5 lysis on for minutes with g 30 3000 twice at washed centrifugation by recovered ihabokn ouin(%lwftml)fr3 iue,clswere cells minutes, 30 for milk) low-fat (3% solution blocking a with ye nhptncbfe 1 MHPSp .,1 MKl . mM 1.5 KCl, mM 10 7.9, pH HEPES mM MgCl [10 buffer hypotonic in lysed 2bas(IM)o c.Tepoensmlswr xrce for extracted were samples antibody. anti-HA protein against The blotted and ice. analysis on blotting western (SIGMA) beads M2 9 9 n c30- (5 Sce3100-R and ) ´ -CCTAGTCCCAGGGCTTTGATT-3 dzadSila (Me Stillman and ndez 2 .4Mscoe 0 lcrl opeepoes inhibitor protease complete glycerol, 10% sucrose, M 0.34 , 9 n c10R(5 Sce180-R and ) 9 -TCTCCCCACACACATGCACTT-3 9 , ,Se10F(5 Sce3100-F ), –% h eaieacmltoso rtisare proteins of accumulations relative The 2–3%. ˚ o 0mnts olwdb h diino 0.125 of addition the by followed minutes, 10 for C 9 -CTTCGGCACCTTTCTCTTCTTTT-3 9 ´ -CGGCGCGGGTCTTGTA-3 dzadSila,20) rel,clswere cells Briefly, 2000). Stillman, and ndez 9 -CCCAGTCATAGCTGTCCCTCTT- m ˚ o iue.Atrtreatment After minutes. 5 for C 9 /l o 0mntsadwere and minutes 10 for g/ml) .Teefcec fDSB of efficiency The ). 3 VO 9 -CATGCCCGAAGGCTA- 4 .Slbemtra was material Soluble ]. 9 -ACGAACTCCAGC- 9 n GAPDH-R and ) 9 ,Se0- (5 Sce600-F ), m fFlag- of g m 9 .A an As ). fFlag of l m 6 Mof 10 m 9 g 6 - osna,I n emaa A. Belmaaza, and I. Cousineau, J. S. Elledge, and A. Ciccia, h ttsia infcneo ifrne a eemnduigStudent’s using determined was differences of significance statistical The using analyzed analyses Statistical then House). were Software (Verity results software CellQuest 3.1 The with LT equipped ModFit Dickinson). cytometer (Becton flow were FACSCalibur software Samples a (Sigma-Aldrich). A on X- RNase Triton processed mg/ DNase-free 0.1% mg/ml 50 containing with 0.5 PBS and stained in 100 was (Sigma-Aldrich) DNA iodide ethanol. propidium 70% ml ice-cold in fixed were Cells analysis cycle Cell (Sigma). M2 anti-Flag and (Milli (Covance) (9E10) anti-Myc Technology), anti-H2BK120ub anti- monoclonal anti-RAD51 (Millipore), and (Sigma), (Abcam), Cruz anti-HA anti-PAF1 anti- anti-H3K4diMet and (Santa (Abcam), Cruz) (Millipore), anti-SUPT16H (Santa anti-H2AX anti-H2B polyclonal (Bethyl), (Bethyl), analysis: anti-RNF20 blotting Biotechnology), western immunoprecipitation immunostaining, and for used were antibodies following The Antibodies hrioa .B,Drh .A,Rzrnv,O . ae .C n Brown, and C. J. Game, V., O. Razorenova, A., J. Dorth, B., Zhang, S. and Chernikova, Y. S. Cheng, S., S. Burkett, M., Yamashita, Y., Tang, M., Blank, B. M. Kastan, and J. C. Bakkenist, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.135855/-/DC1 online available material Supplementary material Supplementary Japan. the Technology, from and Research Science Scientific Sports, for Aid Culture, Grant-in Education, a of by part Ministry in supported was work This Funding the designed manuscript. K.K. respectively. the assay, the co-wrote colony to and and contributed study H.Y. assay the and ChIP to J.K. with contributed proteins. experiment M.O. recombinant H.T. discussion. the and and T.I. using analyses K.N., experiment data micro-irradiation. the laser to with manuscript. contributed experiment the the co-wrote and to data contributed the A.K. analyzed experiments, the performed D.O. contribution Author interests. competing no declare authors The interests Competing technical excellent her work. Yukiko for this and University) in construct, Kyoto assistance Center, reporter Biology DR-GFP New (Radiation and Center, Hayuka SCneo Cancer the Sloan-Kettering providing (Memorial for Jasin York) Maria thank authors The Acknowledgements t es 0 el eecutdfrqatfcto ffcspstv cells. focus-positive of quantification for counted were cells was Fluorescence 200 glass. least slide At FV300). (Olympus a microscope laser-scanning confocal on a mounted with visualized were cells the washing, After and Alexa-Fluor-488 (Invitrogen). the antibodies secondary and Alexa-Fluor-546-conjugated fluorescent antibody, primary appropriate the to exposed eosrosaa . h . odrno .A,Opaie,G,Studitsky, G., Orphanides, A., V. Bondarenko, S., Oh, R., Belotserkovskaya, ts.A -test. eeoyoiyfraBC1tuctn uain eeuae homologous deregulates mutation, BRCA1-truncating a recombination. for heterozygosity knives. with play to elccecekon epnet aito aaei amla cells. mammalian in damage radiation Res. to response checkpoint cycle cell M. J. RNF20. through stability 234. genome and landscape chromatin E. Y. dissociation. dimer and autophosphorylation intermolecular .M n eneg D. Reinberg, alteration. and nucleosome M. V. 506. c tbln(im) niBC1(abohm,anti-phospho-H2AX (Calbiochem), anti-BRCA1 (Sigma), -tubulin 21) uo upesrfnto fSuf soitdwt controlling with associated Smurf2 of function suppressor tumor A (2012). 174 21) eiinyi r1ipishmlgu eobnto earand repair recombination homologous impairs Bre1 in Deficiency (2010). P 558-565. , vleof -value ora fCl cec 21)17 6–7 doi:10.1242/jcs.135855 763–772 127, (2014) Science Cell of Journal elCycle Cell o.Cell Mol. , .5wscniee ob significant. be to considered was 0.05 Science 6 962-971. , 21) h N aaersos:mkn tsafe it making response: damage DNA The (2010). 20) ATfclttstranscription-dependent facilitates FACT (2003). 40 179-204. , 301 20) N aaeatvtsAMthrough ATM activates damage DNA (2003). 20) RA alisfiiny u not but haploinsufficiency, BRCA1 (2007). 1090-1093. , oe,at-3(elSignaling (Cell anti-H3 pore), a.Med. Nat. Nature b atn(Sigma), -actin 421 18 nvitro in Radiat. 499- , 227- , 771

Journal of Cell Science orsn .J,Hgln,J,Koa,N . re-dn . rebat .F., J. Greenblatt, A., Arbel-Eden, J., N. Krogan, J., Highland, J., A. Morrison, i,J,Gemh . cit,R . e,J . ag . in,T A., T. Milne, Z., Tang, S., J. Lee, K., R. McGinty, M., Guermah, J., Kim, G. R. Roeder, and J. Kim, Me Formosa, and J. D. Stillman, H., Xin, A., Olsen, R., Rawlins, L., McCullough, ai . hhbt . emn,H n one,S A. S. Johnsen, and A. H. M. Neumann, Osley, A., and Shchebet, S. V., Berger, Kari, K., Henry, T., Tsukuda, C., Hillyer, F., C. Kao, M. Jasin, and N. Okano, Liu, and D., R. N. Johnson, Seki, Y., Muto, H., Yasuda, R., Iwakami, S., Adachi, K., Ito, alte .A,Mtioi . u,G,Yug .C,Hnzl .J,Mr G., Mer, J., M. Hendzel, C., L. Young, G., Cui, F., Mattiroli, A., F. Mallette, Lukas, J., Bartek, F., Melander, H., Faustrup, S., Bekker-Jensen, E. N., Mailand, J. Cleaver, and M. W. Bonner, E., Giedzinski, L., C. Limoli, ae .C,Wlimo,M . pckv,T n rw,J M. J. Brown, and T. Spicakova, S., M. Williamson, C., J. Game, T. F. Formosa, Thoma, and D. Imholz, M., Johnston, Fink, A., B. Altheim, A., Rowley, Q., Xu, K., N. Brewster, R., D. Evans, ARTICLE RESEARCH 772 A., Tong, G., A. Ianculescu, S., Venkatasubrahmanyam, W., W. Hwang, rjwk,W A. W. Krajewski, odl,M,Sue . ase,M,Hlah . omn . hn,E . Nijmeijer, T., E. Zhang, A., Bowman, F., Halbach, M., Hassler, T., Stuwe, M., Hondele, S. A. Yang, R., M. Lieber, J., Gu, K., Kim, J., Choi, R. H., S. Choi, G. H., Kim, Crabtree, K., Heo, and C. D. Marsh, Hargreaves, and J. A. Gill, A., Clarkson, S., Jackson, A., K. Dickson, A., M. Hahn, ne,J n tlmn B. Stillman, and J. ´ndez, T-eedn hoai eoeigt N aaerepair. damage DNA X. to Shen, remodeling chromatin ATP-dependent and E. J. Haber, hltfr,A,Mi,T .adRee,R G. R. Roeder, and W. T. Muir, A., H2B Shilatifard, histone mediate interactions yeast. Bre1-Rad6 in ubiquitylation finger-independent RING and 3495-3504. h elcce sebyo rrpiaincmlxsi aemitosis. late in complexes prereplication of during Biol. assembly proteins cycle: maintenance cell minichromosome the and cdc6, complex, recognition chaperone. histone FACT the in 835-846. defects suppress that mutants T. ncrmtnsrcuedrn N obesrn ra repair. break double-strand changes DNA dynamic induce during to structure SUPT16H chromatin with in cooperates RNF40 of ligase ubiquitin ubiquitylation through activation transcriptional in role H2B. histone a plays Rad6 (2004). recombination. homologous by breaks 397-399. double-strand DNA of repair RNF8. and ARA54 proteins, RING-finger of Biochem. ubiquitination the mediate Y. 261-266. erdto fKMAJJ2 rges5B1rcutett N damage DNA to recruitment S. 53BP1 Richard, triggers sites. KDM4A/JMJD2A and of K. degradation T. Sixma, proteins. repair of assembly promotes and J. breaks Lukas, and C. USA DNA relocalization. Sci. to Mre11 leads Acad. and variant Natl. formation, -H2AX pigmentosum gamma xeroderma breaks, the double-strand in arrest replication induced eurdfrhsoeHBmnuiutnto n elsz control. size cell D. and monoubiquitination H. H2B histone Madhani, for required and C. Boone, Acta Biophys. Biochim. structure. chromatin N-terminal to H2A cdc68 histone the through repression core-promoter domain. mediates A. pob3 R. Singer, and and C. G. hoai fibers. chromatin in methylation cells. H3K4 stimulates human directly ubiquitylation H2B transcription-coupled 2 eonto yteesnilcaeoeFACT. chaperone essential the by recognition H2B Spt16. of B.,Kotthoff,C.,Rybin,V.,Amlacher,S.etal. ADP-ribosylation and H2AX of Cell phosphorylation 2B by W. regulated histone An, of and maintenance mechanisms. the and genomics for genetics, remodeling: required is and RNF40 monoubiquitination. and RNF20 J. D. RAD18. of independent is that process Genetics radiation RAD51-dependent confers a Saccharomyces through in resistance pathway modification histone RAD6/BRE1 21) nih notemcaimo ulooerognzto rmhistone from reorganization nucleosome of mechanism the into Insight (2011). 20) -emnlyetne ua bqii-ojgtn nye (E2s) enzymes ubiquitin-conjugating human extended N-Terminally (2001). 30 20 21) h uo upesrCC3itrcswt h igfne proteins finger ring the with interacts CDC73 suppressor tumor The (2012). MOJ. EMBO 86-97. , 8602-8612. , Genetics 173 268 21) h oeo ATi aigadbekn nucleosomes. breaking and making in FACT of role The (2012). 1951-1968. , Cell ee Dev. Genes 2725-2732. , 20) ATmdae xhneo itn ain H2AX variant histone of exchange FACT-mediated (2008). 19) feto nvv itn yeaeyaino h tt of state the on hyperacetylation histone vivo in of Effect (1999). 31 .Boo.Src.Dyn. Struct. Biomol. J. 137 150 1865-1878. , 20) N8uiutltshsoe tDAdouble-strand DNA at histones ubiquitylates RNF8 (2007). u.Ml Genet. Mol. Hum. 99 459-471. , 1393-1405. , .Bo.Chem. Biol. J. 1819 233-238. , 19) h es rti ope otiigcdc68 containing complex protein yeast The (1998). 18 20) N8 n am-2Xitrcinlinks interaction gamma-H2AX and INO80 (2004). 184-195. , 247-255. , 20) hoai soito fhmnorigin human of association Chromatin (2000). 20) ietBe-a1cmlxinteractions complex Bre1-Paf1 Direct (2009). 20) osre IGfne protein finger RING conserved A (2003). 21) N8 n RNF168-dependent and RNF8- (2012). o.Cl.Biol. Cell. Mol. 20) otiuino h eie19of 129 serine the of Contribution (2007). 19) amla RC rmtsthe promotes XRCC2 Mammalian (1999). 284 21 16 559-568. , 20582-20592. , 21) tutrlbsso itn H2A- histone of basis Structural (2013). 1097-1106. , 21) T-eedn chromatin ATP-dependent (2011). Nature Cell elRes. Cell 27 20) RAD6-Mediated (2009). 3589-3600. , 499 131 111-114. , 887-900. , Cell 21 21) h H2B The (2011). 396-420. , elCycle Cell Genetics 119 o.Cell Mol. 20) UV- (2002). 20) The (2006). Nature 767-775. , o.Cell. Mol. u.J. Eur. Proc. 188 401 Mol. 10 11 , , , , onhn .E,Ci,J . olr .H n ai,M. R. Jasin, and Murr, H. B. Koller, W., J. Chiu, E., M. Moynahan, Eppink, Y., S. Wang, S., So, G., Mass, M., Gana-Weisz, Y., Lerenthal, L., Moyal, aaih,S,Paa,R,Wlo,S .adSedn M. Smerdon, and H. S. Wilson, R., Prasad, S., Nakanishi, Oliveira, S., Sakamoto, H., Yanagihara, J., Kobayashi, A., Kato, K., Nakamura, A., J. Harper, K., Martinez-Fonts, H., J. Holthoff, B., G. Berner, N., C. Myers, hu .W,Grn .adBrsen .E. B. Bernstein, and A. Goren, W., V. Zhou, I., Takada, S., Takezawa, M., Tanabe, R., Fujiki, H., Kitagawa, K., Yoshimura, T. Formosa, and J. Wittmeyer, g .H,Rbr,F,Yug .A n tul K. Struhl, and A. R. Young, F., Robert, H., H. Ng, rhnds . eo,G,Cag .H,Ls,D .adRibr,D. Reinberg, and S. D. Luse, H., C. Chang, G., Fujigaki, LeRoy, K., G., Orphanides, Suzuki, K., Ohta, K., Ogawa, M., Shimada, K., Murata, H., Niida, ike,D .adLgr K. Luger, and D. D. Winkler, F. J. Ward, ire .J,Jhsn .D,Topo,L .adJsn M. Jasin, and H. L. Thompson, D., R. Reinberg, Johnson, and J., A. A. Shilatifard, Pierce, S., Mandal, P., Trojer, G., Li, B., Zhu, R., Pavri, skd,T,Feig .B,Ncoof .A n se,M A. M. Osley, and A. J. Nickoloff, B., A. Fleming, T., Tsukuda, M. K. Arndt, and P. C. Davis, H., M. Warner, K., M. Mayekar, S., A. Piro, a,B . he,C . ioe .adLe .C. S. Lee, and S. Hirose, T., C. Chien, C., B. Tan, L. J. Workman, and T. Suganuma, N., Sroczynski, S., M. Luijsterburg, A., J. Mateijn, W., W. Wiegant, G., Smeenk, A., Morillon, N., Karabetsou, E., B. Bernstein, R., Schneider, H., Santos-Rosa, III J., R. Sims, and D. Reinberg, oooydrce N repair. DNA double- homology-directed DNA of repair al. timely et for breaks. H2B E. strand histone Shema, of G., monoubiquitylation Shalev, dependent M., Y. Chung, B., fhmlgu eobnto yRF0dpnetHBubiquitination. H2B RNF20-dependent by al. Cell et recombination S. Tashiro, homologous H., Suzuki, of H., that Tauchi, M., Spt16 Shimada, V., D. of regions H3. define histone Spt16 A. with A. interact factor functionally Duina, elongation and transcription N. cerevisiae M. Taylor, S., Alford, mechanisms. oiiain n h ucinlognzto fmmaingenomes. mammalian of organization functional Genet. Rev. the and modifications 9280-9285. protein. a HMG1-like Pob3, with an and to Cdc68/Spt16 similar with protein interacts subunit catalytic alpha polymerase 18369-18374. tutrlsae noioulooe r eurdfreryvru aesesof steps late versus early for repair. required excision are base oligonucleosomes in states structural fCk n h2rdc uorssetblt nvivo. in susceptibility tumour al. reduce and et P. Chk2 M. and mark Hande, Chk1 B., of localized Banerjee, K., a A. Khaw, provides H., II activity. Pol transcriptional elongating recent by of memory methylase histone Set1 ucino hoai eoeigcmlxsta hr omnsubunit, common a share al. that et (WSTF). complexes Y. factor transcription remodeling Furutani, syndrome chromatin Williams T., 2 Kondo, of M., function Yonezawa, I., Yamaoka, reorganization. nucleosome for mechanisms and insights reparability. and Biol. formation, Mol. Res. of mechanisms identities, cells: rmtshmlg-ietdrpi fDAdmg nmmaincells. mammalian in damage Dev. DNA of repair homology-directed promotes II. polymerase RNA by elongation regulate D. nucleosomes. through elongation transcript facilitates 92 that factor a FACT, eoeln taDAdul-tadbekst nSchrmcscerevisiae. Saccharomyces in site break double-strand DNA Nature a at remodelling ewe ATadMMhlcs aiiae ntaino hoai DNA chromatin of initiation facilitates helicase MCM and replication. FACT between modifications. histone SMARCA5/ remodeler signaling. al. damage chromatin et DNA 903. W. the RNF168-dependent Vermeulen, N., to links Mailand, SNF2H A., Poly(ADP-ribosyl)ation Pastink, J., chromatin. (2012). R. with Romeijn, ATPase T., Isw1p Costelloe, the of association mediates Cell T. K4 Kouzarides, Mol. H3 and J. histone Mellor, L., of S. Schreiber, C., Weise, in complex Paf1 complete Chem. for substitute can yeast. in protein 109 ubiquitylation H2B Rtf1 histone global of facilitating region Small 105-116. , 20) itn 2 oobqiiainfntoscoeaieywt ATto FACT with cooperatively functions monoubiquitination H2B Histone (2006). 10837-10842. , 41 13 515-528. , 281 438 2633-2638. , 21) nepa ewe ifrn pgntcmdfctosand modifications epigenetic different between Interplay (2010). ora fCl cec 21)17 6–7 doi:10.1242/jcs.135855 763–772 127, (2014) Science Cell of Journal 12 18) N aaepoue yinzn aito nmammalian in radiation ionizing by produced damage DNA (1988). 23297-23301. , 379-383. , MOJ. EMBO 1325-1332. , 12 d.Genet. Adv. o.Cell Mol. 7-18. , 35 95-125. , uli cd Res. Acids Nucleic 25 nu e.Biochem. Rev. Annu. 3975-3985. , 41 70 529-542. , 101-141. , 21) h itn hprn AT structural FACT: chaperone histone The (2011). 20) eFConcesm dynamics. nucleosome FACTo de (2006). o.Cell Mol. 19) h acaoye eeiieDNA cerevisiae Saccharomyces The (1997). 21) inl n obntra ucin of functions combinatorial and Signals (2011). LSONE PLoS o.Cl.Biol. Cell. Mol. o.Cell Mol. 35 4 511-518. , Cell 4313-4321. , 80 21) uatvrin fteS. the of versions Mutant (2011). 473-499. , rc al cd c.USA Sci. Acad. Natl. Proc. 125 20) agtdrcutetof recruitment Targeted (2003). 20) ucinlcooperation Functional (2006). 6 21) eurmn fATM- of Requirement (2011). 21) oprtv functions Cooperative (2010). e20847. , 11 703-717. , rc al cd c.USA Sci. Acad. Natl. Proc. 21) hrighistone Charting (2011). 709-719. , MOJ. EMBO 17 .Cl.Sci. Cell. J. 19) ra controls Brca1 (1999). 4178-4190. , .Bo.Chem. Biol. J. 20) Methylation (2003). rg uli Acid Nucleic Prog. 21) Regulation (2011). 20) Chromatin (2005). 20) Different (2007). 20) Distinct (2009). 19) XRCC3 (1999). 29 3558-3570. , 126 (1998). (2012). .Biol. J. Genes 889- , 106 286 Mol. Nat. Cell , ,

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