Phase separation directs ubiquitination of gene-body nucleosomes.pdf
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1) Lge1, a scaffold protein involved in H2B ubiquitination, forms condensates through liquid-liquid phase separation due to its intrinsically disordered region. 2) When Bre1, the E3 ubiquitin ligase enzyme, is added, it forms a catalytic shell that encapsulates the Lge1 condensate core. 3) These layered condensates recruit the E2 enzyme Rad6 and nucleosomal substrates, accelerating the ubiquitination of H2B across gene bodies.
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Phase separation directs ubiquitination of gene-body nucleosomes.pdf
- 1. 592 | Nature | Vol 579 | 26 March 2020 Article Phaseseparationdirectsubiquitinationof gene-bodynucleosomes Laura D. Gallego1,3 , Maren Schneider1,3 , Chitvan Mittal2,3 , Anete Romanauska1 , Ricardo M. Gudino Carrillo1 , Tobias Schubert1 , B. Franklin Pugh2 & Alwin Köhler1 ✉ TheconservedyeastE3ubiquitinligaseBre1anditspartner,theE2ubiquitin- conjugatingenzymeRad6,monoubiquitinatehistoneH2Bacrossgenebodiesduring thetranscriptioncycle1 .Althoughprocessiveubiquitinationmight—inprinciple— arisefromBre1andRad6travellingwithRNApolymerase II2 ,themechanismofH2B ubiquitinationacrossgenicnucleosomesremainsunclear.Hereweimplicateliquid– liquidphaseseparation3 astheunderlyingmechanism.Biochemicalreconstitution showsthatBre1bindsthescaffoldproteinLge1,whichpossessesanintrinsically disorderedregionthatphase-separatesviamultivalentinteractions.Theresulting condensatescompriseacoreofLge1encapsulatedbyanoutercatalyticshellofBre1. ThislayeredliquidrecruitsRad6andthenucleosomalsubstrate,whichaccelerates theubiquitinationofH2B.In vivo,thecondensate-formingregionofLge1isrequired toubiquitinateH2Bingenebodiesbeyondthe+1 nucleosome.Ourdatasuggestthat layeredcondensatesofhistone-modifyingenzymesgeneratechromatin-associated ‘reactionchambers’,withaugmentedcatalyticactivityalonggenebodies.Equivalent processesmayoccurinhumancells,andcauseneurologicaldiseasewhenimpaired. Eukaryotic cells require the precisely timed activation of genes, which coincides with chromatin alterations. The fundamental unit of chromatin is the nucleosome core particle (NCP), which is assem- bled from histones and DNA, and iterated to resemble ‘beads on a string’.Post-translationalmodificationsofhistonesaffectchromatin structure and thus gene activity. How histone-modifying enzymes are concentrated in discrete parts of the genome to install histone marks locally is a key question. Histone H2B is monoubiquitinated at lysine 123 (H2BK123ub) in yeast and at lysine 120 in mammals1,2 . Thismarkisfoundacrosspromoter,genicandterminationregionsof mostgenesinSaccharomycescerevisiae4 .H2BK123ubinfluencesDNA transcription,repairandreplication,andRNAprocessing1 .H2BK123ub exertsitstranscriptionaleffectsbyregulatingNCPassembly,stability4 andspecifichistonemethylations1 .AberrantlevelsofH2Bubiquitina- tion are found in various diseases, including cancer1 . It is therefore important to understand how H2BK123ub is established in specific regions of the genome. Ubiquitination is a three-step enzymatic cascade, which requires a ubiquitin-activating (E1), a ubiquitin-conjugating (E2) and a ubiqui- tin-ligating (E3) enzyme. In yeast, H2BK123ub is generated by the E3 Bre1 (an orthologue of the human RNF20 and RNF40 proteins (here- after, RNF20/RNF40)), together with the E2 Rad6 and the E1 Uba15–9 . AcommonviewisthatH2BK123ubisdepositedinacotranscriptional manner1,2 . However, H2B ubiquitination does not strictly depend on Bre1–Rad6bindingtoRNApolymerase(Pol) II10 .Thus,themechanism of H2B ubiquitination across genic NCPs is unclear. Tounderstandhowtheubiquitinationmachineryisenrichedatthe correct genomic positions, we considered the Large 1 (Lge1) protein, whichhasanas-yetunknownbiochemicalrole,copurifieswithBre1and is essential for H2B monoubiquitination in yeast7 . Except for a short C-terminalcoiledcoil,Lge1ispredictedtoconsistentirelyofanintrin- sically disordered region (IDR) (Fig. 1a, Extended Data Fig. 2a, b). The human Bre1 orthologues RNF20/RNF40 interact with WAC, a protein that is similar to Lge1 in its IDR and coiled-coil domain architecture11 (ExtendedDataFig. 2b).SomeproteinsthatcontainanIDRparticipate inmultivalentinteractions,whichcanleadtoliquid–liquidphasesepa- ration(LLPS)andtheformationofbiomolecularcondensates3,12,13 .LLPS has previously been implicated in chromatin organization14 , hetero- chromatincompaction15 aswellaspromoterandenhancerfunction16–19 . HerewedefineadirectroleofLLPSinstimulatinghistoneubiquitina- tionspecificallyacrossgenebodies.Lge1,Bre1,Rad6andnucleosomes together form spatially organized reaction chambers that stimulate H2B ubiquitination. Lge1promotesBre1oligomerization WefirstexploredinteractionsbetweenLge1andBre1,andtheircapacity to phase-separate. Bre1 consists of a series of coiled-coils, flanked by an N-terminal Rad6-binding domain and a C-terminal RING domain20 (Fig. 1a).Bre1associateswithLge1whenpurifiedfromyeast(Extended DataFig. 1a).Lge1taggedwithglutathione S-transferase(GST)purified from Escherichia coli bound the ‘middle’ part of recombinant Bre1, which we therefore call the Lge1-binding domain (LBD; comprising residues 308–632)(ExtendedDataFig. 1b,c,SupplementaryFig. 2,Sup- plementaryTable 1).TheLge1coiledcoilalone(Lge1(CC),comprising aminoacids 243–332)wasnecessaryandsufficientforbindingtoBre1 (Extended Data Fig. 1d). Thus, coiled-coil contacts mediate a direct interaction between Lge1 and Bre1. https://doi.org/10.1038/s41586-020-2097-z Received: 10 June 2019 Accepted: 11 February 2020 Published online: 11 March 2020 Check for updates 1 Max Perutz Labs, Medical University of Vienna, Vienna Biocenter Campus (VBC), Vienna, Austria. 2 Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, Pennsylvania State University, University Park, PA, USA. 3 These authors contributed equally: Laura D. Gallego, Maren Schneider, Chitvan Mittal. ✉e-mail: alwin.koehler@mfpl.ac.at
- 2. Nature | Vol 579 | 26 March 2020 | 593 We next analysed the oligomerization state of Bre1 and how this statemightbeinfluencedbytheIDRofLge1.Bre1showedabroadpeak in the low-molecular-weight region of a sucrose gradient, whereas Lge1 sedimented in a distinct high-molecular-weight region. When mixedwithBre1,Lge1shiftedasubstantialportionofBre1intothehigh- molecular-weightregion(Fig. 1b,SupplementaryFig. 1).TheLge1(CC) alonesedimentedinthelow-molecular-weightregion,whereastheLge1 IDR alone (Lge1(IDR), comprising amino acids 1–242) sedimented in the high-molecular-weight region (Extended Data Fig. 1e). Lge1(IDR) (whichdoesnotbindBre1)leftBre1inthelow-molecular-weightregion. Thus, Lge1 is a scaffold protein that—via its IDR—promotes Bre1 oli- gomerization. Lge1phase-separatesin vitro Lge1hasthehallmarksofproteinsthatundergoLLPS3 ,includingintrin- sicdisorder,lowsequencecomplexityandblocksofalternatingcharges (ExtendedDataFig. 2a,b).Recombinantfull-length6×His-taggedLge1 indeed formed condensates, which grew in size to micrometre-sized spheres by fusion (Fig. 1c, Extended Data Fig. 1f–h, Supplementary Video 1). Phase separation occurred at 0.1–0.5 μM under physiologi- cal salt concentrations without crowding agents, and was inhibited by 1,6-hexanediol (Extended Data Fig. 4a, b). The sedimentation of Lge1 in sucrose gradients could thus be explained by LLPS (Extended Data Fig. 1k). Deletion of the IDR of Lge1 abolished LLPS, whereas the Lge1(IDR)alonewassufficientforLLPS(Fig. 1c,ExtendedDataFig. 1g). The N terminus (amino acids 1–80) of Lge1 is enriched in tyrosine (Y) andarginine(R)residues(ExtendedDataFig. 2a).TruncatingthisY/R- rich region strongly decreased LLPS (Fig. 1c, Extended Data Fig. 1g). Conversely, the Y/R-rich region was sufficient for LLPS and showed LLPS when fused to Lge1(CC), albeit with a lower efficiency. In sum, a shortY/R-rich‘sticker’regionattheLge1N terminusmayactasaseed for LLPS. ABre1catalyticshellencapsulatesLge1 Bre1 does not phase-separate on its own (Extended Data Fig. 4c, d); Lge1thereforebehavesasascaffoldandBre1asaclient.Whenweused recombinant Bre1 tagged with monomeric green fluorescent protein (mGFP–Bre1)atequimolarratiotoLge1,Bre1penetratedtheLge1con- densateonlytoacertaindepthandformedashellaroundanLge1core. This shell grew in thickness with increasing concentrations of Bre1 (Fig. 1d,ExtendedDataFig. 1j).Tounderstandhowthecore–shellarchi- tecture is established, we added mGFP–Bre1 to condensates formed by Lge1(IDR) that lacked the coiled coil. In these experiments, Bre1 no longer assembled a shell and instead diffused into the condensate (ExtendedDataFig. 1i,j).TheBre1(LBD)wassufficientforshellforma- tion (albeit with lower efficiency), and this specifically depended on the coiled coil of Lge1 (Extended Data Fig. 3a, b). To further test the roleoftheLge1–Bre1interactioninshellformation,wecreatedhybrid condensates composed of varying ratios of full-length Lge1 mixed withLge1(IDR).Bre1couldpenetratedeeperintothecorewhenfewer Lge1coiledcoilswerepresent(ExtendedDataFig. 3c,d).Thissuggests that the coiled-coil domain of Lge1 captures and organizes Bre1 as it infiltrates the structure from the periphery. Notably, the Bre1 shell inhibited condensate fusion (Extended Data Fig. 3e, Supplementary Video 2) and thereby restricted condensate growth (Extended Data Fig. 4d). Thus, Lge1 and Bre1 form a distinct membraneless compart- mentwithacatalyticE3shellthatencapsulatesaliquid-likeLge1core. Thematerialpropertiesofacondensatedeterminewhichmolecules areincorporatedintoorexcludedfromit.Wecharacterizedtheeffec- tivemeshsizeusingfluorescentlylabelleddextransthatrangedfrom 35 to 2,000 kDa in size21 . None of the probes was excluded, and the partitioningratiosweresimilarwithorwithoutaBre1shell(Extended DataFig. 4e,f).The2,000-kDaprobehasahydrodynamicradius(Rh)of about27 nmindiluteaqueousbuffer,similartoa16-unitnucleosomal array(ExtendedDataFig. 4e,g).Thus,Lge1–Bre1condensatespermit the penetration of large molecules. A key question about LLPS is how particular interactions shape the mesh and function of a condensate3,22 . Both Y>A and R>K mutations in the Lge1 sticker region strongly decreased LLPS (Extended Data Fig. 5a–c).Inthecontextoffull-lengthLge1,Y>Amutationsinthesticker region decreased LLPS, and three additional Y>A mutations within aminoacids 1–102ofLge1eliminatedLLPS.TheR>Kmutationsinfull- length Lge1 showed only a modest decrease in LLPS. In sum, a multi- valentnetworkunderliesthedynamicbehaviourofLge1condensates, and tyrosines have a key role. LLPSacceleratesH2Bubiquitination Tounderstandthefunctionofthecore–shellcondensates,weexplored theirinfluenceonH2Bubiquitination.FluorescentlylabelledRad6was rapidlyrecruitedtotheBre1shellofpreformedLge1–Bre1condensates (Fig. 2a). Subsequently, Rad6 diffused further into the core, and co- enrichment with Bre1 in the shell still persisted. When Bre1 was omit- ted,Rad6didnottransientlyaccumulateintheoutershell,butinstead became rapidly distributed throughout the condensate (Extended Data Fig. 6a). The recruitment of Rad6 to the Bre1 shell depended on a direct interaction, as shown by the fact that the Bre1(LBD), which lacks the RING domain and the Rad6-binding domain, did not con- centrateRad6intheshell(ExtendedDataFig. 6b).Thisdemonstrates thatLge1condensatescanco-enrichboththeE3Bre1andtheE2Rad6 in a catalytic shell. Forthecondensatestoqualifyasareactionchamberforubiquitina- tion, nucleosomes should also become concentrated. A 16-unit NCP array,reconstitutedfromyeasthistonesona16×Widomsequence,was recruited to the catalytic shell of the Lge1–Bre1 condensate (Fig. 2b, ExtendedDataFig. 6c,d).SimilartoRad6,thechromatinsubstratedif- fusedintothecondensateovertime.Asimilarpartitioningbehaviour a Lge1 IDR 243 332 1 CC RING RBD LBD 700 Bre1 1 CC 632 308 210 462 403 d 6×His–Lge1 (1.5 μM) mGFP–Bre1 (μM) 1.5 3 5 DIC mGFP–Bre1 Merged c b 45 % 5% Sucrose Bre1 80 70 50 40 Coomassie Strep– Bre1 Lge1 80 70 50 40 GST– Lge1 Bre1 + Lge1 6 7 8 9 10 11 12 13 1 2 3 4 5 80 70 50 40 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 Mr (×103) Strep– Bre1 GST– Lge1 6×His–Lge1 DIC 243 332 1 80 CC N IDR Y/R -rich 242 1 IDR Y/R -rich 1 80 Y/R -rich 6×His–Lge1 243 332 CC IDR 81 243 332 CC 1 80 243 332 CC Y/R -rich Fig.1|Lge1formscore–shellcondensateswithBre1.a,OrganizationofBre1 andLge1.CC,coiled-coildomain(green;non-coiled-coilregions,grey);LBD, Lge1-bindingdomain;RBD,Rad6-bindingdomain;RING,reallyinterestingnew genedomain.b,SucrosegradientanalysisofStrep-taggedBre1(Strep–Bre1) andGST–Lge1.Arrowheadslabelpeakfractions.SeeSupplementaryFig. 1for uncroppedgels.c,LLPSassaywith6×His–Lge1constructs(1.5 μM)(cartoons). Scalebar,10 μm.SeeSupplementaryVideo 1forcondensatefusionevents. d, Reconstitutionofcore–shellcondensateswithincreasingamountsof mGFP–Bre1addedtopreformed6×His–Lge1condensates.Arrowheadslabel theshellindifferentialinterferencecontrast(DIC)images.Scalebar,2 μm.
- 3. 594 | Nature | Vol 579 | 26 March 2020 Article was observed for mononucleosomes (Extended Data Fig. 6e). When the Bre1 shell was missing, chromatin immediately diffused into the interior of the Lge1 condensate (Extended Data Fig. 6f). DNA alone showednostrongaccumulation(ExtendedDataFig. 6g).Insum,LLPS spatiallyorganizestheubiquitinationmachineryanditssubstrateinto a distinct core–shell structure. To test the functional importance of these reaction chambers, we reconstituted NCP ubiquitination from defined components under phase-separatingconditions(Fig. 2c).Theadditionoffull-lengthLge1 acceleratedH2Bubiquitination.Importantly,theeffectwasabolished by removing the IDR of Lge1, leaving only the coiled-coil domain that interacts with Bre1. The Lge1(IDR) alone, which undergoes LLPS but does not assemble a Bre1 shell (Extended Data Fig. 3b), could not enhancethereaction.Thestimulatoryeffectoffull-lengthLge1required the sticker region—specifically, the tyrosine residues that promote LLPS. Thus, H2B ubiquitination was accelerated by conditions that correspond to the formation of Lge1–Bre1 core–shell condensates. EndogenousLge1andBre1formlargecomplexes To determine whether Lge1–Bre1 condensates exist in yeast cells, we analysedthesedimentationofLge1,Bre1andPol IIatendogenouslevels inwhole-cellextractsusingsucrosedensitygradients.Bre1taggedwith haemagglutinin was found throughout the gradient, and overlapped withthebroadpeakofLge1taggedwithatandemaffinitypurification taginthehigh-molecular-weightregion(Fig. 3a,ExtendedDataFig. 6h, i).Pol IIsedimentedinthelow-molecular-weightregion,whichsuggests thatLge1–Bre1multimerizationisnotduetoanassociationwithPol II. WhenLGE1wasdeleted,Bre1relocalizedintothelow-molecular-weight regionofthegradient,consistentwithourreconstitutionexperiments (Fig. 1b).DeletionoftheIDRofLge1wassufficienttoshifttheremaining Lge1(CC) and Bre1 into the low-molecular-weight region. Removing only the sticker region had an effect that was similar to—but weaker than—removingtheentireIDRofLge1.ConsistentwithLge1(IDR)con- densatesformingintheabsenceofBre1in vitro(Fig. 1c,ExtendedData Fig. 1e), Lge1(IDR) alone sedimented in the high-molecular-weight regionbutdidnotshiftBre1intothisregion.Overall,themultimeriza- tionstateofLge1andBre1incellextractsisconsistentwiththeforma- tion of core–shell structures that we observed in vitro (Fig. 1d). TovisualizeLge1andBre1incells,weco-overexpressedthemunder a strong promoter. Lge1 and Bre1 colocalized into prominent nuclear foci (Fig. 3b, Extended Data Fig. 7a, b). When expressed from their endogenouspromoters,Lge1andBre1werehomogenouslydispersed intheyeastnucleus(ExtendedDataFig. 7c–f).Toselectivelyvisualize Bre1 that is bound to Lge1, we used bimolecular fluorescence com- plementation (BiFC) (Fig. 3c). Bre1 fused to the C-terminal fragment of Venus exhibited BiFC with full-length Lge1 fused to the N-terminal fragmentofVenus(Fig. 3d,ExtendedDataFig. 7h,i),confirmingtheir interaction.TheBiFCsignalshowedmultiplepuncta(Fig. 3d,Extended Data Fig. 7g), which may reflect condensates. We quantified puncta formation by measuring the heterogeneity (coefficient of variation) offluorescentintensitieswithinthenucleus(Fig. 3e).DeletingtheLge1 IDR or the Lge1 sticker region (resulting in Lge1(Δ1–80)) reduced the formation of BiFC puncta (Fig. 3d, e, Extended Data Fig. 7i). However, a homogenous BiFC signal was maintained (Fig. 3d), consistent with the presence of the coiled-coil domain of Lge1, which interacts with Bre1. As in the other assays, the effect of deleting the entire IDR was stronger than deleting the sticker region only (Figs. 1c, 3a). Together, thesedatasuggestthatthenuclearBiFCpunctacorrespondtothepool of Bre1 that undergoes LLPS when bound to Lge1. Lge1IDRaffectsgene-bodyubiquitination To address how LLPS contributes to H2B ubiquitination in cells, we measured global H2BK123ub levels. Deleting the IDR of Lge1 strongly reduced H2B ubiquitination (Extended Data Fig. 8a), consistent with H2B ~Ub H2B H2B ~Ub H2B H2B ~Ub H2B H2B ~Ub H2B H2B ~Ub H2B 3 a b Time (min) 2 5 60 Rad6–PacB Rad6* (3 μM) mGFP–Bre1 (3 μM) DIC mGFP–Bre1 2 5 15 16×NCP* (0.5 μM) mGFP–Bre1 (3 μM) DIC mGFP–Bre1 Time (min) NCP–Hoech 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 μm 1 2 3 4 μm n = 22 n = 24 n = 29 n = 29 n = 24 n = 22 16×NCP* mGFP–Bre1 4 5 3 2 0 0 1 0 4 5 3 2 0 1 4 5 3 2 0 0 1 4 5 3 2 0 0 1 4 5 3 2 0 0 1 4 5 3 2 0 0 1 1 2 Rad6* mGFP–Bre1 1 2 3 4 1 2 μm 4 6 2 0 4 6 2 0 0 0 4 5 3 2 0 0 1 2 3 4 μm 4 5 3 2 0 0 1 1 n = 26 n = 26 1 2 1 2 3 4 4 6 2 0 0 4 6 2 0 0 n = 39 n = 39 n = 25 n = 25 c CC IDR Y/R -rich 0 3 6 9 – – – – – + + + + + 12 0 3 6 9 12 30 25 15 Mr (×103) Time (min) Time (min) Time (min) Time (min) Time (min) Lge1 0 Time (min) 3 6 9 12 2.0 0 0.5 1.0 1.5 n = 3 Lge1 CC 30 25 15 0 3 6 9 12 0 3 6 9 12 Lge1(CC) 0 Time (min) 3 6 9 12 2.0 0 0.5 1.0 1.5 n = 3 Lge1(CC) IDR Y/R -rich 30 25 15 0 3 6 9 12 0 3 6 9 12 Lge1(IDR) 0 Time (min) 3 6 9 12 2.0 0 0.5 1.0 1.5 n = 3 Lge1(IDR) CC IDR 30 25 15 0 3 6 9 12 0 3 6 9 12 Lge1(Δ1–80) Time (min) 0 3 6 9 12 2.0 0 0.5 1.0 1.5 n = 3 Lge1(Δ1–80) 30 25 15 0 3 6 9 12 0 3 6 9 12 CC IDR Lge1(17×Y>A, 1–102) 0 Time (min) 3 6 9 12 2.0 0 0.5 1.0 1.5 n = 3 Lge1 Y>A Relative H2B~Ub Fluorescence intensity (FU) Fluorescence intensity (FU) Relative H2B~Ub Relative H2B~Ub Relative H2B~Ub Relative H2B~Ub Fig.2|Lge1–Bre1condensatespromoteH2BK123ub.a,Recruitmentof Pacific-Blue-labelled6×His–Rad6(heredenoted‘Rad6*’)toLge1condensates withaBre1shell.Fluorescenceintensitieswerequantifiedacrosssingle condensates.FU, arbitraryfluorescenceunits.n,numberofcondensates. Scalebar,5 μm.b,PartitioningofHoechst(Hoech)-labelled,reconstituted yeastoligonucleosomearrays(heredenoted‘16×NCP*’)intoLge1–Bre1 condensates.Scalebar,5 μm.c,Time-resolvedin vitroH2Bubiquitination assayperformedunderLLPSconditions.±,theindicatedLge1constructsused atequimolarconcentration.IntensityoftheH2B~Ubbandwasquantifiedand normalizedto12-mintimepointwithoutLge1foreachassay.Lge1(17×Y>A, 1–102),Lge1withmutationswithinaminoacids1–102(see‘Proteinpurification’ inMethodsfordetailsofthesubstitutions).n,independentexperiments. Mean ands.d.areindicated.
- 4. Nature | Vol 579 | 26 March 2020 | 595 LLPSacceleratingtheenzymaticreactionin vitro(Fig. 2c).Deletionof thestickerregionhadaweakereffectthanremovaloftheentireIDRof Lge1. Lge1(IDR) alone did not promote H2B ubiquitination, as it is not connectedtoBre1(seealsoFig. 3a,d).Todeterminethegenome-wide effectofLge1–Bre1LLPS,weanalysedthelevelsofH2BandH2BK123ub by sequencing exonuclease-treated chromatin immunoprecipitates (ChIP-exo)23 ,whichresolvedbothsubunitsofH2Bwithineachaveraged nucleosome position (Fig. 4a, left). A lge1Δ strain showed increased nucleosome occupancy (approximated by H2B) downstream of the first (+1) gene-body nucleosome. Lge1(CC) was sufficient to sustain proper nucleosome occupancy and positioning, which suggests that Lge1–Bre1, but not Lge1-directed LLPS, is involved in these aspects of chromatin organization. In examining H2BK123ub, we separated all genes into three well- studied groups—ribosomal-protein, SAGA-dominated and TFIID- dominated genes—that have distinct types of regulation24,25 (Fig. 4a, Extended Data Figs. 8b, c, 9d, e). As expected, a lge1Δ bre1Δ strain showed substantial loss of H2BK123ub. An lge1Δ strain also showed reduced H2BK123ub across all gene-body nucleosomes. The greater loss in thelge1Δ bre1Δ strain indicates that Bre1 retains some activ- ity in the absence of Lge1 (see also Extended Data Fig. 8a). When the Bre1-interactingcoiled-coildomainofLge1wasremoved(NLS-Lge1IDR strain), the H2BK123ub pattern resembled that in the lge1∆ strain, confirming the general importance of the Bre1–Lge1 interaction in regulating H2B ubiquitination in vivo (Extended Data Fig. 8a, b). By contrast, when the IDR of Lge1 was deleted, H2BK123ub levels were mainly reduced downstream of +1 nucleosomes (Fig. 4a). This sug- geststhatLLPSisespeciallyimportantforubiquitinationwithingene bodies. ToexaminewhetheranyIDRcouldexecutetheLLPSfunctionofLge1, wetestedtwoIDRsfromdistantspecies.TheIDRoftheCaenorhabditis elegans P-granule component LAF1 (amino acids 1–169) undergoes LLPS26 , but has no relation to H2B ubiquitination. Homo sapiens WAC (aminoacids1–318)waspickedastheprobablefunctionalcounterpart of Lge111 (Extended Data Fig. 2b). Using ChIP-exo, we found that the IDR of human WAC, but not the IDR of the unrelated LAF1, partially substituted for the IDR of yeast Lge1 when fused to Lge1(CC) (Fig. 4a, Extended Data Figs. 8b, 10a, b, d–g). This was further confirmed by measuring global H2BK123ub levels with immunoblotting (Extended Data Fig. 8a), which suggests that the IDRs of human WAC and yeast Lge1 have analogous functions. The genome-wide binding profile of Lge1 showed enrichment at all gene classes, but was strongest at ribosomal-protein and SAGA- dominatedgenes(ExtendedDataFig. 9a,b).Enrichmentwasmaximal downstreamof+1nucleosomes(ExtendedDataFig. 9c),consistentwith theeffectofdeletingtheIDRofLge1onH2BK123ub.Thegenome-wide distribution of Lge1 was largely unaffected by loss of Bre1 (Extended Data Fig. 9b, c). Thus, intrinsic features of Lge1 (or as-yet unknown factors) may guide Lge1 to its target locations. These results support anucleosome-position-specificfunctionofLge1condensates,down- streamofthe+1nucleosome.Ubiquitinationatthe+1positiondepends on Bre1, but not through Lge1-directed LLPS. e c a 1.2 MDa 2.0 3.2 40S 60S 80S Polysomes 5 % Y/R Lge1 IDR CC g 1 ge1 IDR CC N C Bre1 80 243 332 -rich b Lge1–mCh Merged mGFP–Bre1 bre1Δ lge1Δ d VC–Bre1 Lge1 (IDR)– VN Lge1 (Δ1–80)– VN Lge1– VN Lge1 (CC)– VN BiFC BiFC Nup188– mCh bre1Δ lge1Δ 0 10 20 30 80 200 320 440 560 0 10 20 80 200 320 440 560 Fluorescence intensity 0 80 200 320 440 560 10 50 30 0 Pixel frequency Pixel frequency Pixel frequency Pixel frequency 80 200 320 440 560 10 6 7 8 9 10 12 1 2 3 4 5 11 13 WT lge1Δ lge1CC lge1IDR lge1Δ1–80 HA– Bre1 Lge1– TAP RNA Pol II HA– Bre1 RNA Pol II Lge1– TAP HA– Bre1 RNA Pol II Lge1– TAP HA– Bre1 RNA Pol II Lge1– TAP HA– Bre1 RNA Pol II Lge1– TAP 0 0.5 0.4 0.3 0.2 0.1 VC–Bre1 *** *** ** n = 60 Coefficient of variation L g e 1 – V N L g e 1 ( C C ) – V N L g e 1 ( Δ 1 – 8 0 ) – V N 45% Sucrose VC VN Fig.3|EndogenousLge1andBre1formlargecomplexesandnuclearpuncta. a,Sucrosegradientanalysisofcellextractsfromlge1∆cellstransformedwith theindicatedplasmids.Ribosomalspeciesserveassizemarkers.Gradient fractionswereanalysedbyimmunoblotting.Peakfractionsarehighlighted (arrowheads).HA,haemagglutinin;TAP,tandemaffinitypurificationtag;WT, wild-typeLGE1.b,Liveimagingofbre1∆ lge1∆cellsthatcoexpresstheindicated constructsfromastrongGPDpromoter.Scalebar,2 μm.mCh,mCherry.c,BiFC design.VNandVCdenotethecomplementaryVenusfragments(N-terminal andC-terminalfragments,respectively).d,Liveimagingofbre1∆ lge1∆cells expressingVC–Bre1andtheindicatedLge1–VNconstructsfromtheir endogenouspromoters.ArrowheadslabelnuclearBiFCpuncta,Nup188– mCherrylabelsthenuclearenvelopeanddashedlinerepresentsthecell contour.Histogramsrepresentpixelfrequenciesoffluorescenceintensity values.Scalebar,2 μm.e,CoefficientofvariationofnuclearBiFCfluorescence- intensityprofiles(ExtendedDataFig. 7i).Thehigherthecoefficientof variation,thegreatertheheterogeneityoftheBiFCsignal.Medianand interquartilerangeareindicated.n,numberofanalysedcells.**P = 0.0037, ***P < 0.001,determinedbytwo-sidedMann–Whitneytest.
- 5. 596 | Nature | Vol 579 | 26 March 2020 Article Lge1IDRaffectsviabilityinhtz1Δcells We next asked whether Lge1–Bre1 condensates affect cell physiol- ogy. Both LGE1 and BRE1 are essential for viability in cells that lack the conserved histone variant H2A.Z (yeast Htz1)7 (Fig. 4b, Extended Data Fig. 7f). Htz1 assembles into +1 nucleosomes27 —the only gene- body nucleosomes that do not depend on Lge1 LLPS for ubiquitina- tion. A truncation of the sticker region of Lge1 (either Lge1(Δ1–80) or Lge1(Δ1–40)) showed a synthetically enhanced growth phenotype whencombinedwithhtz1∆(ageneticinteractionresultinginasyner- gistic growth defect), as did Y>A mutations in the N terminus of Lge1 (aminoacids1–102)(Fig. 4b,ExtendedDataFig. 10h,i).Thedeletionof the middle region of Lge1 (amino acids 81–242) displayed no obvious growthdefectwithhtz1∆.Notably,however,deletionoftheentireIDR was synthetically lethal with htz1∆—despite the presence of the Bre1- interactingcoiled-coildomainofLge1.ExpressionofLge1(IDR)alone, which cannot bind Bre1, was similarly synthetic lethal (Fig. 4b). The IDR of human WAC—but not that of LAF1—partially compensated for the deletion of the IDR of Lge1 (Extended Data Fig. 10c, e), consistent with their effect on H2B ubiquitination of gene-body nucleosomes. Overall,thesegeneticassayssuggestthatLge1LLPSbecomesessential forviabilitywhenHtz1isabsentfrom+1nucleosomes,reinforcingthe physiological importance of Lge1–Bre1 condensates. We propose that the Bre1 shell has a direct catalytic role, whereas the core concentrates the E2 Rad6 and the chromatin substrate. By confining the reactants in a small space at high concentration, LLPS increases the opportunity for productive interactions many fold. Moreover, by avidity, the strength and specificity of multiple simul- taneous Bre1–NCP interactions will be greater than the sum of indi- vidualbindingevents.ThehumanorthologuesofBre1(RNF20/RNF40) copurify with WAC, which may perform a function analogous to that ofLge1inhumancells11 .Despitenoobvioussequencesimilarity,WAC andLge1shareasimilardomainorganization(ExtendedDataFig. 2b). OurresultsprovideanentrypointtostudywhetherLLPScontributes to DeSanto–Shinawi syndrome, a neurodevelopmental disorder that is caused by mutations that truncate WAC28 (Extended Data Fig. 2b). In conclusion, we have discovered that LLPS augments the catalytic activity of the chromatin modifier Bre1. We propose that core–shell condensatesformdynamichistoneubiquitinationhubs,whichtarget gene-bodynucleosomes(Fig. 4c)andtherebyshapegenearchitecture and expression. Onlinecontent Anymethods,additionalreferences,NatureResearchreportingsum- maries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author con- tributions and competing interests; and statements of data and code availabilityareavailableathttps://doi.org/10.1038/s41586-020-2097-z. Occupancy 2,500 2,000 1,500 1,000 500 3,000 3,500 H2B All genes 1,500 1,000 500 3,000 2,000 2,500 +1 +2 +3 NFR 1,500 1,000 500 3,000 2,500 2,000 +1 Nucleosome distance (bp) 1,500 1,000 500 3,000 2,500 2,000 +1 +2 +3 NFR WT lge1Δ 50 100 150 200 50 100 150 200 1,500 1,000 500 2,500 2,000 SAGA dom. Ribosomal protein TFIID dom. H2B K123Ub WT lge1Δ bre1Δ 50 100 150 200 50 100 150 200 500 2,500 2,000 1,000 1,500 +1 +2 +3 NFR WT lge1CC 50 100 150 200 1,500 1,000 500 2,500 2,000 50 100 150 200 WAC- lge1CC WT 50 100 150 200 50 100 150 200 1,500 1,000 500 2,500 2,000 –400 –200 200 0 400 –400 –200 200 0 400 –400 –200 200 0 400 –400 –200 200 0 400 +1 +2 +3 NFR a c Bre1 NFR TSS Htz1 Ub +1 –1 +2 Ub Ub Ub Ub Ub Ub Ub Ub +3 Ub Lge1 b lge1Δhtz1Δ SDC–His 5-FOA LGE1 lge1Δ lge1Δ1–40 lge1Δ1–80 lge1IDR NLS-lge1IDR lge1CC lge11–80, CC 243 332 1 80 CC N IDR Y/R -rich CC IDR CC IDR CC IDR CC IDR Y/R -rich Y/R -rich Y/R -rich Fig.4|TheIDRofLge1enhancesH2BK123ubingenebodiesandisrequired forviabilityinhtz1Δcells.a,H2B(left)orH2BK123ub(right)ChIP-exotag5′ endswereplottedrelativetothe+1nucleosomeofallgenesforwildtype(grey trace)andtheindicatedlge1mutants(redtraces).Thefirstthreegenic nucleosomesarelabelled+1,+2and+3.TwoH2Bpeaksareobservedper nucleosomeposition.H2BK123ubpatternsareshownseparatelyforeachgene class29 .dom.,dominated;NFR,nucleosomefreeregion.b,Geneticinteraction analysis.Doubledeletionstrains containingawild-typeLGE1 coverplasmid (Uramarker)werecotransformedwiththeindicatedplasmids(Hismarker). Growth(tenfoldserialdilutions)wasfollowedonSDC–His(control)andon SDC + 5-fluorooroticacid(5-FOA),whichshufflesouttheUracoverplasmid. c,LLPS-basedubiquitinationmodel.TheHtz1-containing+1NCPisdepicted outsideofthe‘reactionchamber’asitisubiquitinatedindependentlyofLge1 LLPS.Forsimplicity,Rad6andtheE1areomittedandonlyasinglelayerofBre1 moleculesintheshellisshown.TSS,transcriptionstartsite.
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- 7. Article Methods No statistical methods were used to predetermine sample size. The experimentswerenotrandomizedandinvestigatorswerenotblinded to allocation during experiments and outcome assessment. Proteinpurification Allrecombinantproteinsusedinthisstudywereexpressedaspublished20 . ProteinswereclonedbyPCR-basedmethodsfromgenomicDNA.Muta- tions and deletions were generated by PCR-based methods and con- firmedbysequencing.StrepII–Bre1constructswerepurifiedona5-ml Strep-TactinSuperflowcolumn(GEHealthcare)aspreviouslydescribed20 . 6×His–Lge1–StrepII constructs were purified on Ni-NTA Sepharose 6 FastFlow beads (GE Healthcare) coated with Co2+ (Co(NO3)2) at room temperature.Lysiswasperformedinabuffercontaining10mMTris,pH 8.0,100mMNaH2PO4,1mMTCEPand8Murea,followedbyawashing stepinwashingbuffer(10mMTris,pH7.4,100mMNaH2PO4,1mMTCEP and 8 M urea). A linear gradient to the final buffer (containing 20 mM Tris,pH7.4,500mMNaCl,1mMTCEP,3Mureaand10%v/vglycerol)was applied.Additionalwashingstepsin10mMTris,pH5.9,100mMNaH2PO4, 1 mM TCEP, 3 M urea and 10% v/v glycerol, and the same buffer with pH4.5wereperformed.Proteinswereelutedin10mMTris,pH7.2,300mM or 1M NaCl, 1 mM TCEP and 1 M imidazole, and directly used for phase separationassaysinbuffercontaining100mMNaCland100mMimidazol. TheLge1mutantwithR>Ksubstitutions(Lge1(11×R>K))(R11K,R18K, R20K, R27K, R28K, R30K, R39K, R48K, R63K, R70K and R77K) was ordered from Invitrogen. Mutant Lge1(17×(Y>A), 1–102) (Y4A, Y9A, Y12A,Y23A,Y38A,Y39A,Y45A,Y49A,Y53A,Y68A,Y69A,Y73A,Y79A, Y80A, Y87A, Y95A and Y102A) was ordered from GenScript. StrepII–mGFP and StrepII–mGFP–Bre1 constructs were affinity- purifiedona5-mlStrepII-TactinSuperflowcolumn(GEHealthcare)in buffercontaining50mMTris,pH7.5and100mMNaCl.Proteinswere elutedin5mMdesthiobiotinandstoredin10%v/vglycerolat−80 °C. 6×His–TEV–Rad6 was cloned, expressed and purified as previously described20 . Where stated, purified 6×His–TEV–Rad6 was labelled with Pacific Blue C5-maleimide (ThermoFisher) as follows. Rad6 was incubated with Pacific Blue dissolved in DMSO for 20 min at 4 °C, before dialysis (18 h) in 50 mM Tris, pH 8.0, 50 mM NaCl, 50 mM KCl, 10 mM MgCl2 and 1 mM TCEP. Unbound fluorophore was removed by size-exclusionchromatographyonaSuperdex20016/60column(GE Healthcare) equilibrated with the same buffer. Purified protein was stored in final 10% v/v glycerol at −80 °C. LAF1(residues1–169)wasamplifiedbyPCRfromaplasmidcontain- ingfull-lengthLAF1(obtainedfromthelaboratoryofC. Brangwynne) and fused with Lge1(CC) in an expression vector. The coding region forWAC(aminoacids1–318)wasamplifiedbyPCRfromhumancDNA andwasfusedtoLge1(CC)inanexpressionvector.6×His–LAF1(1–169)– Lge1(CC)–StrepIIand6×His–WAC(1–318)–Lge1(CC)–StrepIIwerepuri- fied in urea as described for 6×His–Lge1–StrepII, followed by buffer exchange to 10 mM Tris, pH 8.0, 0.2 mM EDTA and 100 mM NaCl. In vitrobindingassays Purified GST–Lge1–StrepII constructs were mixed with StrepII–Bre1 constructs in a 1:3 molar ratio in binding buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 0.5 mM DTT and 50 μg of BSA) and incubated with glu- tathione-sepharose 4B beads (GE Healthcare) for 1 h at 4 °C. Beads were washed with binding buffer (without BSA) and bound proteins were eluted in 0.006 mg/ml glutathione. After trichloroacetic acid (TCA) precipitation, samples were analysed by SDS–PAGE (4–12% gel, MOPS buffer) and Coomassie staining, or by immunoblotting with anti-StrepII antibody (Qiagen, no. 34850). Sucrose gradientsedimentationassay Forrecombinantproteins,30μgofpurifiedproteinswereappliedtoa 12-ml5–45%sucrosegradientin20mMTris,pH7.5,10mMKCland5mM MgCl2,andsedimentedat27,000rpmfor15hat4 °CinaSW40-Tirotor (BeckmanCoulter).Gradientswerefractionatedin13×1-mlfractionsby pumpingFluoriniertFC-40(3M)intothebottomofthegradientwhile collecting the fractions from the top using a Brandel gradient frac- tionationsystem.FractionswereprecipitatedwithTCA,washedtwice withacetoneandanalysedbySDS–PAGE(4–12%gel,MOPSbuffer)and Coomassiestaining.FortheBre1–Lge1complex,30μgofeachprotein was incubated for 30 min at 4 °C before loading the gradient. For the fractionationofcelllysates,cellsweregrownexponentially,collected andresuspendedinbuffer20mMTris,pH7.5,10mMKCl,5mMMgCl2 and 1mM DTT. Cells were lysed by vortexing with glass beads. Lysates wereclarifiedbycentrifugationat14,000rpmfor5minat4 °C.Thetotal proteinamountwasquantifiedbyBradfordassay(BioRad)andabout 350μgwerelayeredonto12-ml5–45%sucrosegradients,sedimented andfractionatedasdescribed.Ribosomalprofileswererecordedasa reference by monitoring absorbance at an optical density of 254 nm. After TCA precipitation, samples were analysed by immunoblotting withanti-HA(Sigma,no.11666606001),anti-proteinA(Sigma,P3775) and anti-RNA polymerase II (Rpb1; Covance, no. MMS-126R). Input samples were also analysed with anti-Pgk1 (Abcam, no. Ab113687) as loading control. Lge1phase-separationassay PurifiedLge1proteinsinatotalvolumeof20μlwereplacedina16-well glass-bottom ChamberSLIP slide (Grace, BioLabs). Slides were pre- treatedwith1%(w/v)pluronicF-127(PF127,SigmaAldrich)andwashed twicewithbuffer25mMTris,pH7.5,50mMNaCl,1mMDTTand10%v/v PEG6000.Afinalbuffercompositionof20mMTris,pH7.5,100mMNaCl and1mMDTTwasusedforthedilutionoftheproteins.1,6-Hexanediol was used at a final concentration of 5 or 10% (w/v) (Sigma Aldrich, no. 240117).Finalproteinconcentrationandincubationtimesfortheexperi- ments are detailed in the corresponding figure legends. DIC imaging was performed on a temperature-controlled DeltaVision Elite micro- scope (GE Healthcare) at 20 °C. Images were acquired with a 60× oil immersionobjectivecoupledtoaCoolSNAPHQ2charge-coupleddevice camera (Photometrics). Image deconvolution was carried out using the SoftWoRx software (GE Healthcare). Images were processed with ImageJ. Quantification of the total area (in μm2 ) of randomly selected condensates was analysed in ImageJ. One hundred condensates were measured for each condition. Statistical significance was evaluated bytwo-sidedMann–WhitneytestusingtheGraphPadPrismsoftware. Phase-separationassayswithfluorescentproteins 6×His–Lge1constructs(1.5μM)weremixedwithfluorescentproteins (StrepII–mGFP,StrepII–mGFP–Bre1constructs,6×His–Rad6*,1×NCP*, 16×NCP* or 16× 601 Widom DNA*) in pretreated and washed 16-well glass-bottom ChamberSLIP slides at 20 °C in a final volume of 20 μl. Abuffercompositionof20mMTris,pH7.5,100mMNaCl(50mMNaCl forNCPassays,1mMDTT)wasused.Theconcentrationoffluorescent constructs and incubation times for each experiment are described in the figure legends. Samples were either directly imaged or, where stated, the experiment was followed over time. Image acquisitions parameters for images of the same experiment are always identical. Images were processed with ImageJ. The peak fluorescence intensity of the condensates with fluorescent-tagged proteins was measured by drawing a line across at least 25 randomly selected condensates. Line-scan graphs were generated in ImageJ. For Bre1 shell formation, 6×His–Lge1–StrepII (1.5 μM) and StrepII–mGFP–Bre1 constructs (3μM)werepreincubatedinpretreatedandwashed16-wellglass-bottom ChamberSLIP slides for 15 min at 20 °C. Fluorescent shell thickness wasquantifiedasfollows:ontheline-scangraphs,backgroundsignal insidethecondensatewassubtractedfromthemaximumfluorescent intensityofthepeaks.Finalthicknessvaluescorrespondtothelength (in μm) of the resulting baseline of the peaks. Statistical significance was evaluated by two-sided t-test analysis with the GraphPad Prism
- 8. software.Imageacquisitionsparametersforimagesofthesameexperi- mentarealwaysidentical.Thenumberofanalysedcondensates(n)is indicated in each figure. Turbidityassay Two hundred microlitres of each purified protein (buffer: 20 mM Tris pH7.5,100mMNaCl,100mMimizadoland1mMDTT)weremeasured at450nmonabottom-clear96-wellplate(GreinerBio-One)inaVictor Nivo plate reader (Perkin Elmer). Dextranexperiments Tetramethylrhodamineisothiocyanate(TRITC)-labelleddextranofan average molecular weight of 35–45 (TdB Consultancy AB, no. TD40), 65–85 (Sigma Aldrich, no. T1162), 155 (Sigma Aldrich, no. T1287) or 2,000kDa(ThermoFisher,no.D7139)wasdissolvedin20mMTris,pH 7.5 and 100 mM NaCl. The dextrans with a final concentration of 0.05 mg/mlwereaddedtosamplescontaining6×His–Lge1–StrepII(1.5μM), 6×His–Lge1(IDR)–StrepII(1.5μM)or6×His–Lge1–StrepII(1.5μM)with apreformedBre1shell,eachin20μl.Thefinalbuffercompositionwas 20 mM Tris, pH 7.5, 100 mM NaCl and 1 mM DTT. Samples were incu- batedfor15minat20 °Cinpretreatedandwashed16-wellglass-bottom ChamberSLIPslides.SampleswereimagedsimultaneouslyinDIC,FITC andTRITCchannels.Partitionratioswerecalculatedonthebasisofthe background-correctedTRITCfluorescentintensityinsideandoutside ofacondensatequantifiedwithImageJ.Atotalof60randomlyselected condensates were considered for each condition. Dynamiclightscattering The hydrodynamic radius and molecular weight of recombinant pro- teins were measured at 20 °C in a ProteinSolution DynaPro-99-E50 DLS module (Wyatt). Nucleosomearrayreconstitutionandlabelling Mononucleosomes(1×NCP)werereconstitutedaspreviouslydescribed20 . Forchromatinreconstitution(16×NCParray),DNAconsistingof16repeats ofthe167-bp601WidomsequencewasgeneratedfromapUC19-16x601Wi- domplasmid(agiftfromT.Richmond)bytripledigestionwithPstI,BamHI andDpnIfor20hat37 °C.Fortypercent(w/v)PEG6000–NaClprecipita- tionwasusedtoseparatethedigestedplasmidfromarrayDNA,which wasprecipitatedinEtOHandfinallyresuspendedin10mMTris,pH8.0, 0.1 mM EDTA. Chromatin was reconstituted with the purified histone octamer and the 16× 601Widom DNA, in a histone octamer:DNA ratio (mg:mg)of10,followedbyKCldialysisandMonoQion-exchangechroma- tography,aspreviouslydescribed20 .Sampleswerethendialysedin20mM Tris,pH7.5,1mMEDTAand1mMDTTfor18h.Histonestoichiometrywas assessedbySDS–PAGEandCoomassiestaining.Nucleosomeoccupancy wastestedbyScaIdigestionandelectrophoresisina1%agarosegel.Both1× and16×NCPswerelabelledwithHoechst33258(SigmaAldrich)(1μg/ml) byincubationfor30minat4 °C,followedbydialysisin20mMTris,pH 7.5 and 1 mM DTT for 20 h at 4 °C. Labelled 1×NCP* and 16×NCP* were storedat4 °C.The16×DNAwaslabelledwithHoechst(final1μg/ml)by incubationfor18hat4 °Candstoredat−20 °C. Negative-stainelectronmicroscopy Thequalityofthe16×NCParraywasalsoassessedbyelectronmicros- copy.Samplesin20mMTris,pH7.5,wereadsorbedonglow-discharged electron microscopy grids coated with carbon film and stained with 2%uranylacetate(Merck),pH4.0.Electronmicroscopysampleswere examinedonaFEIMorgagni268DTEMoperatedat80kV.Digitalimages were acquired using an 11-megapixel Morada charge-coupled device camera from Olympus-SIS. NCPubiquitinationassay Forthein vitroH2BubiquitinationassaywithLge1–Bre1condensates, 6×His–Lge1(0.5μM),Bre1(3μM),155-kDadextran(0.65μM),1×NCP(1μM), E1 (100 nM), Rad6 (3 μM) and ubiquitin (36 μM) were incubated in 20 mM Tris HCl, pH 7.5, 75 mM NaCl and 1 mM DTT. Reactions were startedbyadditionofATP(3mM)andcarriedoutfor12minwithshak- ingat300rpmat30 °C.Reactionswerestoppedattheindicatedtime pointsbyadding4×SDSloadingbufferandboiling(5minat95 °C),and analysed by SDS–PAGE and immunoblotting with anti-Flag antibody (Sigma Aldrich, no. F1804). Each experiment was reproduced at least three times. Condensate formation was verified by microscopy. Yeaststrainsandtandemaffinitypurification Allyeaststrainsusedinthisstudyarelistedinthe SupplementaryTable. Genesinyeastweretaggedordeletedbyastandardone-stepPCR-based technique.Microbiologicaltechniquesfollowedstandardprocedures. Cells were grown in yeast-extract peptone dextrose (YPD) or, when transformed with plasmids, in selective synthetic dextrose complete (SDC) drop-out medium. Tandem affinity purifications (TAPs) from yeast were performed according to standard protocols. Live-cellimagingofyeast Exponentially growing cells were immobilized on microscope slides with agarose pads and imaged on a DeltaVision Elite microscope (GE Healthcare).Imageswereacquiredwitha60×oilimmersionobjective and recorded with a CoolSNAP HQ2 charge-coupled device camera (Photometrics). Deconvolution was carried out using the SoftWoRx software (GE Healthcare). Images were processed with ImageJ. Cell contours were marked with a dashed white line based on bright-field imaging.Forthe1,6-hexanediolexperiment,cellswereincubatedwith 10%(w/v)1,6-hexanedioland10μg/mldigitonin(oronly10μg/mldigi- tonin as control) for 10 min at 30 °C before imaging. To record Lge1–Bre1 BiFC intensity in a histogram, a contour was drawnaroundthenucleusandfluorescenceintensitieswereanalysed usingtheImageJ‘Histogram’function.Forquantificationofthemean nuclear BiFC intensity, mean fluorescence-intensity values from the histogramswereused.Thecoefficientofvariationvaluesrepresentthe ratio of the standard deviation to the mean (both values determined from histogram data). To quantify the corrected total-cell fluorescence, a contour was drawn around a cell and the integrated density was measured using ImageJ. To quantify the fluorescence intensity of Lge1 constructs in cells, a line was drawn across the nucleus and line-scan graphs were obtained by ImageJ. The number of analysed cells (n) is indicated in eachfigure.Statisticalsignificancewasevaluatedbytwo-sidedMann– Whitney test using the GraphPad Prism software. Yeastgeneticinteractionanalysis Doublemutantstrainscontainingawild-typeLGE1coverplasmid(Ura marker)werecotransformedwithplasmidscarryingLGE1,WACorLAF1 (His marker). Growth was followed on SDC–His (loading control) and onSDC + 5-FOAplatestoshuffleouttheUracoverplasmid.Cellswere spotted in tenfold serial dilutions and incubated for 2 (SDC–His) or 3 days (5-FOA) at 30 °C. Proteinexpressionlevelsinwhole-cellextracts Yeast lysates were prepared, normalized for protein concentration and analysed by western blotting according to standard procedures. Antibodies were used according to the manufacturer’s instructions: anti-mCherry(Abcam,no.Ab125096),anti-proteinA(Sigma,no.P3775) and anti-Pgk1 (Abcam, no. Ab113687). GloballevelsofH2Bubiquitination LGE1 was deleted in a strain carrying Flag-tagged H2B. This strain was then transformed with plasmid-based versions of wild-type LGE1- mCherry, empty vector and the indicated mutants, all of which were expressedfromtheendogenousLGE1promoter.Celllysatesweresub- jectedtoanti-Flagimmunoprecipitation(M2beads,Sigma,no.A2220).
- 9. Article RecoveredproteinswereanalysedbySDS–PAGEandimmunoblotting with an anti-Flag antibody (Sigma, no. A8592). ChIP-exoversion5.0 The antibodies rabbit monoclonal antibody against ubiquityl- H2B(Lys120)(CellSignaling,no.5546),andrabbitpolyclonalantibody againstH2B(Abcam,no.ab1790)10 wereusedtoprobeforthespecific ubiquitinmarkandtheunderlyingnucleosome,respectively.ForTAP- taggedstrains,rabbitIgG(Sigma)conjugatedtoDynabeadswasused. The protein A module of the TAP tag was the target. IndicatedmutantstrainsweregrowninselectiveCSM − Hismedium, andthewild-typestrainsweregrowninCSM + allmediumtoanoptical densityat600 nmof0.8at25 °C.Cellswerecrosslinkedwithformalde- hydefor15minatroomtemperatureatafinalconcentrationof1%,and quenchedwithglycinefor5minatafinalconcentrationof125mM.Cells were then centrifuged and washed with ST buffer (10 mM Tris-HCl, pH 7.5and100mMNaCl)at4 °C.Supernatantwasremovedandcellpellets were flash-frozen and stored at −80 °C until further use. Fifty-millilitre culture aliquots were resuspended and lysed in 750 μl FA lysis buffer (50mMHepes-KOH,pH7.5,150mMNaCl,2mMEDTA,1%TritonX-100, 0.1% sodium deoxycholate and complete protease inhibitor (Roche)) alongwith1mlof0.5-mmzirconiabeadsbybead-beatingfor4cyclesof 3-minon/7-minoffinaMini-Beadbeater-96machine(Biospec).Whole- celllysatesweretransferredtomicrocentrifugetubesandspunat14,000 rpmfor3minat4 °Ctoobtainchromatinpellets.Thesupernatant(repre- sentingthecytoplasmicfraction)wasdiscardedandthechromatinpellet wasresuspendedin200μlFAlysisbuffersupplementedwith0.1%SDS and75μl0.1-mmzirconiabeads.Theresuspendedchromatinsamplewas sonicatedinaBioruptor(Diagenode)for4cyclesof30-son/30-soff.The tubeswerecentrifugedat14,000rpmfor10mintocollectthesonicated chromatin fraction. Chromatin obtained from 50 ml culture was used for a single ChIP experiment. A Reb1–TAP-tagged strain (Open Biosys- tems) was used a positive control to determine the success of the ChIP experiments.Appropriateantibodies(5μgperChIP)wereconjugatedto proteinAmagneticsepharoseresin(GEHealthcareLifeSciences)at4 °C for6–8h.Unconjugatedantibodywasremovedandchromatinsample wasaddedandincubatedat4 °Covernighttoallowforimmunoprecipi- tation.Allstepsafterimmunoprecipitationwereessentiallyperformed aspreviouslydescribed23 .Preparedlibrariesweregel-purifiedandwere sequencedinpaired-endmodewithaNextSeq500.Sequencereadswere alignedtotheS. cerevisiaegenome(sacCer3)usingbwa-mem(v.0.7.9a)30 . AlignedreadsobtainedwerefilteredtoremovePCRduplicatesandany non-unique alignments. ChIP-exo read-1 5′ ends had their coordinates shifted by 6 bp in the 3′ direction, to reflect the offset of exonuclease stopsandthesiteofcrosslinking.Atleasttwobiologicalreplicateswere performedforChIP-exoexperiments,unlessotherwiseindicated.These methodsandinformationondataanalysisandscriptscanbeaccessed online(https://github.com/CEGRcode/Gallego_2019). Structuralbioinformatics Charge profiles for Lge1 and WAC protein sequences were generated usingscriptswrittenforthispurpose,withawindowof10aminoacid residuesusedforsmoothing.HydrophobicityprofilesforLge1andWAC protein sequences were generated and superimposed using VOLPES server31 (http://coil.msp.univie.ac.at/app.html). Statisticsandreproducibility TheexperimentsinFig. 1b,c,dwereperformed4,2and3times,respec- tively;thequantificationofcondensatesisincludedinExtendedData Fig. 1j.ExperimentsinFig. 2a,bwereperformed3times.Experimentsin Fig. 3a,b,dwereperformed2,3and3times,respectively;quantificationis includedinExtendedDataFig. 7i.TheexperimentshowninFig. 4aisrepre- sentativeof2replicates.TheexperimentinFig. 4bwasperformed6times. Experiments were performed the following numbers of times: 2 (ExtendedDataFigs. 1i,3c,4a,g,6a,d,g,10a),3(ExtendedDataFigs. 1k, 3a,b,4b,e,6b,7a–d,f–h,8a,10d,f),4(ExtendedDataFigs. 1d,e,5a,c,6e, f,h,10c,e,h,i),5(ExtendedDataFig. 1b,c)or6(ExtendedDataFig. 1a) In Extended Data Fig. 1f, Lge1(1–80) was purified 2 times, Lge1(1– 80, CC) was purified 3 times and the other constructs were purified >10 times. The time lapse for droplet fusion in Extended Data Fig. 1h wasrecordedin2independentexperiments.InExtendedDataFig. 2b, sequenceswereanalysedasdescribedinthefigurelegendandin Supple- mentaryMethods.ThetimelapseinExtendedDataFig. 3ewasrecorded in2independentexperiments.TheexperimentinExtendedDataFig. 4c wasperformed10timesascontrol.InExtendedDataFig. 6c,thepurifi- cationofmononucleosomes(1×NCP)wasperformed29timesandthe purificationof16×NCPwasperformed11times.Theexperimentshown in Extended Data Fig. 6i corresponds to Fig. 3a (performed 2 times). ThenumbersofrandomlyselectedcellsinExtendedDataFig. 7ewere: Lge1 = 22;Lge1bre1Δ = 33;Lge1(Δ1–40) = 24;Lge1(Δ1–80) = 25;Lge1(1– 80,CC) = 25;Lge1(IDR) = 28;NLS–Lge1(IDR) = 21;andLge1(CC) = 27.The quantification for Extended Data Fig. 7h is shown in Extended Data Fig. 7i. Data shown in Extended Data Fig. 8b, c are representative of 2 replicates. In Extended Data Fig. 9a, the dot spots were performed 4 times,andwholecellextractsandwesternblottingwereperformed2 times.DatashowninExtendedDataFig. 9b–earebasedon2replicates. Reportingsummary Further information on research design is available in the Nature Research Reporting Summary linked to this paper. Dataavailability ChIP-exo sequencing files can be accessed at NCBI Gene Expression Omnibus(https://www.ncbi.nlm.nih.gov/geo/),withtheaccessionnum- ber GSE131639. All source data (that is, uncropped gels and western blots)associatedwiththepaperareprovidedinSupplementaryFigs. 1,2. Codeavailability Data analysis software, scripts and parameters used to analyse ChIP- exo datasets can be accessed via https://github.com/CEGRcode/Gal- lego_2019. 30. Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at https://arxiv.org/abs/1303.3997 (2013). 31. Bartonek, L. & Zagrovic, B. VOLPES: an interactive web-based tool for visualizing and comparing physicochemical properties of biological sequences. Nucleic Acids Res. 47, W632–W635 (2019). 32. Armstrong, J. K., Wenby, R. B., Meiselman, H. J. & Fisher, T. C. The hydrodynamic radii of macromolecules and their effect on red blood cell aggregation. Biophys. J. 87, 4259–4270 (2004). 33. Yassour, M. et al. Ab initio construction of a eukaryotic transcriptome by massively parallel mRNA sequencing. Proc. Natl Acad. Sci. USA 106, 3264–3269 (2009). Acknowledgements We thank B. Zagrovic and A. Polyansky for critical insights into the multivalency of Lge1 LLPS, and Lge1 and WAC sequence analyses; O. R. Abdi for technical support; and G. Warren and D. Gerlich for discussions. A.K. was funded in part by a NOMIS Pioneering Research Grant, L.D.G. by a L’Oréal-UNESCO-OeAW Austria Fellowship and A.R. by an OeAW DOC Fellowship. B.F.P. and C.M. were funded by the National Institutes of Health grant HG004160. Author contributions L.D.G. and M.S. performed all biochemical and genetic experiments with support from R.M.G.C. C.M. conducted ChIP-exo and analysed the data with B.F.P. T.S. contributed to sucrose gradient analyses. A.R. performed all cell biology experiments. A.K. designed the study and wrote the paper with input from all authors. Competing interests B.F.P. has a financial interest in Peconic LLC, which uses the ChIP-exo technology implemented in this study and could potentially benefit from the outcomes of this research. The remaining authors declare no competing interests. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020- 2097-z. Correspondence and requests for materials should be addressed to A.K. Peer review information Nature thanks Fred van Leeuwen, Tanja Mittag and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Reprints and permissions information is available at http://www.nature.com/reprints.
- 11. Article ExtendedDataFig.2|Lge1structuralproperties,andcomparisonwith WAC. a,AminoacidsequenceofS. cerevisiaeLge1.TheIDR(aminoacids1–242) ishighlightedinlightorange;thepredictedcoiled-coildomain(aminoacids 281–332)ishighlightedinindarkorange;andtheY/R-richstickerregion(amino acids1–80)isunderlined.ThemutatedYandRresiduesarelabelledinmagenta andblue,respectively.b,ComparativesequenceanalysesofyeastLge1(left) andhumanWAC(WW-domain-containingadaptorproteinwithcoiled-coil) (right).CartoonshowspredictedLge1andWACdomainorganization; boundariesaredrawntoscale.WW,protein–proteininteractiondomainwith twotryptophans(W).Asterisksindicateresiduesthemutationofwhichhas previouslybeenimplicatedinthepathogenesisofDeSanto–Shinawi syndrome28 .Thisneurodevelopmentaldisordercausesadevelopmentaldelay anddysmorphicfacialfeatures.Itiscausedbymutationsthatarepredictedto truncatetheIDRofWAC,andthereforedisrupttheWACinteractionwith RNF20/RNF40(thatis,nonsenseandframeshiftmutationsleadingto nonsense-mediateddecayorproteintruncation)28 .Fordisorderprediction, disorderscoreswerecalculatedwiththeIUPredalgorithm.BothLge1andWAC showextensiveintrinsicdisorder.For coiled-coildomain predictions,the COILSsoftwarewasused,showingputativecoiled-coilelementsatthe C terminiofbothproteins.Forhydrophobicityanalysis,theVOLPESwebserver wasusedtoplothydrophobicityprofilesofproteinsequences31 (furtherdetails areprovidedintheMethods).Lge1andWACdisplayasimilarhydrophobicity patternintheirN-terminalregions.Theregionwiththebestmatchis highlightedwithagreyrectangle.Similarityinthisregionisindicatedby Pearsoncorrelationcoefficient(R = 0.68)androot-mean-squareddeviation (r.m.s.d. = 0.16).Intheirchargedistributionprofile,bothLge1andWACexhibit alternatingblocksofnegativeandpositivecharge.Thesequencecharge densitywascalculatedusingacustom-madescript.Awindowoftenresidues wasusedforsmoothing.Forcondensateformation,thecatGRANULE algorithmwasusedtopredictthepropensityforcondensateformation.Top scoresareindicated.Thehigh-scoringsequenceinLge1corresponds approximatelytotheY/R-richregion.
- 12. ExtendedDataFig.3|MechanismofBre1shellformation. a, b, Reconstitutionofcondensateswithcore–shellarchitecture.Recombinant mGFP-taggedproteins(1.5 μM)wereaddedtopreformed6×His–Lge1 condensatesor6×His–Lge1(IDR).Sampleswereincubatedfor15 minbefore imagingbyDICandfluorescencemicroscopy.Scalebar,2 μm. c,Reconstitutionofhybridcondensates,withvaryingratiosof6×His– Lge1:6×His–Lge1(IDR)showadifferentialpartitioningofmGFP–Bre1intothe core.Proteinsweremixedattheindicatedmolarratiosandincubatedfor 15 minat20 °C.mGFP–Bre1(0.5 μM)wasaddedtothepreformedcondensates andincubatedfor15 minbeforeimagingbyDICandfluorescentmicroscopy. Fluorescentintensitieswerequantifiedacrosssinglecondensates.Cartoons indicateputativeassemblystateofhybridcondensates,withadeteriorationof thecore–shellstructureuponreductionofavailableLge1coiled-coildomains. n,numberofcondensates.Scalebar,2 μm.d,QuantificationofmGFP–Bre1 shellthicknessinc.Box-and-whiskerplotshowsmedian,interquartilerange, andminimumandmaximumvalues.****P < 0.0001,determinedwithtwo-sided t-test(t = 9.4,degreesoffreedom = 62).n,numberofcondensates;n.d.not determinable. e,Analysisofcondensatefusion.6×His–Lge1condensates (1.5 μM)withanmGFP–Bre1shell(1.5 μM)werefollowedovertimeby microscopy.Condensatescollidebutdonotfuse(SupplementaryVideo 2). ComparetoExtendedDataFig. 1hforfusiondynamics.Scalebar,2 μm.
- 15. Article ExtendedDataFig.6|Lge1–Bre1condensatesrecruittheE2Rad6and chromatin.a,RecruitmentofPacific-Blue-labelled6×His–Rad6(heredenoted ‘Rad6*’)(3 μM)ormGFP–Bre1(3 μM)toLge1condensates.Experimental conditionsasinFig. 2a.Fluorescentintensitieswerequantifiedacrosssingle condensates.n,numberofcondensates.Scalebar,5 μm.b,Therecruitmentof Rad6tocondensateswasexaminedbyaddingRad6*to6×His–Lge1 condensatesinthepresenceandabsenceofanmGFP–Bre1(1.5 μM)ormGFP– Bre1(LBD)(15 μM)shell.TheBre1(LBD)constructhasaweakeraffinitytoLge1 thanfull-lengthBre1,andthereforerequiresahigherconcentrationinthe assay.MicroscopywasperformedimmediatelyafteraddingRad6*(3 μM)and followedovertime.Fluorescentintensitieswerequantifiedacrosssingle condensates.n,numberofcondensates.Scalebar,5 μm.c,Reconstituted mononucleosomes(1×NCP)andoligonucleosomes(16×NCP)wereanalysedby SDS–PAGEandCoomassiestainingtoassesspurityandstoichiometry. d,Negative-stainelectronmicroscopywasperformedtoassessthestructure ofoligonucleosomes.White arrowheads labelindividualnucleosomes,blue arrowheadsindicatethelinkerDNA.Scalebar,100 nm.e,Recruitmentof mononucleosomestoLge1condensateswithanmGFP–Bre1shell. Hoechst- labelled,reconstitutedmononucleosomes(1×NCP*,0.5 μM)wereaddedto 6×His–Lge1condensateswithanmGFP–Bre1shell(3 μM),andimagedovertime (min).n,numberofcondensates.Scalebar,5 μm.f,Recruitmentof oligonucleosomestoLge1condensates.The16×NCPs*(0.5 μM)wereaddedto 6×His–Lge1condensatesandimagedovertime(min).n,numberof condensates.Scalebar,5 μm.g,Diffusionandretentionof601WidomDNAinto Lge1condensates.SamesetupasinfbutwithHoechst-labelled16×601Widom DNA(16×DNA*)addedto6×His–Lge1condensates.n,numberofcondensates. Scalebar,5 μm.h,Traces(opticaldensityat254 nm)ofthesucrose gradient sedimentationassays(5–45%)showareproduciblefractionationpatternfor cellextractspreparedfromtheindicatedstrains.Thedifferentribosomal speciesandfractionsareindicatedandcorrespondtothefractionsinFig. 3a. i,Proteinlevelsincellextractsusedforsucrosegradientassays.Anti-Pgk1 servesasaloadingcontrol.SeeSupplementaryFig. 2foruncroppedgelsand western blots.
- 16. ExtendedDataFig.7|AnalysisofLge1andBre1in vivo.a,Galleryof representativeimagesofbre1∆ lge1∆cellsco-expressingtheindicated constructsfromthestrongGPDpromoter.Scalebar,2 μm.b,1,6-Hexanediol treatment(10%,w/v)reducestheformationofLge1–Bre1puncta,butalso affectsnuclearimportandthuscomplicatestheinterpretationofhexanediol effectsincells.Whiteasterisklabelsthevacuole;dashedwhitelineshowsthe cellcontour.Scalebar,2 μm.c, d,Liveimagingofbre1∆ lge1∆cellsexpressing mGFP–Bre1(c)oranemptyvector(d),andtheindicatedLge1–mCherry constructs.Dashedwhitelineshowsthecellcontour.Thefluorescence intensityofLge1–mCherryconstructswasquantifiedacrossalinespanningthe nucleus.Forcomparison,thearbitraryfluorescenceunitvalue = 1ismarked withahorizontaldashedline(exceptford,inwhichthedashedlineindicatesa valueof0.5).n,numberofrandomlyselectedcells.Scalebar,2 μm. e,Quantificationofbackground-correctedtotalcellfluorescence(CTCF)of mCherryinc, d.Dotplotsshowmedianandinterquartilerange.f,Comparison ofprotein-expressionlevelsofdifferentLge1–mCherryconstructsinc.Cell lysateswereanalysedbySDS–PAGEandimmunoblottingwithanti-mCherry antibody.Anti-Pgk1servesasaloadingcontrol.Asteriskindicatesa degradationproduct.RedarrowheadsindicateLge1constructsaccordingto theirpredictedsizes.SeeSupplementaryFig. 2foruncroppedwesternblot. g,Galleryofrepresentativeimagesofbre1∆ lge1∆cellsexpressingVC–Bre1and Lge1–VNorLge1(CC)–VN.Nup188–mCherrymarksthenuclearenvelope.Scale bar,2 μm.h,Liveimagingofbre1∆ lge1∆cellsexpressingVC–Bre1andthe indicatedLge1–VNconstructsfromtheirendogenouspromoters. Nup188– mCherrymarksthenuclearenvelope,andthedashedlinemarksthecell contour.Histogramsrepresentpixelfrequenciesoffluorescenceintensity values.Scalebar,2 μm.i,QuantificationofmeannuclearBiFCintensityinhand Fig. 3d.Medianandinterquartilerangeareindicated.n,numberofcells. ***P < 0.001,determinedbytwo-sidedMann–Whitneytest.ns,notsignificant.