progression igogJ,HiiH,FnYn,Jpn un agYn,LaginJag aynQa,BihnJiang, Baichun Qian, Yanyan Jiang, Liangqian Yang, Yang Yuan, Jupeng Yang, Fan Hu, Huili Ji, Qinghong complex SIN3A-HDAC the repress coordinates to and with interacts CRL4B ARTICLE RESEARCH ß eevd1Arl21;Acpe 6Ags 2014 August 16 Accepted 2014; April 1 Received ([email protected]) correspondence for *Author Medicine, of School of China. University Institute 250012, Shandong Education, Jinan, Genetics, of 1998; and Ministry Medicine Teratology, histone Allis, Molecular Experimental of and Laboratory enzymes, Key (Kuo state of acetylation (HDACs), types reversible deacetylases the histone two control The and accessibility. of (HATs) chromatin binding acetyltransferases regulate creating activities through by that or either proteins antagonistic DNA the expression, for for histones gene sites of affinity of of the opening activation the altering to linked and is H4, 2011). and chromatin the H3 al., on histones residues of et lysine tails Tan at N-terminal mostly modifications 2004; occurring histones, Laniel, These of post-translational and Acetylation 2009). (Peterson sumoylation of Gasser, ubiquitylation and acetylation, and methylation, Smith, variety phosphorylation, and Attikum include Iizuka 2007; van a (Berger, response 2003; transcription damage to configuration, DNA the chromatin and affect subject that modifications are Histones INTRODUCTION CRL4B, SIN3A-HDAC WORDS: KEY and CRL4B repression. between transcriptional in action epigenetic complexes findings coordinated Our to a SIN3A-HDAC. of contributing reveal function also deacetylation directly CRL4B target the monoubiquitylation, to of facilitates H2AK119 promoters catalyzing addition by the in silencing on SIN3A-HDAC Thus, of genes. for retention required not stable H4. is the CRL4B and of H3 SIN3A- function acetylated ubiquitylation of of the retention Interestingly, levels we decreased the increased a and to components co-occupy Furthermore, led HDAC CUL4B and interact of Lack proteolysis. complexes other promoters. SIN3A-HDAC each through and with CRL4B transcriptional not through that p21 demonstrate but of function the repression, regulate negatively cyclin- to the by (encoded of p57 and upregulation CDKN1A CUL4B p21 (CKIs) the of inhibitors G1 (CDK) by kinase loss causes accompanied dependent and that proliferation arrest, cell show cycle in cell We reduction significant progression. a in cycle results cell CUL4B of driving role ubiquitin crucial in a of CRL4B demonstrate spectrum we broad the Here, a processes. of assembles biological regulation the that in participates protein complex, ligase scaffold a CUL4B, ABSTRACT Gong* Yaoqin and Shao Changshun Wang, Yan Zou, Yongxin 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,47–61doi:10.1242/jcs.154245 4679–4691 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. and CDKN1C CDKN1A CDKN1A epciey.Srknl,CLBwsfound was CUL4B Strikingly, respectively). , I3-DC elcycle Cell SIN3A-HDAC, , CDKN1A n rv elcceprogression cycle cell drive and and CDKN1C aaaa 01 aet ta. 97 age l,2009b). al., et and Wang 1997; generally (Hayakawa al., are et repression they Laherty and transcriptional 2011; – Nakayama, with complex CoREST-HDAC associated complex the including BCH10-containing – and date. the complexes to deacetylase NuRD-HDAC, HDACs of of number SIN3A-HDAC, best-characterized members a the Ruijter in HDAC2, of (de function and They IV two HDAC1 class represent them, and I, III Among class class 2003). II, al., class are et I, and the characterized class been date, into catalyze have To classified isoforms proteins. HDACs HDAC acetylated from whereas mammalian moieties 18 proteins, acetyl the other of removal and acetylation the histones catalyze HATs of 2007). Grunstein, and Shahbazian ol lc h erdto fWR,acr uui fthe of subunit core a WDR5, ablation of CUL4B degradation regulators. the epigenetic block al., could important et as Liu elucidated. CRL4B, 2012; be to al., need et still mechanisms al., Jiang underlying 2012; the et yet embryonic al., have Li 2012), et groups for 2004; (Chen independent al., development dispensable that three et from be Hu demonstrated studies 2008; to However, al., 2006). seems et CKIs (Abbas it and by development CDT1 (encoded cells, targeting p27 by and been cultured cycle has p21 cell CUL4A as Although the such 2013). regulate al., to Nakagawa et 2011; proved Zou al., et 2011; and Li Xiong, 2012; (ROS) and al., species et neuronal oxygen (Hu function regulation reactive repression, microRNA to to transcriptional response reported the in regulation, recently the gene CUL4A on been of from members has focused distinctly of CUL4B studies substrates family, of earlier common CUL4 variety and Although function broad 2007). redundant a (Higa Zhang, processes regulates for and controlled developmentally thus or and degradation and physiologically CRL4 proteasomal complexes modification, 2006). al., for ligase et protein substrates Scott ubiquitin 2007; different Zhou, E3 various and targets Lee CRL4 a 2006; recruits al., distinct in et which (Higa of resulting family (DDB1), proteins), DNA N- large (DCAF adaptor 1 The substrate proteins 2009). protein the substrate-recognition to ubiquitin- Xiong, for binding binds and ready E2 CUL4 damage (Jackson ubiquitin of the substrate domain a terminal recruits the with ROC2 to CUL4 charged or transfer (E2) RBX1) RBX2)], enzyme as as conjugating known RING (also known small a ROC1 (also with [either end) protein C-terminal its finger (at interaction By CRL4. 2010). al., et Zupkovitz 2010; al., et Yamaguchi kinase by (encoded cyclin-dependent p57 CDKN1C and the p21 (CKIs) of inhibitors arrest (CDK) upregulation cycle cell by G1 show HDAC2 accompanied and HDAC1 both lacking Cells oevr eetsuishv salse R4 especially CRL4, established have studies recent Moreover, complex ligase E3 the of scaffold the as acts (CUL4) 4 Cullin epciey Gie l,20;Wlige l,2010; al., et Wilting 2004; al., et (Gui respectively) , Cul4b nl mro r mardin impaired are embryos -null CDKN1B o rtoyi in proteolysis for ) CDKN1A 4679 and

Journal of Cell Science hra U4 a eetda oprbelvl between levels comparable at detected Cul4b was CUL4A whereas fMF aedfeeta rlfrto,w eddeulnumbers equal seeded we of proliferation, differential have MEFs of Cul4b nrae ihicesdpsaenme Fg B,indicating 1B), (Fig. number significantly passage that were increased cells different with CUL4B-positive increased at of percentages cells observed the We CUL4B. that CUL4B-positive of staining of immunofluorescent by passages fractions the monitored R2cmlxo ihDAmtytaseae P and by HP1 methylation methyltransferase, DNA suppressors or DNA methylation histone with regulate tumor to or the a SUV39H1 either complex as with of functions coordinating PRC2 and CRL4B H2AK119 that co-repressor monoubiquitylating shown have Xiong, and transcriptional we (Nakagawa Recently, upregulation their 2011). to gene leading neuronal thus some promoters, on and (H3K4me3) complex, trimethylation methyltransferase H3K4 (H3K4) increase 4 lysine H3 histone ARTICLE RESEARCH 4680 of (qRT- level transcript RT-PCR with the quantitative that obtained a indicated middle were assay Importantly, 2A, PCR) (Fig. p21 panels). results cells of lower HeLa Similar accumulation and MEFs. and significant panel). HEK293 in a CUL4B-knockdown upper CKI in resulted 2A, this (Fig. CUL4B that of showed of of immunoblotting expression effect report, loss previous the the the examined with next on p21 Consistent we of depletion Thus, accumulation 2012). the CUL4B al., in et in results (Liu expression line protein CUL4B cell of silencing extra-embryonic that an shown has report recent A of manner upregulation independent in results depletion CUL4B wild- prepared then as designated we are we and mice, development, and transgenic type Sox2-cre embryonic to mice during CUL4B viable which survival al., by generated et mechanism cell Zou the 2012; understand regulates al., further et To (Jiang in 2007). against heterozygotes mouse selected arrest and severely developmental human are to cells lead CUL4B-deficient proliferation can cell reduced apoptosis Cul4b that increased shown have and MEFs results in previous proliferation cell Our inhibits CUL4B of Lack RESULTS CUL4B upregulation that the show to we C owing Here, proliferation of 2013). cell al., inhibit et can Yang depletion 2012; al., et (Hu i.1,n U4 rti a eetdin detected was protein CUL4B no 1A, Fig. hs eut niaeta U4 oiieyrgltscl cycle cell regulates positively CUL4B progression. that together, Taken indicate S 1F). and results (Fig. G2 these cycle, MEFs in cell wild-type cells with the of compared of proportion phases the phase in G1 decreases in concomitant cells with of accumulation increased an OT-nuil r rngncmc Fg CE.As,as Also, cytometry, 1C–E). flow (Fig. of results mice the by transgenic shown Cre obtained (OHT)-inducible were results from MEFs Similar the with 1C–E). of (Fig. that littermates with wild-type compared reduced significantly was d elpoieainasy.Tepoieainof proliferation The assays. proliferation cell EdU xr t ersieeffect. to repressive SIN3A-HDAC its with exert coordinates and interacts complex CRL4B Cul4b Cul4b dkn1a f/Y f/Y nl mro Jage l,21) eas oe that noted also We 2012). al., et (Jiang embryos -null Cul4b and and Cul4b f/Y -/Y and nl Esgnrtdwt 4-hydroxytamoxifen with generated MEFs -null Cul4b Cul4b and Eswr eetdaant h rlfrto of proliferation The against. selected were MEFs nl uieebyncfbolss(Es,which (MEFs), fibroblasts embryonic murine -null Cdkn1c Cul4b -/Y Cul4b Cul4b -/Y Es odtriewehrtetotypes two the whether determine To MEFs. Eswsfrhreautdb T and MTT by evaluated further was MEFs f/Y motnl,w eosrt htthe that demonstrate we Importantly, . nl ieb crossing by mice -null -/Y and Esot elct ltsand plates replicate onto MEFs Cul4b -/Y epciey ssonin shown As respectively. , Cul4b Cdkn1a -/Y Cul4b Cdkn1a Cul4b Esshowed MEFs Cul4b nvivo in -/Y nap53- a in -/Y which , -floxed MEFs, MEFs in U4 rmtscl rlfrto tlatprilythrough partially least that idea at of the transcription proliferation the supporting repressing cell depletion, promotes CUL4B CUL4B by caused S1, Fig. defects material C supplementary and of 2F knockdown Fig. in shown As siRNA. -rnfrs GT uldw xeiet eeperformed were experiments pull-down glutathione complexes, (GST) SIN3A-HDAC is S-transferase and complex CRL4B the CRL4B between the the that complex. SIN3A-HDAC that suggest the co-immunoprecipitated with revealed results associated be physically with could also These complexes immunoblotting 3B). DDB1 two (Fig. by the or in followed CUL4B proteins RBP1 against co- with or antibodies SAP130, be SAP30 SAP180, immunoprecipitation SIN3A, could SAP45, HDAC2, Reciprocally, control. HDAC1, DDB1 against negative SIN3A-HDAC 3A). antibodies and the (Fig. a of CUL4B components complex as that the RING1B with showed immunoprecipitated with (also results RBP1 ARID4A), and includingThe ARID4B), SAP30 as SDS3), as complex, as known known known SIN3A-HDAC (also (also SAP45 SAP180 the SAP130, SIN3A, HDAC2, of HDAC1, components against antibodies with key co-immunoprecipitation immunoblotting by followed to from was DDB1 extracted or subjected CUL4B physically were against were antibodies lysates with Immunoprecipitation experiments. and complexes protein total cells SIN3A-HDAC end, CUL4B-mediated HEK293 this and To for interact. CRL4B required of whether the whether is regulation test To transcriptional 2005; 2010). complex al., al., et et Yamaguchi SIN3A-HDAC Dannenberg 2006; 2005; al., al., et Doyon et (Cowley complex HDAC The SIN3A- the with complex associated HDAC physically is complex CRL4B The U4 ih ucint ers h aa rncito of that transcription demonstrate basal the results cells repress these to control significant function CDKN1A together, a might and reveal Taken CUL4B not CUL4B-knockdown did 2E). (Fig. synthesis, between protein medium difference new culture decay the inhibit and p21 to cycloheximide of to also adding CUL4B-overexpressing examination by Furthermore, performed between rates, 2D). We degradation, (Fig. difference cells not protein p21 p53. did control MG132 the CUL4B-mediated the inhibitor on of to proteasome narrow (stress-free) the CUL4B due with independent of treatment basal not because effect is was negative the level the p21 that that of demonstrated of suggesting that with compared 2C), upregulation as the (Fig. cells in blotting CUL4B-deficient difference controls no in Western was p53 there cells. of that level CUL4B-deficient showed assays in qRT-PCR and p53 of of activation the whether transcription examined next we increased that p53, panels), of Given repress target lower level. transcriptional major transcriptional might and the middle CUL4B at 2B, expression that (Fig. suggesting CUL4B-knockdown cells in respectively, HeLa higher and 1.8-fold and HEK293 2.0-fold the and were panel), levels in upper 2B, higher (Fig. MEFs 2.3-fold wild-type was p21, encodes 2,w efre eceeprmnsi hc etransfected we which of in upregulation experiments the Cul4b rescue by performed mediated we was p21, cells CUL4B-deficient in ofrhrivsiaetemlclrbssfrteinteraction the for basis molecular the investigate further To odtriewehrtecmrmsdpoieainobserved proliferation compromised the whether determine To CDKN1A -/Y ora fCl cec 21)17 6949 doi:10.1242/jcs.154245 4679–4691 127, (2014) Science Cell of Journal Eswt iN targeting siRNA with MEFs . eehsbe hw ob agtdb h SIN3A- the by targeted be to shown been has gene dkn1a ol atal eceteproliferation the rescue partially could CDKN1A Cdkn1a CDKN1A . Cul4b Cdkn1a a u oincreased to due was efrtdetermined first we , nl Esta in than MEFs -null CDKN1A rwt control with or CDKN1A CDKN1A mRNA sa is

Journal of Cell Science EERHARTICLE RESEARCH oan(I)adoehgl osre ein(C) among (HCR), region conserved highly one and interaction (HID) deacetylase histone domain one paired (PAH1–4), four including helices domains, amphipathic conserved six through co-regulators 3C). SIN3A- other (Fig. the tested with components not but HDAC SIN3A, DDB1 with and interact CUL4B positive directly are both the could that which as revealed serving of experiments SIN3A) these both with controls, interact respectively, physically RBBP4, to and (also known SAP180, RbAp48 RBBP7 and as RbAp46 SAP45, With known HDAC2. SIN3A, and HDAC1 including SAP130, and complex, proteins SIN3A-HDAC DDB1 GST-fused or vitro CUL4B GST-fused using I3 neat ihalrenme ftasrpinlfcosand factors transcriptional of number large a with interacts SIN3A rncie n rnltdidvda opnnso the of components individual translated and transcribed in n I3-DC ihSNAsriga linker. CRL4B a as between serving association SIN3A with physical Collectively, SIN3A-HDAC, a 3D). and S2 (Fig. demonstrate DDB1 results the and CUL4B these that SIN3A, to of bind domains directly demonstrated HID could and PAH3 experiments 658– the contained acids which pull-down fragment, (amino S3 GST and 401–657) SIN3A 1269). acids GST-fused (amino S2 (amino three 1–400), S1 acids GST–SIN3A generated To domains, interact different we 1990). contained directly that DDB1, can al., constructs protein and et as SIN3A CUL4B the Wang such with in complex, 2005; region a co-repressor which Ekwall, as determine the and serve of (Silverstein HID subunits HDACs and other PAH4 for PAH3, various scaffold with whereas interactions for factors, reserved transcription are PAH2 and PAH1 which ora fCl cec 21)17 6949 doi:10.1242/jcs.154245 4679–4691 127, (2014) Science Cell of Journal infcn difference. significant ** needn xeiet) * experiments); independent mean the show A– C,E in data Quantitative experiments. independent three of are representative shown data number, The cell respectively. and content DNA show h ecnae fEdU-positive of percentages The (D). shown are photomicrographs Representative MEFs. nlssi hw.The shown. is analysis cytometric flow by determined cycle of cell distribution The (F) random (E). three fields in scored were cells ( efre nwl-yeand wild-type in performed was assay An incorporation (D,E) EdU assay. MTT the to subjected ( wild-type of expression in Cul4b proliferation cell Reduced 1. Fig. ( Cul4b ( Wild-type (C) proliferation. cell inhibits of Knockout fields (C–E) random (right). three in and counted (left) were shown are cells positively stained of percentages The DAPI. with counterstained and antibody CUL4B-specific the with stained were (P1–P3) passages different Cells from plates. replicate onto seeded sa of growth assay Competitive (B) panel). analysis (lower blotting western and (upper panel) assay qRT-PCR a using qa ubr of numbers Equal Cul4b Cul4b Cul4b P , .1 *** 0.01; -/Y nl MEFs. -null -/Y f/Y -/Y Eswsdtrie by determined was MEFs ) Eswr ie and mixed were MEFs Cul4b n 4OHT and n 4OHT and Cul4b P Cul4b Cul4a f/Y , f/Y .0;NS,no N.S., 0.001; and and ) Cul4b A The (A) 2 6 + nl Esas MEFs -null and Eswere MEFs ) and ) ..(three s.d. Cul4b x Cul4b f/Y Cul4b and Cul4b Cul4b Cul4b and -/Y P , -null y MEFs. 0.05; 4681 in axes -null -null

Journal of Cell Science EERHARTICLE RESEARCH U4 hoaorpi rcin eeicbtdwt calf with 4682 incubated were fractions chromatographic CUL4B CUL4B- affinity-purified complex the associated of determined activities then in deacetylase We left). histone detected 4A, the were (Fig. complexes fractions components SIN3A-HDAC affinity-purified the the and expected, CRL4B affinity histone As both by gel. the prepared of affinity were in M2 extracts using CRL4B expressed cellular purification and was of cells, FLAG–CUL4B HEK293 role end, in this the SIN3A-HDAC To determine activity. and deacetylase to CRL4B us between prompted association physical The histone with activity associated deacetylase is complex CUL4B-containing The nvitro in nraigaonso h purified the of amounts Increasing . fteprfe U4-soitdcmlx niaigta the that indicating complex, CUL4B-associated that found purified activity and deacetylase the reaction the the of reduced an to significantly HDACs) TSA (TSA, II with and A the treatment I trichostatin of class authenticity of added inhibitor the we confirm activity, further CUL4B-associated histone To possesses activity. complex CUL4B-associated deacetylase the supporting decreased that fractions, gradually notion CUL4B the purified were of AcH4K16 amounts increasing and with and AcH4K8 (AcH3) H3 acetylated (AcH4), deacetylation of amounts H4 the histone (right), 4A As Fig. histones. detected for in acetylated shown against then antibodies buffer were with blotting products western the by reaction The in (HDM). histone measurement bulk thymus ora fCl cec 21)17 6949 doi:10.1242/jcs.154245 4679–4691 127, (2014) Science Cell of Journal .. osgiiatdifference. significant no N.S., * with Cdkn1a transfected were MEFs null h mean the represent data fields Quantitative random (right). three in counted were the cells and EdU-positive left of the percentages on shown are photos Representative immunostained. and fixed were cells hours transfection, 48 after At (siNC). siRNA control of defects proliferation the rescue partially Cdkn1a nlss xrsini represented is Expression densitometric analysis. by quantified and blotting (left) western by analyzed proteins were of amounts time- Equal indicated points. the at harvested and in results of upregulation depletion CUL4B 2. Fig. stepretg eann relative to remaining percentage the as ih50 treated with were cells HEK293 control and CUL4B-knockdown (E) western blotting. by analyzed and extracted were proteins and DMSO (MG132-) or MG132 with treated were cells HEK293 overexpressing CUL4B- (D) panels). analysis (lower blot western and (upper panels) qRT-PCR p53 by of level determined basal as the change cells not HEK293 did and MEFs in CUL4B depletion (C) shRNA. control shNC, CUL4B; against (sh)RNA hairpin small- shCUL4B, and levels. mRNA protein the both at cells HeLa and HEK293 MEFs, in p21 the of in upregulation resulted CUL4B of that depletion showed (B) qRT- analysis and PCR (A) blotting Western (A,B) manner. p53-independent a in P , t 5 .5 ** 0.05; rgt.()Kokonof Knockdown (F) (right). 0 seii iN (si siRNA -specific m in /lccoeiie(CHX) cycloheximide g/ml 6 Cul4b Cul4b ..(he replicates); (three s.d. P , .1 *** 0.01; nl MEFs. -null Cdkn1a nl Escould MEFs -null P , expression Cdkn1a 0.001; Cul4b )or -

Journal of Cell Science EERHARTICLE RESEARCH ope.W eteaie h feto U4 eeinon deletion CUL4B of effect the CUL4B-containing examined the next to We TSA- bound complex. to deacetylases attributed is histone left. precipitates the sensitive on CUL4B shown of is molecular activity experiments per M, deacetylase pulldown were bands. experiments GST specific pulldown in indicate GST purified arrows CUL4B–DDB1. and GST–S3 with Black SIN3A and interact left. of GST–S2 directly the GST–S3 CUL4B, GST–S1, to on of and SIN3A of shown staining GST–S2 for staining is Blue required GST–S1, domains experiments Coomassie expressed essential controls. pulldown bacterially the positive GST with of as in Identification the used an purified (D) and were CUL4B–DDB1 control marker. GST–DDB1 th GST GST–RbAp48 between mass and as and and interaction GST–CUL4B IgG GST–RbAp46 RbAp48 Molecular expressed with indicated. RbAp46, (C) bacterially as proteins, DDB1, antibodies. with proteins, indicated indicated performed subunit the the were SIN3A-HDAC against using experiments translated antibodies (WB) pulldown the blotted GST with western subunits. (IP) then SIN3A-HDAC immunoprecipitated were were Immunocomplexes lysates complex. control. SIN3A-HDAC Whole-cell negative the complexes. with SIN3A-HDAC associated physically and is CRL4B complex CRL4B The 3. Fig. nvitro in ctlto eeso ukH n 4wr nrae in increased were H4 and H3 the bulk MEFs, of wild-type levels with acetylation Compared MEFs. in acetylation histone Es(i.4) odtriewehrteei istowards bias a is there whether determine To 4B). (Fig. MEFs rncie n rnltdpoen fCLBo D1 omsi Blue Coomassie DDB1. or CUL4B of proteins translated and transcribed AB oimnpeiiainaayi fteascainbtenthe between association the of analysis Co-immunoprecipitation (A,B) ora fCl cec 21)17 6949 doi:10.1242/jcs.154245 4679–4691 127, (2014) Science Cell of Journal nvitro in rncie and transcribed Cul4b formed 4683 -null e d

Journal of Cell Science EERHARTICLE RESEARCH S.Atog h xrsino a4CLBldt a to a in led gene 4684 reporter Gal4–CUL4B without the of of or expression with the expression stable in cells decrease the 5 with treated remarkable by Although and cells driven cells), TSA. in reporter HeLa (Gal4-UAS luciferase involved into domain a are purpose, then of Gal4–CUL4B expression this HDAC2 We transfected For and we regulation. acetylation. transcriptional HDAC1 is histone CUL4B-mediated complex whether increases CRL4B that examined the and depletion complex, that SIN3A-HDAC CUL4B the indicate with above associated physically shown data by The CUL4B-mediated repressor repression in transcriptional involved are transcriptional HDAC2 that and HDAC1 a suggest as deacetylases. observations histone of function function cells these also the supporting HeLa were might together, results and CRL4B Taken Similar HEK293 4B). 4C). CUL4B-knockdown (Fig. (Fig. with MEFs wild-type obtained of in CRL4B that H4K16 individual and acetylation the H4K8 of of H3K27, levels levels at for increased acetylation significantly residue observed the and examined residues histone also particular we complex, a of deacetylation Cul4b nl Escmae with compared MEFs -null 6 Gal4-binding I3-DCt h a4pooe a o fetd(i.5C). (Fig. affected not histone was of on expressed promoter binding effect Gal4 the was the the although to Gal4–CUL4B Gal4–CUL4B, blocked SIN3A-HDAC by TSA when caused with deacetylation decreased H3 Treatment histones 5C). was (Fig. acetylated and H4 of was HDAC2 H2AK119ub1 that and of HDAC1, whereas level DDB1, increased, the significantly Consequently, of 5C). recruitment (Fig. with SIN3A the an in Compared in resulted Gal4–CUL4B vectors. increase of Gal4–DBD transfection with (Gal4–DBD), transfection control control after promoter or Gal4 a the Gal4–CUL4B to HDAC1, performed in DDB1, SIN3A of and we recruitment assay HDAC2 transcriptional the notion, investigate (qChIP) and to cells CUL4B-mediated this immunoprecipitation Gal4-UAS HDAC1 confirm chromatin in that further quantitative notion involved To the repression. are supporting of a HDAC2 activity 5B), to repressive (Fig. led transcriptional also CUL4B the (HDAC1/2) in combination reduction in significant gene HDAC2 of and CUL4B- knockdown reporter HDAC1 in Furthermore, involved regulation. in 5A), are transcriptional (Fig. mediated HDACs Gal4–CUL4B reduction TSA-sensitive of that expression the suggesting the by attenuate caused treatment expression TSA efficiently previously, observed could as manner dose-dependent ora fCl cec 21)17 6949 doi:10.1242/jcs.154245 4679–4691 127, (2014) Science Cell of Journal hC oto shRNA. control shNC, CUL4B; against shRNA cells. shCUL4B, control with compared (C) cells HeLa and HEK293 CUL4B-knockdown and (B) MEFs in histones acetylated of levels increased showed analysis blotting Western (B,C) control. CON, (right). histones modified or histones the indicated against antibodies using western blotting by analyzed were reaction mixtures The (left). proteins the indicated against antibodies using fractions CUL4B-purified the of analysis blotting Western antibody. FLAG anti- the with immunoprecipitated were and FLAG–CUL4B stably expressing cells HEK293 from obtained were extracts Cellular activity. deacetylase histone possesses complex CUL4B-associated activity. deacetylase histone is with complex associated CRL4B The 4. Fig. A The (A) Cul4b -null

Journal of Cell Science EERHARTICLE RESEARCH oee,cmae ihGl–U4,temtn construct mutant the Gal4–CUL4B, (Gal4– with compared However, 5. Fig. eemn h oeo h 3lgs ciiyo R4 n its and CRL4B of activity ligase E3 the to of ability its role further for To the promoters. dispensable target determine is to complex CRL4B SIN3A-HDAC that of the suggesting recruit activity promoter, ligase the E3 to the SIN3A-HDAC recruit the to retained but ability monoubiquitylation, H2AK119 catalyze to ability e etpg o legend. for page next See D uln nwihtecli oani eee)ls the lost deleted) is domain cullin the which in Cullin, ramn ol atal lc h euto Fg 5D,E). (Fig. reduction the block partially However, could expected, As of treatment reduction TSA. several-fold inhibitor a in HDAC CDKN1A resulted the CUL4B of with overexpression cells the we treated CUL4B- repression, or transcriptional CUL4B in overexpressed activity deacetylase associated ora fCl cec 21)17 6949 doi:10.1242/jcs.154245 4679–4691 127, (2014) Science Cell of Journal xrsina ohRAadpoenlvl,adTSA and levels, protein and RNA both at expression CDKN1A xrsinwsrdcdby reduced was expression D ulni E23clsand cells HEK293 in Cullin , 0 when 50% 4685

Journal of Cell Science Fg F.Ipraty iutnoskokono I3 and knockdown SIN3A individual than of derepression knockdown further in simultaneous resulted derepress CUL4B Importantly, also could 5F). SIN3A (Fig. of knockdown observations, CUL4B- by repression the Fg F.Tkntgte,teerslssgetta R4 might complex. by SIN3A-HDAC CRL4B and the ubiquitylation that with H2AK119 suggest promoting coordinating by results genes target these its together, repress Taken 5F). (Fig. *** oto iN sN) uieaeatvt a esrd(et n the and (left) measured was activity Luciferase negative or (siNC). (siHDACs) siRNA HDAC2 control and HDAC1 encoding against construct siRNA with and transfected Gal4–CUL4B were activity repeats. cells repressive Gal4-UAS-reporter six transcriptional CUL4B. for potential with of the TSA measured impairs without was knockdown or activity HDAC with reporter (B) treated luciferase were Gal4 cells the h. expressing the 24 cells then HeLa and into reporter, transfected Gal4-UAS was Gal4– Gal4–DBD encoding control Construct or CUL4B. CUL4B of activity repressive CUL4B-mediated transcriptional in involved are regulation. HDAC2 transcriptional and HDAC1 5. Fig. ARTICLE RESEARCH 4686 the that results assays The antibodies. first revealed DDB1, appropriate ChIP-Re-ChIP was subsequently with CUL4B, were re-immunoprecipitated immunoprecipitates chromatin proposition, The against SIN3A. experiments, or regions. antibodies HDAC1/2 this these upstream with more In immunoprecipitated test the performed. on were further primarily binds the To to p53 known on is 6A, which sites Fig. p53, of binding profile binding have the examined also We complexes SIN3A- the SIN3A-HDAC and co-occupy these and CRL4B around might CRL4B of that region suggesting of sites the 6A), binding in (Fig. of occupancy the peaked the (TSS) both that HDAC that and site revealed overlap start proteins assay 13-kb transcription qChIP a The on the SIN3A surrounds and CDKN1A HDAC1/2 that DDB1, region CUL4B, of CRL4B pattern the of repression between transcriptional interplay CDKN1A the functional in complexes the SIN3A-HDAC define and to order In SIN3A-HDAC the the at of complex retention stable promotes CUL4B CUL4B- the show data of Quantitative levels shRNA. protein control mean and shNC, RNA analyzed. the were CUL4B- then p21 into (shCUL4B), than transfected cells effect were HEK293 severe siNC knockdown more or Simultaneous in siSIN3A (F) resulted knockdown. control. CUL4B individual CON, and (E). SIN3A blotting of western knockdown and (D) qRT-PCR by TSA. 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P , xrsinwsdetermined was expression .5 ** 0.05; 2 0 pto bp 800 P , 0.01; D ulnor Cullin CDKN1A CDKN1A 2 0 bp 300 rvosyietfe sHA12tre ee nprimary in genes were target that regulators HDAC1/2 cycle as cell ten identified of previously used expression we the examine complexes, CRL4B SIN3A-HDAC between association and functional the understand HDAC1/2 further of To subset a of genes regulation target the in participates CRL4B hw ytetetwt h rtaoeihbtrM12and as MG132 p21, inhibitor of proteasome degradation Meanwhile, the the with MEFs. influence treatment gene control to by of the found shown that not of was with CUL4B transcription compared quantitative MEFs the of our null that report However, p21, 2012). indicates in recent encoding al., assay a p21 et Our RT-PCR the with (Liu 2009). of cause cells consistent could embryonic Xiong, accumulation MEFs is in and p21 increased CUL4B Jackson of of loss accumulation 2007; the that Zhang, different observation a targeting and modification of by protein for (Higa regulation processes or degradation the proteasomal biological for in substrates of participates spectrum CUL4B broad ROC1, and p21. DDB1 at of progression regulation cycle negative in cell the an p21 through promote partially of might by least CUL4B knockdown accompanied that Because conclude arrest, p21. CKI cycle Cul4b the cell cell of reduced G1 significantly show upregulation in We results and progression. MEFs in cycle proliferation CUL4B cell of of in loss CUL4B that arrest of developmental function the crucial and to proliferation lead cell Cul4b could reduced apoptosis that showed increased results previous CUL4B by Our p21 of repression Transcriptional the co-occupied DISCUSSION complexes SIN3A-HDAC that showed and CDKN1C assay ChIP-Re-ChIP CRL4B A CUL4B-overexpressing the 7C,D). (Fig. and significantly cells CUL4B-knockdown HEK293 also of in expression was the on confirmed CUL4B family, of effect negative CKI The Cip/Kip in the upregulated a regulating Interestingly, of in genes. member involved target HDAC1/2 is of complex subset CRL4B the upregulated that were suggesting five examined, genes in ten Among fibroblasts. hs aaspotteagmn htCLBcnrbtsto of contributes retention CRL4B the facilitating that the at by argument SIN3A-HDAC probably Collectively, the deacetylation, right). support histone 6F, p21(Fig. data of these expression to the HDAC1/2 derepressed and SIN3A CUL4B, or CUL4B of DDB1 the recruitment and the SIN3A alter upon CUL4B not of did of recruitment AcH4, increased the the knockdown affect to AcH3, not were However, did including HDAC1/2 6D). AcH4K16, (Fig. histones, reduced knockdown acetylated greatly and was of AcH4K8 H2AK119ub1 those of and level to the bound SIN3A observations, vice and marked HDAC2 and HDAC1, a HDAC2 of the in levels or resulted the CUL4B not in HDAC1 reduction of did SIN3A, knockdown CUL4B of Importantly, of versa. knockdown expression 6C, the Fig. alter in shown As promoter. yasmln h R4 bqii iaecmlxwith complex ligase ubiquitin CRL4B the assembling By Cul4b CDKN1A CDKN1A nl Escudrsu hi rlfrto eet,we defects, proliferation their rescue could MEFs -null the demonstrate we Here, 2012). al., et (Jiang embryos -null CDKN1A ora fCl cec 21)17 6949 doi:10.1242/jcs.154245 4679–4691 127, (2014) Science Cell of Journal nl Escmae ihwl-yeMF Fg 7A,B), (Fig. MEFs wild-type with compared MEFs -null rmtra el(i.7E,F). (Fig. well as promoter rmtr(i.6,lf) ept h atta it that fact the despite left), 6F, (Fig. promoter Cdkn1a Cul4b rmtr(i.6) ossetwt these with Consistent 6D). 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Journal of Cell Science * ncdw fiinyo bp64 n 2 ee eeaaye ywsenbotn.FrDF aarpeettemean t the to represent SIN3A data and D–F, HDAC For CUL4B, blotting. of western by recruitment analyzed the were Left, level (F) p21 SIN3A. and or RbAp46/48 HDAC1/2 of targeting efficiency siRNAs knockdown or (siNC) siRNA control CDKN1A with transfection after cells HEK293 EERHARTICLE RESEARCH DC n DC sHA12.()qhPaayi fteercmn fteidctdpoen tthe at proteins antibodies. (siSIN indicated indicated SIN3A the the targeting targeting of with siRNA shRNA enrichment or cells (shCUL4B) or the HEK293 CUL4B of (shNC) in targeting analysis shRNA performed shRNA qChIP control with were transfected (D) with experiments cells (siHDAC1/2). Re-ChIP in HDAC2 blotting and and western ChIP by HDAC1 (B) analyzed was signal. efficiency bound/input Knockdown of (C) percentage the on the p53 as and the presented SIN3A co-occupy HDAC1/2, complexes DDB1, SIN3A-HDAC CUL4B, and CRL4B 6. Fig. P , .5 ** 0.05; rmtri E23clsatrtaseto ihcnrlsRAo iNstreigRA4 n bp8(ibp64)b CI nlss Right, analysis. qChIP by (siRbAp46/48) RbAp48 and RbAp46 targeting siRNAs or siRNA control with transfection after cells HEK293 in promoter P , .1 *** 0.01; P , .0;NS,n infcn difference. significant no N.S., 0.001; CUL4B CDKN1A E CI nlsso h erimn fteCLBadDB rtist the to proteins DDB1 and CUL4B the of recruitment the of analysis qChIP (E) . eelcs abtnra g evda eaiecnrl h eut fCI nihet are enrichments ChIP of results The control. negative a as served IgG normal Rabbit locus. gene CDKN1A promoter. A CI nlsswr odce odtrietebnigrgo of region binding the determine to conducted were analyses qChIP (A) ora fCl cec 21)17 6949 doi:10.1242/jcs.154245 4679–4691 127, (2014) Science Cell of Journal CDKN1A rmtri E23clsatrtransfection after cells HEK293 in promoter 6 ..(he needn experiments); independent (three s.d. CDKN1A rmtrin promoter A or 3A) 4687 he

Journal of Cell Science opee otre the co-target complexes EERHARTICLE RESEARCH 4688 not but repression, negatively proteolysis. transcriptional CUL4B by by repression. p21 that transcriptional of function in suggest the complexes results regulates SIN3A-HDAC and These CRL4B analysis. the between half-life data association A–C, the For depicting (D). model analysis A blot (G) western shown. and is (C) cells qRT-PCR HEK293 by in determined was (C,D) cells cells. HEK293 control CON) of (shNC, mean that control the with as represent compared well (B) as genes. MEFs cells target knockout CUL4B-overexpressing HDAC1/2 4-OHT-inducible of subset or a (A) represses MEFs transcriptionally CRL4B 7. Fig. 6 ..(he needn xeiet) * experiments); independent (three s.d. CDKN1C rmtr hP()adR-hP()aayi ftercuteto U4,DB,HA12adSNAt the to SIN3A and HDAC1/2 DDB1, CUL4B, of recruitment the of analysis (F) Re-ChIP and (E) ChIP promoter. P , .5 ** 0.05; P , .1 *** 0.01, P ta. 93.Avreyo rncito atr swl as well as factors transcription of variety A 1993). (Xiong level al., transcriptional the at et regulated primarily is p21 cells, AB PRaayi fteepeso fHA12tre ee in genes target HDAC1/2 of expression the of analysis qPCR (A,B) , samjrrgltro elcceporsini mammalian in progression cycle cell of regulator major a As .0;NS,n infcn ifrne EF h R4 n SIN3A-HDAC and CRL4B The (E,F) difference. significant no N.S., 0.001; ora fCl cec 21)17 6949 doi:10.1242/jcs.154245 4679–4691 127, (2014) Science Cell of Journal CDKN1C xrsini U4-ncdw sCLB and (shCUL4B) CUL4B-knockdown in expression CDKN1C Cul4b gene -/Y

Journal of Cell Science rmtr nrae h eeso itn ctlto and of acetylation knockdown histone However, H2AK119ub1. of of levels the the those to in the SIN3A reduction decreased and increased marked HDAC2 a promoter, HDAC1, in of resulted to of recruitment CUL4B promoter led of SIN3A- a the and and knockdown revealed CRL4B on the assays reporter complexes of recruit qChIP sites HDAC occupancy artificial AcH4. the could and of the overlap AcH3 perfect protein of to levels SIN3A fusion decreased and Gal4–CUL4B HDAC1/2 a Accordingly, ectlto ttre ee uhas such genes The target HDACs. histone at and and higher-order deacetylation monoubiquitylation a SAP30 H2AK119 form catalyzing with thus in complex complexes SIN3A SIN3A-HDAC responsible of and also is CRL4B association which SIN3A, the in containing domains for HID fragment further and the PAH3 and by We SAP30, the CRL4B bridged the HDAC2. is SIN3A, between complexes interaction and SIN3A-HDAC direct including HDAC1 the that SAP45, complex, demonstrated SAP18, SIN3A-HDAC SAP130, the components both the with that associated of physically showed are DDB1 assay SIN3A-HDAC and CUL4B and co-immunoprecipitation CRL4B A the complexes. between interplay the examined on hti h bec fetiscsrs 5 ciaini not is we activation study, p53 this stress p53 extrinsic In of 2010). of absence al., the independently et in as Yamaguchi that p21 such found 2000; factors, al., upregulate many et can repair, (Huang to DNA damage inhibitors, expression and DNA arrest HDAC p21 to cycle response of cell in induced been ensure regulation mechanism be p53-dependent can the p21 a have Although by in 2005). Radhakrishnan, involved co-repressors) and (Gartel be and to reported (co-activators co-regulators ARTICLE RESEARCH ope a ensont agtthe target to shown SIN3A been the repressor Because sequence-specific has 2008). of with Seto, complex and variety interacting (Yang by factors a part their transcription acquire in in which REST, activities present and regional NuRD SIN3A, are as such HDAC2 complexes and HDAC1 SIN3A- the complex with HDAC CRL4B of association functional and Physical of reduction partially the could the treatment TSA in Fourth, by block cells. detected wild-type blocked was of that 4 be with acetylation and increased to 3 could significantly histones that of Third, found TSA. activity an inhibitor was deacetylase HDAC Second, are a complex complex, HDAC2. CRL4B possess and CUL4B-associated the HDAC1 of affinity-purified with components associated key physically the DDB1, of and CUL4B- evidence repression in of involved transcriptional also lines are mediated several HDAC2 and presented HDAC1 we that p21 indicating study, of this co-repressors In well-documented regulation. are HDAC2 and HDAC1 of CUL4B-mediated repression in transcriptional HDAC2 and HDAC1 of process. Involvement this in Instead, role CUL4B. crucial by a expression play p21 of HDACs regulation the in involved hte te DC r novdi CUL4B-mediated in involved CUL4B-mediated are in evaluate HDACs to repression. involved transcriptional that needed other are idea are studies whether the More HDAC2 support repression. Lastly, transcriptional results and these in overexpression. CUL4B-containing reduction HDAC1 together, the significant Taken of a CUL4B activity complex. to repressive led transcriptional by also the HDAC1/2 of caused knockdown expression CDKN1A CDKN1A Cul4b xrsinadreporter and expression CDKN1A CDKN1A CDKN1A nl Escompared MEFs -null CDKN1A is,CUL4B First, . and Importantly, . CDKN1C ee we gene, CDKN1A . rmtr,laigt nrae 2K1 monoubiquitylation H2AK119 increased to leading promoters, n erae itn ctlto n,cneunl,to consequently, and, progression. cycle cell acetylation driving thus CKIs, histone of silencing epigenetic decreased and eeto fSNAHA othe stable CRL4B to and that recruitment CRL4B–SIN3A-HDAC SIN3A-HDAC the reveals of promotes study in of and retention SIN3A function our with and conclusion, involved interacts future identity In the Clearly, elements functionality. could determine promoters. to that to additional needed domains recruitment SIN3A- are their nor DNA-binding studies cycle to CRL4B cell contain contribute neither impaired subunits Notably, to HDAC 7G). leading (Fig. become their accumulate, CKIs and progression inhibits depleted, and and monoubiquitylation is CUL4B derepressed p57 of H2AK119 When and promoters deacetylation. catalyzing p21 the histone as occupies by such that expression CKIs complex encoding large genes we a the together, SIN3A- form and catalyzing CRL4B HDAC Taken facilitates conditions, physiological SIN3A-HDAC. by under that, also propose of silencing function CRL4B to epigenetic addition deacetylation monoubiquitylation, in to Thus, H2AK119 CRL4B. histone contributing of depend function not directly ubiquitylation promotes did SIN3A-HDAC the the and on Furthermore, CRL4B promoters. CRL4B gene between of target interaction retention that at and/or HDAC2 CUL4B recruitment and of HDAC1 the suggesting influencing recruitment by the deacetylation DDB1, affect not did and HDAC1/2 or SIN3A tnadpooos nuil odtoa ncot eecetdby to created according were embryos knockouts conditional (E)13.5–14.5 crossing Inducible day protocols. embryonic standard from obtained GX4- xrsinvco.UigteFA–I3 osrc sa the as into construct RbAp48 FLAG–SIN3A the or Using RbAp46 vector. DDB1, expression CUL4B, pGEX-4T-3 full-length created inserting were GST–RbAp48 by GST– and pCMV-Tag2B. GST–RbAp46 into GST–DDB1, HDAC2 and CUL4B or HDAC1 SAP180, SAP30, SIN3A, SAP45, and full-length SAP130, inserting by FLAG–HDAC1 generated were FLAG–SAP180, FLAG–SAP30, FLAG–HDAC2 FLAG–SIN3A, FLAG–SAP45, 2012). al., FLAG–SAP130, et (Hu previously described Gal4– and Gal4–CUL4B FLAG–DDB1, FLAG–CUL4B, construction Plasmid supplementary in listed S2. are was primers GAPDH Table All of material control. expression internal The an machine. as 480 Scientific, and isolated determined Roche performed was a (Thermo was was using genes by Transcriptase target cDNA RNA analyzed of Reverse quantification total (Invitrogen). Relative MMLV IL). and Rockford, the instructions reagent, with manufacturer’s Trizol prepared the with lysed following were Cells RT-PCR quantitative Real-time respectively. (Invitrogen), 2000 (Polysciences to Lipofectamine CUL4B precipitation according or phosphate PA) performed with calcium Warrington, was al., using siRNA to cells et protocols or h (Zou standard HEK293 plasmids 48 previously of for described and Transfection as MO) 2009). were Louis, HeLa overexpression St or recombinase. (Sigma, knockdown 4-OHT Cre nM 200 activate units/ with 100 treated FBS, (Gibco, were 10% 100 with medium and CA) penicillin Eagle’s Carlsbad, ml modified Invitrogen, NY; Dulbecco’s Island, in Grand cultured were MEFs transfection and culture Cell carrying Mice 2012). al., et (Jiang previously of generation The of Generation METHODS AND MATERIALS necosdwt Sox2-Cre with intercrossed ora fCl cec 21)17 6949 doi:10.1242/jcs.154245 4679–4691 127, (2014) Science Cell of Journal Cul4b flox/flox Cul4b Cul4b eaemc oCGCeEs aemice. male CAG-Cre/Ers1 to mice female m nl MEFs -null /lsrpoyi.Teidcbekoku cells inducible-knockout The streptomycin. g/ml foe ieadgntpn eedescribed were genotyping and mice -floxed +/ 2 aemc n rmr Eswere MEFs primary and mice male CDKN1A Cul4b and D ulnwr as were Cullin flox/flox CDKN1C 4689 were

Journal of Cell Science als X,rbi nip1(elSgaigTcnlg,Beverly, Technology, Signaling (Cell anti-p21 rabbit TX), Biotechnology, Dallas, Cruz (Santa (Sigma), anti-DDB1 anti-FLAG rabbit mouse (Sigma), were anti-CUL4B study rabbit this in used antibodies primary The GST–S3 reagents and and Antibodies 401–657) GST–S1 acids i.e. (amino SIN3A, 658–1269). GST–S2 of acids GST-fusion (amino 1–400), domains express acids different that (amino to constructs GST corresponding three proteins generated we template, ARTICLE RESEARCH 4690 for buffer the CUL4B- in histones the bulk with then incubated were immunoprecipitates cells, antibody.The anti-FLAG 293 the with immunoprecipitated HEK was complex into associated transfected was FLAG–CUL4B System vitro Assay In triplicate. Reporter in analyzed were Dual-Luciferase samples All the Wisconsin). Madison, using (Promega, ( activities luciferase pRLTK determined and h, ng 48 were 40 after prepared with were lysates vectors cell Total control. assay Gal4-CUL4B were of fusion ng (Gal4-UAS) Gal4-DBD 50–800 reporter with or transfected Gal4-UAS and plates the 24-well in expressing cultured stably The cells software. HeLa assays CellQuest analyzed reporter with Luciferase was equipped suspension times. cytometer three repeated single-cell flow was iodide a experiment A propidium and using were solution. 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Table from HDAC1/2 purchased material were (CHX) human (supplementary cycloheximide and against Sigma MG132 TSA, by siRNA S1). synthesized and were p21 RbAp46/48 murine Science. against Applied Cruz Roche siRNA from was cocktail (Santa inhibitor Amersham Protease from Biosciences. purchased were anti-RING1B beads Protein-A/G–Sepharose glutathione–Sepharose Texas). and SAP45,CL-4B Montgomery, (Bethyl, SAP130, rabbit RBP1 SAP180, and against SAP30 antibodies Biotechnology), rabbit and anti-p53 Biotechnology) Cruz rabbit (Sigma), anti-RbAp46/48 (Santa rabbit rabbit China), (Abcam), Kong, anti-HDAC2 Hong (Abcam, anti-HDAC1 anti- rabbit Biotechnology), mouse MA), nuae ihEUfr4hbfr en ie ih4 omleyefor formaldehyde 4% with fixed being were by before cells h followed the min, 4 assay, for 20 EdU EdU for the with lysed For incubated values. then absorbance were the cells of the measurement and well per DMSO of 0mnadicbtdwt gm lcn o i.Te,05 Triton 0.5% Then, min. 1 and 5 permeabilization for for glycine used mg/ml was 2 X-100 with incubated and min 30 t2 ,4 ,7 n 6h h T egn a de ta at added was reagent MTT the h, 96 incubated were cells and the and h medium cell 72 37 the at to mg/ml h, 5 48 of concentration h, 24 at ial,46daiio2peyidl DP)wsue osanthe stain to used was (DAPI) nuclei. 4,6-diamidino-2-phenylindole Finally, 6 10 ˚ 3 o nadtoa .Terato a tpe yadn 100 adding by stopped was reaction The h. 4 additional an for C nl n idtp Esa asg eehretdby harvested were 3 passage at MEFs wild-type and -null e eli 6wl ltswt ih eet o ahgop Then, group. each for repeats eight with plates 96-well in well per itn ectlto assay deacetylation histone Cul4b b m atn(no,rbi niSNA(at Cruz (Santa anti-SIN3A rabbit (Anbo), -actin g/ml. nl n idtp Eswr ie n plated and mixed were MEFs wild-type and -null 9 doyrdn EU proliferation (EdU) -deoxyuridine 6 ecinccti a added. was cocktail reaction Renilla sthe as ) m l itn ectlto esrmn HM 0m rs 0m C,5mM 5 KCl, mM MgCl 50 Tris, mM 50 (HDM; measurement deacetylation histone ta. 02 age l,21) rel,1 Briefly, 2013). al., (Hu et Yang previously 2012; described al., et as performed were assays Immunoprecipitation pulldown GST and Immunoprecipitation upeetr aeilaalbeoln at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.154245/-/DC1 online available material Supplementary material Supplementary and 8133005 numbers [grant China of and Foundation 81321061]. Science 2013CB910900]; of and Natural Program 2011CB966200 National Research numbers the Basic [grant National Program) the from (973 grants China by supported was work This Funding Y.G. and C.S., H.H., and Q.J., manuscript. Y.Z. discussion; the B.J., and Y.Q., wrote of L.J., assistance Y.Y., J.Y., majority technical F.Y., the provided H.H., performed analysis. C.S. Q.J. data project. and the experiments of the conceived Y.G. and Y.W. H.H., Q.J., contributions Author interests. competing no declare authors The interests Competing Student’s unpaired two-tailed a using mean the represent Data analysis Statistical 5 Briefly, 2004). al., et Zhang 2009a; previously described al., as et performed were (Wang assays ChIP-Re-ChIP and ChIP ChIP-Re-ChIP and ChIP nteratoswr hnaaye ywsenbotn ihteindicated the with blotting western by analyzed antibodies. then were reactions the in rslne ih1 omleye oiae,pecerdadincubated and 5–10 pre-cleared sonicated, with formaldehyde, 1% with crosslinked TTSses rmg) nteGTpldw assay, pulldown GST the In Promega). the lysate Systems; reticulocyte in rabbit (TNT PBS with accomplished cold The were mixture. in experiments inhibitor translation sonication protease by the prepared of presence were assay. lysates blot bacterial western BL21 crude in to expressed subjected were constructs were fusion complexes GST immune the and buffer ed eepenuae nbnigbfe 08 S nPSi the in PBS in BSA 4 (0.8% at mixture) buffer inhibitor protease binding the of in glutathione–Sepharose presence on preincubated immobilized were proteins beads fusion GST Then, PBS. nuaigwt rncie n rnltdpout t4 at products translated and transcribed with incubating ed eecletd ahdfv ie ihwsigbfe and buffer washing with times five washed collected, were beads eedtce ihseii nioisb etr blotting. western by antibodies specific with detected were 30 in resuspended iho ihu S o 2ha 37 at h 12 for TSA without or with ihcl B n hnlsdwt yi ufra 4 at buffer lysis with lysed then and PBS cold with sn rmr pcfcfrec agtgn rmtr l rmr are primers All promoter. PCR S3. gene Table conventional target material by supplementary each analyzed in for The listed was 8.0). specific template (pH primers buffer DNA using elution the using performed of reaction. ChIP was immunoprecipitation enrichment with step second elution 30-fold a with final diluted to beads The then subjected the and were For buffer from eluates precipitated. dilution The eluted and dithiothreitol. were extracted mM complexes was 20 DNA immune the assays, and Re-ChIP buffers, salt high and ihteatbd o t4 bind at to h added 2 for and antibody prepared the with were beads CL-4B Protein-A/G–Sepharose /–ehrs L4 ed eepeae n de obn ihthe with 4 bind at to h added 2 and for prepared antibody were beads CL-4B A/G–Sepharose oclua xrcsadicbtda 4 added at incubated was (IgG) and extracts G immunoglobulin cellular mouse to or rabbit normal or antibodies Gaha otae a ig,CA). Diego, San Software, (GraphPad ehrs ed t4 at beads 30 Sepharose with incubated were proteins fusion GST appropriate 2 %gyei,1m T,1MPS,ajs Ht . ihHCl), with 8.5 to pH adjust PMSF, 1mM DTT, mM 1 glycerin, 5% , ora fCl cec 21)17 6949 doi:10.1242/jcs.154245 4679–4691 127, (2014) Science Cell of Journal m fatbd e ecina 4 at reaction per antibody of g m ˚ ˚ lof2 o ,floe yetniewsigwt cold with washing extensive by followed h, 1 for C .Baswr ahdfu ofv ie ihlysis with times five to four washed were Beads C. 6 6 .. ttsia infcnewseautdby evaluated was significance statistical s.d.; ˚ D-AElaigbfe.Poenbands Protein buffer. loading SDS-PAGE .Te,cmlxswr ahdwt low with washed were complexes Then, C. ˚ ,adtelvl faeyae histones acetylated of levels the and C, ˚ vrih.Sbeunl,Protein- Subsequently, overnight. C 6 t ts yuigGaha Prism GraphPad using by -test 10 7 ˚ vrih.Subsequently, overnight. 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Journal of Cell Science i . u . e . hu . i,Q,Wn,Y,Sa,C n og Y. Gong, and C. Shao, Y., Wang, Q., Liu, H., Zhou, F., He, D., Lu, X., Li, i . i,N,Kpr .adCu,K T. K. Chun, and R. Kapur, N., Jia, B., Li, in,B,Za,W,Ya,J,Qa,Y,Sn . o,Y,Go . hn . Shao, B., Chen, C., Guo, Y., Zou, W., Sun, Y., Qian, J., Yuan, W., Zhao, B., Jiang, Y. M. Xiong, and M. S. Jackson, Smith, and M. Iizuka, e,J n hu P. Zhou, and J. Lee, ba,T,Svpaa,U,Tri . mdr . aao .adDta A. Dutta, L. and S. M. Berger, Pagano, V., Amador, K., Terai, U., Sivaprasad, T., Abbas, References ARTICLE RESEARCH un,L,Sw,Y,Ski .adPre,A B. A. Pardee, and T. Sakai, Y., Zou, Sowa, X., Li, L., Q., Huang, Liu, J., Yuan, R., Liu, B., Jiang, W., Zhao, Q., Ji, Y., Yang, H., Hu, Co J., A. Landry, M., Ullah, C., Cayrou, Y., Doyon, aet,C . ag .M,Sn .M,Dve .R,St,E n Eisenman, and E. Seto, R., J. Davie, M., J. Sun, M., W. Yang, D. D., C. C. Laherty, Allis, and H. M. Kuo, u . cal .M,Ot,T n in,Y. Xiong, and T. Ohta, M., C. McCall, J., Hu, H. Zhang, and H. 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