MBD4 Interacts with and Recruits USP7 to Heterochromatic Foci

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MBD4 Interacts with and Recruits USP7 to heterochromatic foci

Citation for published version: Meng, H, Harrison, DJ & Meehan, RR 2014, 'MBD4 Interacts with and Recruits USP7 to heterochromatic foci', Journal of cellular biochemistry, vol. 116, no. 3, pp. 476–485. https://doi.org/10.1002/jcb.25001

Digital Object Identifier (DOI): 10.1002/jcb.25001

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Published In: Journal of cellular biochemistry

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Download date: 06. Oct. 2021 3 Uni 2 2XU, EH4 Edinburgh, of Edinburgh, 1 HuanMeng foci to heterochromatic USP7 Recruits and with Interacts MBD4 Article SupportingInformation Additional may inbefound theversion online ofthis article. doi: as article this cite Please Record. of Version to the lead and may [10.1002/jcb.25001] version which process, this proofreading between and differences pagination typesetting, copyediting, the through † Grant sponsor:MRC, IMI-MARCAR andthe BBSRC [email protected] 2XUMolecularEdinburgh,Hospital,EH4 Medicine, TheGeneral UniversityWestern Edinburgh,of UK *CorrespondenceProfessor to: Richard R. Meehan, MRC Human Unit, Genetics The Institute of Genetic and

This article has been accepted for publication and undergone full peer review but has not been been not has but review peer full undergone and publication for accepted been has article This MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University University Medicine, Molecular and Genetics of Institute Unit, Genetics Human MRC School of Medicine, University of St Andrews, St Andrews, StAndrews, Andrews, ofSt University School ofMedicine, e Lbrtr o Mdcl el ilg, iity f dcto, hn Medical China Education, of Ministry Biology, Cell Medical of Laboratory Key Accepted Article 110001, China Shenyang versity, ReceivedSeptember5 Revised2014; 8 October Accepted2014; 1,2

, , DavidJ.Harrison ; Tel: +44Tel: (0)131 332 2471 UK

October23 †

; Email: 2014 5001

WORDS: KEY can that factor additional potentially activity regulate an Dnmt1 proliferation. during cell as it implicates chromocenters to USP7 recruit and with directly interactto MBD4 of ability The Dnmt1. methyltransferase, maintenance DNA the of stability the regulate enzyme, de-ubiquinating a USP7, and ligase, ubiquitin E3 a UHRF1, Both MBD4. for partners interaction new demethylation. two as relevant USP7 and and DNA UHRF1 development identified We in elusive. and remains processes diseases these repression to MBD4 of transcriptional contribution precise apoptosis, the However, of initiation the response, damage DNA repair, DNA including pathways nuclear of number a with associated been has It domain. glycosylase C-terminal a possesses that protein binding methyl-CpG only the is MBD4 ABSTRACT Accepted Article

MBD4, UHRF1, USP7, Heterochromatin Replication and Formation and Heterochromatin Replication USP7, UHRF1, MBD4,

[ Fas- as replication DNA and proliferationduring cell apoptosis with repair DNA and genome surveillance link such proteins interacting MBD4 potentially (DNMT1) 1 Indeed,methyltransferase DNA and MLH1 (FADD), protein stability. domain death associated genome to contribute can that MBD4 apoptosis MMR-dependent [ during roles additional have may sites heterochromatin to 1) [ homolog (MutL MLH1 protein 1998 2011 al., et [ interaction physical their by mediated possibly MBD4, including levels, or agents 2003 al., et Sansom damaging DNA to exposure from [ Dnmt1 methyltransferase, resulting maintenance the of inactivation response apoptotic the mediating in role a [ tumorigenesis MMR-dependent affect not that of MLH1[ USP7 of 2007 2011 al., et [ activity deubiquitylase its via UHRF1 of stability the regulates [ HAUSP) as known also virus-associated, forms UHRF1 which during large-scale undergo that 2008 al., histones) modified (via chromocenters [ regions heterochromatin at clustering progressive and reorganization the to proliferation cell during [ observed was sites CpG at an create mutation G:T in increase fold 2-3 a although to MSI; increased or rates, survival reduced tumorigenesis, targeted mice mutant However, [ (MMR) with repair associated mismatch generally (MSI), in instability defects microsatellite exhibit that carcinomas other and colon [ formation 5-methylcytosine for affinity DNA its to similar is that DNA (5hmC)-containing hydroxymethylcytosine daughter of [ methylation facilitate to strands DNA hemi-methylated to DNMT1 recruits and with interacts lack protein, truncated a in results 2003 al., et Screaton [ 2003 demethylation al., et Cortellino DNA and repression transcriptional apoptosis, of initiation response, damage DNA the repair, DNA including pathways nuclear of number a with associated been M Ruzov et al.,2009 1999 al., et Bellacosa UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1, also known as Np95 and ICBP90) and Np95 as known also 1, domains finger RING and PHD with (Ubiquitin-like, UHRF1 BD4 is the only methyl-CpG binding protein that possesses a C-terminal glycosylase domain. Ithas glycosylasedomain. C-terminal a possesses that protein binding onlymethyl-CpG the BD4is ; ; Felle et al., 2011 al., et Felle uo e a. 2009 al., et Ruzov [ aauaa t l, 2011 al., et Rajakumara ]. UHRF1 is preferentially expressed in the cells undergoing proliferation [ proliferation undergoingcells the in preferentiallyexpressed is UHRF1 ]. per se per Accepted 2007 al., et Bostick Article ; ], and modulates the enzymatic activity of Dnmt1 on the UHRF1 platform [ platform UHRF1 the on Dnmt1 of activity enzymatic the modulates and ], aat t l, 2008 al., et Papait Ruzov et al., 2009 al., et Ruzov , is generally diffuse [ generallydiffuse is , Ruzov et Ruzov al., 2009 ; ]. Depletion of DNMT1 in cancer cell lines can result in decreases in MMR protein MMR decreasesin in result can lines cell cancer in DNMT1 of Depletion ]. Screaton etal., 2003 ; Thillainadesan et al., 2012 al., et Thillainadesan ; ; ; Hendrich et al., 1999 al., et Hendrich Cortellino et al., 2003 al., et Cortellino Qin et al., 2011 al., et Qin , n hs lo en hw t pyial itrc wt ad eri MMR recruit and with interact physically to shown been also has and ], Cortellino et al., 2003 al., et Cortellino Millar et al., 2002 al., et Millar a ; ]. MBD4 strongly associates with heterochromatin [ heterochromatin with associates strongly MBD4 ]. complex with Dnmt1 and USP7 (ubiquitin specific peptidase 7, herpes 7, peptidase specific (ubiquitin USP7 and Dnmt1 with complex ; aat t l, 2007 al., et Papait ], which contrasts with thestrong which with contrasts ], andchromocenter- heterochromatin . HF i srnl lne t htrcrmtn elcto and replication heterochromatin to linked strongly is UHRF1 ]. hrf t l, 2007 al., et Sharif ing its intervening region and glycosylase domain, glycosylase and region intervening its ing Holowaty et al., 2003 al., etHolowaty ae e a. 1999 al., et Bader ; ]. ]. Sharif et al., 2007 al., et Sharif

Felle et al., 2011 al., et Felle Sansom et al., 2004 al., et Sansom ; ; ; Meng et al., 2011 al., et Meng Ruzov et al., 2009 al., et Ruzov Wong et al., 2002 al., et Wong ]. A naturally occurring frameshift mutation in MBD4 in mutation frameshift occurring naturally A ]. ], possibly through unknown protein associations of associations protein unknown through possibly ], Mbd4 , hc my eed n t seii localization specific its on depend may which ], . t lo a a idn seiiiy o 5 for specificity binding a has also It ]. ul uain i nt xii increased exhibit not did mutation null ; ; ]. However, the nuclear distribution pattern distribution nuclear the However, ]. van der Horst et al., 2006 al., et Horst der van Loughery et al., 2011 al., et Loughery ae e a. 2007 al., et Bader ; Ma et al., 2012 al., et Ma ; Felle et al., 2011 al., et Felle Sansom et al., 2003 al., et Sansom ; ]. These findings suggest that MBD4 MBD4 that suggest findings These ]. Rai et al., 2008 al., et Rai ]. Loss of MBD4 function also does also function MBD4 of Loss ]. Nishiyama et al., 2013 al., et Nishiyama Laget et al., 2014 al., et Laget ; Qin et al., 2011 al., et Qin elcs e a. 1999 al., et Bellacosa ; ; ico t l, 1999 al., et Riccio ; ; Ma et al., 2012 al., et Ma Papait et al., 2008 al., et Papait Ruzov et al., 2009 al., et Ruzov Ruzov et al., 2009 al., et Ruzov Hendrich and Bird, and Hendrich ], however it plays it however ], ] and is similar to similar is and ] occurs in human human in occurs Bostick et al., et Bostick ; ; Loughery Papait et Papait ]. USP7 ]. ; Qin ]. ]. ] 3 - ; ; ; ,

[ their on mutual engagement[ onchromatin depend to suggested been has complex USP7/UHRF1/DNMT1 trimeric the of stabilization Spectrometry a Mass of Andrews lane St. control to sent and were experimental chunks the to These thensubjected were cubes. cut 1mm and into excised was analysis. gelservicesfor The chunk gel from The excised weights. were molecular weights corresponding of molecular chunks Several diverse Invitrogen). (NuPAGE, of Blue Colloidal bands with stained were gels analysis, MS For SPECTROMETRYMASS (MS) ANALYSIS separated, and (BioRad) SDS-PAGE 10% large a sample on in loaded followed byanalysis. Western Blot boiling were by samples eluted eluted were proteins the Bound and PBS. buffer, ice-cold pure with once containing and buffer extract NP40, nuclear 0.05% ice-cold with times three washed were Beads 4°C. at hoursrotation 2 with for incubated and samples to added were (ChromoTek) beads GFP Flag- or to (Sigma) conjugated mAb specific covalently beads sepharose 50ul supernatants. nuclear post 30 get for to 20,000g at twice centrifuged minutes was sample the and added, is MNae if digestion chromatin the stop to 10 EDTA-free x KCl, 30 mM 7.4, used was EDTA 20 5mM of concentration final A chapter. result the in indicated as Roche) (NucleaseS7; pH NaCl, mM (Roche), 300 cocktail HEPES, inhibitor mM protease (20 complete buffer extract nuclear a in lysed were cocktai inhibitor x protease 30 complete 7.4, and pH EDTA, (Roche), mM 5 KCl, mM 10 MgCl2, mM 1.5 HEPES, mM [ 10 buffer(0.05%lysedwith ahypotonic NP-40, lysis and were werewith SF-TAP-hMBD4 transfected to according cells HEK293T from prepared was extract Nuclear PREPARATION IMMUNOPRECIPITATION NUCLEAR AND EXTRACT FCS, 10% Pen, and1000U/ml 650μg/ml Strep. with supplemented Invitrogen) (DMEM; Medium Eagle Modified Dulbecco's in cultured p21b-6his- the to cloned then were mutants MBD4 Human (Stratagene). kit mutagenesis site-directed QuikChange the using vector, of upstream 6HIS- the into make induced was tag To 6-HIS (aa) NotI. acid amino and EcoRI using vector SF-TAP to cloned [ Ueffing) Marius (Dr SF-TAP below: laboratories the by provided kindly were plasmids following The PLASMID AND CULTURE CELL CONSTRUCTION METHODS AND MATERIALS in MBD4 for role direct a recruiting tochromocenters. USP7 demonstrate and MBD4 of novel region a intervening characterized the We in USP7. domain and interaction UHRF1 with interacts specifically MBD4 that found we and [ UHRF1 2013 al., et Zaman [ proliferation [ cell partners protein nuclear fociits by recruited relocated to and during UHRF1 of expression associated F-MD (r din id [ Bird) Adrian (Dr GFP-mMBD4 2009 al., [ Spada) Fabio and Leonhardt Heinrich (Drs MCherry-USP7 hp e a. 2008 al., et Thapa Gloeckneral., et2007 In this study we performed biochemical isolation and identification of MBD4 associating proteins, associating MBD4 of identification and isolation biochemical performed we study this In ], FLAG-p75 (Drs Madapura M. Pradeepa and Wendy A. Bickmore) [ Bickmore) A. Wendy and Pradeepa M. Madapura (Drs FLAG-p75 ],

Accepted Article 2011 al., et Felle , n peiu suis ae ugse a iet neato bten S7 and USP7 between interaction direct a suggested have studies previous and ], . o ae LGhB4 S-A-MD) hmn ul egh B4 was MBD4 length full human (SF-TAP-hMBD4), FLAG-hMBD4 make To ]. ], HA-UHRF1, FLAG-UHRF1 (Dr John Peter[ McPherson) John (Dr HA-UHRF1, FLAG-UHRF1 ], 10 6 cells/ml). Cytosolic fraction was discarded and the separated cell nuclei nuclei cell separated the and discarded was fraction Cytosolic cells/ml). ; ; TF a t l, 2012 al., et Ma -UB vector using BamhI and XhoI. CMT93 and HEK293T were were HEK293T and CMT93 XhoI. and BamhI using vector -UB Felle et al.,2011 edih n Br, 1998 Bird, and Hendrich

. oee, hs neato my e rnin and transient be may interaction this However, ]. ]. ]. , p21b- ], Daubeuf et al., 2009 al., Daubeuf et Qin et al., 2011 al., et Qin TF uia e a. 2013 al., et Dunican -UB site of the original p21b- original the of site -UB 6 el/l wt o wtot MNase without or with cells/ml) rdea t l, 2012 al., et Pradeepa TF TF - U vco (r lSo Park) Il-Seon (Dr vector -UB UB hB4 uat, h 18- the mutants, -hMBD4 ]. GFP-MLH1 [ GFP-MLH1 ]. Pradeepa et al., 2012 al., et Pradeepa ; Mistryet al.,2008 Everett et al., 1997 Everett et . S7 a be can USP7 ]. . HEK293T ]. Ruzov et Ruzov TF -UB -UB ] ] 4 l ; , ,

lsi wr mtd ih sri pernfre wt a os ebync a 1. cN library cDNA 11.5 day int clone embryonic library and Bait mouse (Clontech). pGADT7 a into cloned with pretransformed strain a with mated were each plasmid carrying strains Yeast (Clontech). pGBKT7 construct BD-Bait GAL4 a into cloned was MBD4 [ described previously as performed were assays Y2H The TWO-HYBRID YEAST fluorescence II Axioplan (Chroma set Zeiss filter a pass band Photonics), using triple #83000 Technology),'IP andLab'was analysis. image software usedfor (Hamamatsu taken Chroma and were camera objectives Plan-neofluar images CCD with microscope Fluorescence AG Orca pre-mixed). media Hamamatsu mounting DAPI 'Vectashield' (250ng/ml in mounted Laboratories) were Coverslips (Vector solution. block in and 1:1000 diluted slides were on α Goat (Invitrogen); grown conjugate Red Alexafluor was CMT93, F rabbit F1804, mouse (Sigma; antibody line, anti-FLAG cell [ cancer described previously as (pFa) paraformaldehyde colon mouse The IMMUNOFLUORESCENCE necessary) toremove imidazole. on 6xH protocol The standard 4°C. a at by 15,000g purified at was min 30 protein for tagged centrifugation after recovered was fraction soluble The thenlysedbenzenesulfonylcells(AEBSF).The were fluoride by sonication, 4- mM Ex 1 containing glycerol] 30% (2-ME), 2-mercaptoethanol mM Protein 10 imidazole, mM BugBuster® ml 10 NaCl, mM 300 7.4, pH Na2HPO4/NaH2PO4, mM [50 20 A buffer or (Novagen), NucleaseBenzonase either in resuspended and harvested were Cells instructions. manufacturer’s with accordance in Technologies) (Agilent cells 6-HIS- respective The FROM COLI E. EXPRESSIONPURIFICATION AND OFPROTEINS Human GeneticsUnit). 11814460001), MRC Seawright, Anne from (gift mouse antibody anti-6HIS antibody[5F8]), RFP anti-MCherry(Chromotek, (Roche, anti-GFP F7425), rabbit F1804, mouse (Sigma; antibody anti-FLAG antibodies: primary following the with probed were blots Western loading analysis. blotting western in for buffer mixture boiled immediately the then was agarose °C, Ni-NTA The 4 PBS. ice-cold at pure by incubation followed buffer hour 1 After wheel. onto loaded rotating was beads agarose a containing on °C 4 at overnight (Invitrogen) 6HIS- purified For with mixed were instructions. GFP-Mlh1 or FLAG-UHRF1 containing manufacturer’s that plasmids with cells transfected 293T transiently with were from digestion) chromatin accordance (without lysates in nuclear cell assays, used pull-down reciprocal was (Invitrogen) 2000 Lipofectamine WESTERN AND ASSAY ANALYSIS BLOT PULL-DOWN RECIPROCAL TRANSFECTION, TRANSIENT Feb database 2011 (12852469 sequences) withrestriction. nospecies NCBInr the against UK) London, Science, (Matrix engine search 2.1 Mascot the using 150 containing 8.0, pH buffer, phosphate-based or Tris- mM (50 buffer against dialyzed was protein eluted the yieldpurity, and judge SDS After required. if glycerol and beta-mercaptoethanol, mM 1 NaCl, mM 300 phosphate a or Tris- a in either with eluted generally [ protocols standard using MI) in Accepted ArticleArbor, Ann Solutions, (Genomic robot digestion in-gel Investigator ProGest a using digestion, -gel TF U hmn B4 uat ta wr peicbtd ih iNA agarose Ni-NTA with pre-incubated were that mutants MBD4 human -UB TF

-UB hMBD4 mutants were transformed in transformed were mutants hMBD4 -UB Shevchenko et al., 1996 al., et Shevchenko – 0 m NC ad m bt-ecpotao, n gyeo as glycerol and beta-mercaptoethanol, mM 1 and NaCl mM 300 a column, followed by extensive washes (4 X) with RIPA RIPA with X) (4 washes extensive by followed column, -mouse IgG-Alexafluor Green conjugate (Invitrogen)) conjugate Green IgG-Alexafluor -mouse Pradeepa et al., 2012 al., et Pradeepa -based buffer containing 200 mM imidazole, 150 imidazole, mM 200 containing buffer -based 7425). Secondary antibodies (Goat α (Goat antibodies Secondary 7425). ]. Th ]. ikl grs (nirgn. rtis were Proteins (Invitrogen). agarose Nickel Dellaire et al., 2002 al., et Dellaire rcin egn (oae) ih 1μl/ml with (Novagen) Reagent traction e MS/MS data file generated was analyzed was generated file data MS/MS e eraction was identified by β by identified was eraction to BL21 ]. The primary antibodies were antibodies primary The ].

or -CodonPlus®(DE3)-RIPL ]. The full length mouse length full The ]. with with we – BugBuster reagent. PAGE analysis to analysis PAGE e ie i 4% in fixed re (2 -Galactosidase -Galactosidase -aminoethyl) -rabbit IgG- -rabbit is 5 – -

n te nevnn rgo (mn ai 1 acid (amino region intervening the and UHRF1 epitope-tagged FLAG with interacting domains the identify to proteins performed purified were the experiments using pull-down vitro in Reciprocal lower). 2B (Fig. mutants respective the for collected were elutions individual E.coli; from purified and induced vector, expression prokaryotic [ proteins fusion soluble C its at ubiquitin as well as Factor’ 6xHis- were mutants These 2A). (Figure associations protein partner for responsible be may that regions interacting the minimize the MBD,glycosylase domains ofMBD4,and tooverlap the and region, intervening withto each other represent to designed were MBD4 of versions truncated four The B). & 2A (Figure (156-455) region 408 acid (amino region interaction we complex, UHRF1 with association 1 acid (amino the domain MBD the mediating containing proteins MBD4 MBD4recombinant the purified of domains protein the identify To ITSINTERACTS UHRF1THROUGHINTERVENINGMBD4 WITH COMPLEX REGION [ reports previous Collectively, that MBD4 show can interactUHRF1 our with data and USP7. the with consistent control), negative1B. (Figure MBD4 with precipitated not was p75 control negative the while control), positive A lane). lower UHRF1 addition, In mouse. and human between conserved and specific is interaction their suggesting lanes), three upper UHRF1 1B. USP7 (Figure UHRF1 and lanes) three of versions upper mouse and human with IP by UHRF1 interaction similar a showed MBD4 1B. and USP7), 1B. (Figure (Figure UHRF1 with affinity similar exhibited MBD4 mouse and Human binding. chromatin from independent and direct are interactions these suggesting lanes), two upper UHRF1 1B. (Figure digestion chromatin without and with conditions both in USP7 and UHRF1 of co-precipitation showed IP MBD4 analysis. Blot Western by tested was IP reciprocal a in MBD4 as well as USP7 UHRF1, of presence The lane). lower UHRF1 1B. (Figure extracts nuclear In right). USP7 & digested left MNase in UHRF1 human epitope-tagged FLAG with out carried was IP reciprocal a addition, UHRF1 1B. (Figure digestion chromatin without extracts nuclear in MBD4 mouse epitope-tagged GFP with as well as extracts, nuclear digested MNase in MBD4 human tagged interaction ch specific the verify To are associations their brackets).if clarify to and USP7, and UHRF1 partners two protein two the and MBD4 between 1A. (Figure regions mass excised by proteins the interacting of novel two spectrometry as USP7 and UHRF1 (MNase) identified nuclease We micrococcal 1A). with (Figure digestion prepared extracts nuclei using cells 293T from MBD4 tagged epitope- FLAG of purification affinity out carried we partners, interaction MBD4 identify to order In WITH USP7 UHRF1MBD4 COMPLEXES AND FORMS RESULTS were picked forcolonies and followingrescue colony plasmid PCR followed by sequencing confirmation. Confirmed restreaking. of confirmation and selections dropout appropriate and assays Accepted Article epitope- FLAG with (IPs) immunoprecipitations preformed we dependent, romatin-interaction

Thapa et al., 2008 al., et Thapa co -IP of MBD4 and Mlh1 confirmed their positive interaction (Figure 1B. 1B. (Figure interaction positive their confirmed Mlh1 and MBD4 of -IP agd uin rtis ht ae temsal poen ald ‘Trigger called protein thermostable a have that proteins fusion tagged – -terminal region (Fig. 2B upper), which allows for the isolation of isolation the for allows which upper), 2B (Fig. region -terminal 580), and the intervening region and the downstream interaction downstream the and region intervening the and 580), a ]. The four versions of human MBD4 were cloned into the into cloned were MBD4 human of versions four The ]. – eircl P f HF peiiae MD (iue 1B. (Figure MBD4 precipitated UHRF1 of IP reciprocal 0) te lcsls dmi ad t kon upstream known its and domain glycosylase the 408), elcs e a. 1999 al., et Bellacosa ; rdea t l, 2012 al., et Pradeepa – 156), the MBD MBD the 156), ] 6 .

AI rgt pt i te ulu (iue A lf & ht arrows white & left 3A. (Figure nucleus the in spots bright DAPI with coincident was that signal strong a exhibited protein MBD4 mouse epitope-tagged GFP Ectopic as sites heterochromatin prominent othersites. at heterochromatin has that line each with cell associate might UHRF1 and MBD4 that hypothesis our cancer test to staining, DAPI by evidenced colon a cells, We CMT93 3). mouse (Figure distribution used subcellular and co-localization potential their determine to microscopy IN CHROMOCENTERS AT 2008 UHRF1 [ cells mouse in WITH sites heterochromatic to localize respectively UHRF1 and MBD4 that found have studies Previous COLOCALIZES HETEROCHROMATIN REPLICATIONFORMATION AND TIGHTLY MBD4 the and UHRF1, with interact directly can MBD4 that interveningregion canassociation. mediate ofMBD4 the show studies vitro in arrow). our black together, 2A, Taken (Figure domain glycosylase C-terminal active catalytically the losing to addition in MBD4 of profile interaction proteinprotein the affect major MMR-deficient to potential in the a has 313 presumably acid carcinomas constitutes human amino MBD4 at truncation of MBD4 region occurring naturally intervening The the region. interaction that view the supports This right). 2E. (Figure 2) upper 2E. (Figure assay reciprocal recombinant the in affinity binding weak very the MBD4 to contrasts which left), upper of binding the to (similar an UHRF1 contain co-precipitated not successfully does MBD4 of region intervening [ domain functional obvious The lower). 2E. (Figure region intervening its were domains to MBD4 glycosylaseof regioninteraction the map to able were we Therefore, and right). upper 2E. (Figure weakvery MBD containing mutants other the co-precipitated contrast, by strongly MBD4, UHRF1 of of 2 of mutant IP capable Reciprocal not left). were upper respectively, 2E. (Figure domain UHRF1 glycosylase with and interacting MBD the while containing vitro, 3, in and UHRF1 1 co-precipitated efficiently mutants MBD4 of 4 and 2 Mutants 2E). (Figure UHRF1 [ Mlh1 415 with acid region interaction amino which in study the with Y2H consistent was previous This cartoon). lower 2D. (Figure association and the for 4 requirement and minimum 3 mutants between region overlapping the to corresponds 410-455 in the resides Mlh1 with association the for responsible MBD4 of region interaction the that reveal data with IP These right). upper 2D (Figure 4 An and 3 2, recombinants MBD4 with interactionan indicated also left). Mlh1 upper 2D (Figure Mlh1 co-precipitated efficiently MBD4 of 4 mutant particularly co-transf performed we organization, chromatin as such phenotypes cellular particular affects association their if and heterochromatin, at UHRF1 with space cellular same the occupies MBD4 if determine To maintenance. and regulation heterochromatin was Mlh1 with association its for responsible identified MBD4 previously of region interaction positive as the selected because was Mlh1 control, epitope-tagged GFP 2C). (Figure cells 293T from purified complex nevnn rgo ad lcsls dmi o MD (iue D lwr cartoon). lower 2D. (Figure MBD4 of domain glycosylase and region intervening ; Accepted2011 al., et Nady Article uia e a. 2013 al., et Dunican in ; peiu yat w-yrd YH suy [ study (Y2H) two-hybrid yeast previous a Papait et al., 2007 al., et Papait Meng et al., 2011 al., et Meng ilr 2002 Millar, ; eao t l, 2014 al., et Gelato ; . e hn etd h itrcin ein f B4 with MBD4 of region interaction the tested then We ]. Ruzov et al., 2009 al., et Ruzov ], in addition, our data show that MBD4 recombinant 4 recombinant MBD4 that show data our addition, in ], 40 f B4 a mpe a MD minimum MBD4 as mapped was MBD4 of -420 cin olwd y muoloecne (IF) immunofluorescence by followed ection ; edih n Br, 1998 Bird, and Hendrich ], suggesting they may be involved in involved be may they suggesting ], ilr 2002 Millar, in ih mgiiain; this magnification); right iey ersns the represents likely ; Karagianni et al., al., et Karagianni ]. uat and 3 Mutant Amino acids acids Amino 7

ept dfeet ellr ytm n cniin ue, prev used, conditions and system cellular different Despite 4A at the vicinity with USP7 USP7. ofthe domainof TRAF-like binding interacts (Figure directly MBD4 USP7 which through of route one be domain may this TRAF-like that suggesting shadow), C-terminal gray vertical with overlaps it and sequence, coding USP7 to aligned was USP7 of fragment cDNA The direct. is USP7 and MBD4 possibility between interactionthe the supporting that 4A), (Figure bait as protein Mbd4 mouse using assay Y2H a an in as interactor USP7 identified also we USP7, with interacts directly MBD4 that finding our with Consistent MBD4 DIRECTLY TO CHROMOCENTERS RECRUITS USP7 of (Figureinvolved inlarge-scaleheterochromatin black reorganization arrows 3Ciii. inDAPI lane). Decondensin square). or circle). circle or square white lane, DAPI iii, black i, vs. ii & lane iii, & ii i, DAPI middle, 3C (Figure UHRF1 andMBD4 spots, of co-localization tightthe changesin to lead not did but bright DAPI in intense less by Merge manifested was chromocenters & at remodeling or UHRF1 heterochromatin MBD4, of lanes 3C. (Figure formation and Merge replication heterochromatin during and dynamics chromocenter perturb UHRF1 may which vs. MBD4 iii space cellular same ii, the occupied tightly UHRF1 i, and MBD4 Specifically, 3C 1B). Figure Supplementary (Figure with cells co-localized tightly CMT93 MBD4 in ectopic cases, chromocenters all at In B). UHRF1 & A vs. marked C a 3 (Figure with size concomitant cell are in phenotypes increase cellular such with cells The sites. co-localization their 3C, (Figure reformation heterochromatin characterized we specifically, More left). 4B. (Figure diffused as USP7 of distribution reportednuclear MCherry 2006 al., et Horst der van [ staining cytoplasmic some as well as nuclei cell within USP7 of distribution arrows). black 3Ciii. (Figure chromocenters at staining strong their to adjacent are that clusters ring-like forming arrows); white 3Ciii. (Figure nuclei cell dividing of indicative be may that UHRF1 and MBD4 of localization at occurring events reorganization large-scale marked 3C, (Figure clustering major or middle), 3C. (Figure fragmentation left), 3C. in resulted MBD4 (Figure bridging either by manifested magnification), lower & upper 3C. (Figure sites heterochromatic and UHRF1 of [ cells proliferating in levels high [ proliferation cell regulate to demonstrated been has UHRF1 [ MeCP2 or UHRF1 of overexpression for reported previously phenomenon cellular a arrows); white B, & 3A (Figure clustering heterochromatin 2014 al., [ proteinUHRF1 endogenous of location the resemblingmagnification), also that distribution nuclear condensed a showed UHRF1 human epitope-tagged FLAG greenstaining). light MBD4 3A. (Figurecells CMT93 [ MBD4 for ligand methylatednatural a DNA, satellite methylated to correspond spots bright DAPIThe resembl uo t l, 2009 al., et Ruzov es ]. In CMT93 cells, ectopic expression of either MBD4 or UHRF1 induced minor minor induced UHRF1 or MBD4 either of expression ectopic cells, CMT93 In ]. epitope-tagged USP7 USP7 epitope-tagged Accepted Article[ distribution MBD4 mouse endogenous the ]. Additional non-heterochromatic staining of ectopic MBD4 is also observed in observed also is MBD4 ectopic of staining non-heterochromatic Additional ]. This may represent an intermediate transition, in which MBD4 and UHRF1 are UHRF1 and MBD4 which in transition, intermediate an represent may This . e eemnd h sbellr oaiain f coi epeso of expression ectopic of localization subcellular the determined We ]. in the CMT93 cell model, and out data is consistent with the previously consistent cellin the CMT93 model,and out data is Papait et al., 2007 al., et Papait co et o pras elcto (iue 3C, (Figure replication perhaps or left) Brero et al., 2005 al., et Brero -localized with DAPI bright dots (Figure 3B. left & right & left 3B. (Figure dots bright DAPI with -localized ]. Consistently and intriguingly, co-overexpression co-overexpression intriguingly, and Consistently ]. . Interestingly, we observed some moderate co- moderate some observed we Interestingly, . Hendrich and Bird, 1998 Bird, and Hendrich ; Jenkins et al., 2005 al., et Jenkins Papait et al., 2008 al., et Papait ious studies have reported a diffused diffused a reported have studies ious Dunican et al., 2013 al., et Dunican right); possibly implying implying possibly right); Holowaty et al., 2003 al., et Holowaty ; ; ], and is expressed at expressed is and ], Ruzov et al., 2009 al., et Ruzov Papait et al., 2007 al., et Papait ide ocr at occurs middle) ; Gelato et Gelato & ] ] 8 ; . .

process interaction via its [ well as Dnmt1 perhaps and UHRF1 [ of platform UHRF1 fromphosphorylation USP7 expels phase-specific UHRF1 M and cycle, cell the of phases S and G1 during ubiquitinylation chromocenters. heterochromatin-associated [ at dependent cycle cell platform be UHRF1 the to to has shown been USP7 deubiquitylase and Importantly,UHRF1 theregulation interactionbetween of USP7 recruit and with [ activitydeubiquitylase its through Dnmt1 as well as UHRF1 of stability protein the regulate can USP7 [ patterns methylation DNA of inheritance faithful facilitate to platform recruitment a as act may UHRF1 proliferation, cell During DISCUSSION during chromocenters to USP7 with concomitant is recruits this large-scale ofheterochromatin. reorganization and and co-localized tightly with be also can interacts UHRF1 and directly MBD4 where interphase, MBD4 that marked show and data arrows), arrows),our white lane, white Collectively, DAPI arrows). white lane, lane, DAPI ii iii 4C. (Figure DAPI chromocenters at 4C. remodeling heterochromatin i (Figure fragmentation 4C. and (Figure We linkage iii). diminution chromocenter & and ii 4C. clustering in arrows heterochromatin black by observed indicated spots heterochromatin heterochromatin disappearing of & lane degree DAPI a to leads also middle vs. right & left upper, 4C (Figure transfectionssingle the in chromocentersevident not was that reorganization at co-localization their and relocation USP7 MBD4-induced the 3, Figurein UHRF1 and MBD4 of co-localization of observations above the arrows white Merge, and tested USP7 MBD4, cells 4C. (Figure CMT93 co-transfected all chromocenter- in chromocenters Strikingly, green to USP7 bound. recruit tightly 4C. to be able was to (Figure MBD4 binding 3 Figure chromocenters in at shown was exclusively UHRF1 which detected to staining), was MBD4 epitope-tagged this address (Figure GFP MBD4 ectopic To 4C). of presence chromocenters. the in the USP7 of to localization subcellular USP7 UHRF1/Dnmt1/USP7 the studied of the we question, recruitment in participate the to facilitating ability by the complex possess trimeric might MBD4 that us to suggested This 1A). Figure (Supplementary model cell CMT93 the in phenomenon same the observed have we [ cells MEF in chromatin heterochromatic with associated are that spots bright DAPI at accumulate to MLH1 diffused of re-distribution the to leads it which by function recruitment 2009 al., et [ response immunity innate and apoptosis, repair, inand damage DNA as such processes cellular participate effectively to partners, protein its with interactions physical via loci nuclear particular additional require to relocated and recruited be can may USP7 that shown have studies of USP7 number a Indeed, bound. tightly is implying arrows), white UHRF1 where chromocenters the to magnification, recruited be to partner(s) interaction heterochromatin-associating right & left USP7 of 4B. distribution nuclear the (Figure from excluded largely were spots bright DAPI prominent the that Accepted2012 al., et Ma Article ; Everett et al., 1997 al., et Everett ; Sharif et al., 2007 al., et Sharif

with all three components. with allthree components. a t l, 2012 al., et Ma Bostick et al., 2007 al., et Bostick ; Zaman et al., 2013 al., et Zaman ]. In this report, we have shown that MBD4 can directly interact interact directly can MBD4 that shown have we report, this In ]. Felle et al., 2011 al., et Felle ; HF i epesd n poetd y S7 rm auto- from USP7 by protected and expressed is UHRF1 ]; Ma et al., 2012 al., et Ma ; Sharif et al., 2007 al., et Sharif ]. Moreover, MBD4 has been reported to possess a possess to reported been has MBD4 Moreover, ].

& ; Ma et al., 2012 al., et Ma Supplementary Figure 1C). In agreement with agreement In 1C). Figure Supplementary ], leading to proteasomal degradation of degradation proteasomal to leading ], ]. Mounting evidence indicates that indicates evidence Mounting ]. ]. MBD4 may have a role in this in role a have mayMBD4 ]. Ruzov et al., 2009 al., et Ruzov Daubeuf ], and ], 9

NestorY2H (Linköping work. thankColm We themanuscript. for University) comments on GeneticsBickmoreIGMM, Human University MRC andfor Unit, Edinburgh) lab, advice inAP/MS of of(Queensland Technology,Brisbane,University Qld,Australia) and (Wendy M.Pradeepa Madapura Heidi issupported G.Sutherlandscholarships. thank We D.J.H. by EUFP7 programme. CASyM UniversityCollegeBritish Edinburgh and government research funding of ofMedicine overseas labissupportedIMI inR.M.’s Work by theMRC, ACKNOWLEDGEMENTS cancers[ relevant in modifications histone and remodeling, chromatin interaction organization, nuclear on impact This also may chromocenters. at patterns methylation genome-wide maintaining in as well as loci defined at patterns methylation specific targeting to respect with functions novel have DNMT1 partner identified previously the and USP7 UHRF1, with interaction protein MBD4 the if address to required [ outcomes patient poor and metastasis with associated is which genes, target specific at methylation DNA reprogrammed in results that overexpression RON 2010 al., et [ outcome poor a with correlate often which cancers, human of variety a in USP7, and UHRF1 proteins, partner MBD4 of overexpression the documented have studies Recent of theglycosylase ofMBD4. domain los to addition in region intervening of function interaction the deconstruct presumably would which 313, acid amino at truncation protein MBD4 human in resulting deficiency MMR exhibiting cancers human of number a in found MBD4 in mutations frameshift recurrent to link vertebrates, might This interactions. higher and functional new lower attract may latter between the in acquired motifs conserved and/or structure protein additional poorly suggesting are region intervening the of sequences [ functions glycosylase repair which novel interactions, for potential significant protein a possess may region this that suggests strongly characterization our viewed MBD4Theregion was intervening within previously study current pathways proliferation, stress ofcell responseDNA ormethylation machinery. Our pathways. these in participate also related in rolesand interactions MBD4new these of studies functionalfuture for foundation a provides might USP7 and UHRF1 that suggest data 2009 al., et [ pathways death cell or repair DNA in participate may they where damage, oxidative following survival cell for [ essential stress is protein MBD4 the that indicates evidence Recent ae e a. 2014 al., et Laget

AcceptedCunha et al.,2014 Article ; ]. Although cell models and physiological conditions are different, our MBD4 interaction MBD4 our different, are conditions physiological and models cell Although ]. Unoki et al., 2009 al., et Unoki ]. MBD4 and DNMT1 can be recruited at sites of oxidation-induced DNA oxidation-induced of sites at recruited be can DNMT1 and MBD4 ]. ; Jones, 2012 Jones,

Menget al.,2011 a aget B4s well MBD4’s augment may ]. In addition, MBD4 activation has been shown to be a consequence of consequence a be to shown been has activation MBD4 addition, In ].

]. ]. ; Millar, 2002Millar, -MARCAR and the BBSRC. H.M.was and supported theBBSRC. by-MARCAR uh e a. 2014 al., et Cunha caatrzd ehlCG idn and binding methyl-CpG -characterized ; as a functional desert[ a functional as Screaton et al., 2003 Screaton et Mudbhary et al., 2014 al., et Mudbhary Laget et al., 2014 al., et Laget . uue tde wl be will studies Future ]. ]. ]. Meng et al.,Meng 2011 et Motif and primary and Motif ; ; Ruzov Unoki 10 ] s ,

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- 52. DNA demethylation zebrafishin involves - 12. Np95 implicatedis in -86. - 106. - 13 8.

Accepted Article BiolCell 33:4971-84. deubiquitination by the TRIM27-USP7 complex regulates tumor necrosis factor alpha-induced apoptosis. Mol Zaman Nomura MM, T, Takagi Okamura T, T, W, Jin Shinagawa T, Tanaka Y,Ishii S. 2013. Ubiquitination- gastrointestinal tumor formation. Proc Natl Acad Sci U SA 99:14937-42. Edelmann 2002. W. Mbd4 inactivation increases Cright-arrowT transition mutations promotes and Wong E,Yang Kuraguchi K, M, Werling U, Avdievich E,Fan FazzariK, M, Jin B, Brown AM, Lipkin M, Nat Cell Biol 8:1064-73. Burgering BM. 2006. FOXO4 transcriptional activity regulated is by monoubiquitination and USP7/HAUSP. van Horst der A, de Vries-Smits AM,Brenkmanvan AB, MH,van Triest Broek den N, F,Maurice Colland MM, 14

screen ( screen of mapping and alignment for scheme Figure 4. Figure vector). Empty (ctrl: analysis MS by ( orepitope-tagged GFP extract) nuclear (MNase treated ectopic FLAGepitope-tagged with proteins identified ( indicated. are (M) were marker weight molecular FLAG-MBD4-IP the and MBD4 Precipitated the for specific USP7 ( MCherry-USP7 by transfected and coverslips on grown were domain SMART ( fragment. cDNA interacting integrated with overlapping Blast searching by identified are domains are (USP7 database domains Protein USP7. of domains parallelbars, 10μ white line. Scale ( right ( In immunostaining andheterochromatin replicationininterphase. reformation mayheterochromatin,bewhich of indicative the In cells. CMT93 in ( chromocenters in images at other each with colocalized exclusively ( ( cells. CMT93 analyzed and visual antibodies was secondary counterstaining conjugate Red IgG-Alexafluor anti-rabbit goat and primary rabbit anti-FLAG with immunostained or (A), (IF) immunofluorescence by directly ( FLAG-UHRF1 and GFP-MBD4 by transfected ( transfectedGFP-MBD4 andcoverslipsby on were grown cells CMT93 the for responsible 3. Figure domains interacting the identified are MBD4 ( Mlh1 associations with of representation by schematic labelled regions The blot. each of left the on indicated is weight molecular the of E ( Mlh1 ( UHRF1 and & ( assays.(co-IP) reciprocalco-immunoprecipitation a together pooled were mutants MBD4 individual of elutions three The left. the on loaded and storage, pre-purified from was with lanes lysate only of TFUB containing control) left TFUB 0. as the (labeled control vector on empty The weight. molecularindicated their are lanes marker dye The indicated. as shown are elutions protein their and 1-4 mutants MBD4 of cells E.coli transformed and of factor lysates total trigger Respective with ubiquitin. fused proteins MBD4 purified the of staining comaissie and mutants, MBD4 site truncation ( MMR-deficiencycarcinomas. human protein in 313 MBD4 occurring natural the indicates arrow black A proteins. mutant MBD4 are domains (MBD4 MBD4 of domains 2. Figure loaded. Thethemolecular migration of onthe weightleft. indicated is IP were (Lane 2) antibodycoupled and (Lane the input 1) ofthe analysis. 3% Blot detected by Western were proteins Co-precipitated antibodies. anti-GFP or anti-FLAG with extract) nuclear treated MNase MNase with extracts SDS nuclear to subjected from and immunoprecipitated treatment was MBD4 MBD4. of partners interaction 1. Figure Legends Figure ) and the antibody coupled IP are indicated with antibodies used for immunodetection. The migration The immunodetection. for used antibodies with indicated IPare coupled antibody the and ) E C ) Co-IP assay to determine the interaction of recombinant MBD4 mutants with Mlh1 ( Mlh1 with mutants MBD4 recombinant of interaction the determine to assay Co-IP ) . A ), the cells appear to be undergoing orderly division into two daughter cells. The insets on the on insets The cells. daughter two into division orderly undergoing be to appear cells the iii), D i

) USP7 coding sequence ( sequence coding USP7 ) . 6HIS IP) or FLAG-UHRF1 ( FLAG-UHRF1 or IP) 6HIS . & MBD4 directly interacts with and recruits USP7 to chromocenters. ( chromocenters. to USP7 recruits and with interacts directly MBD4 B4 ihl c-oaie UR1 t hooetr. snhoosy rwn mouse growing Asynchronously chromocenters. at UHRF1 co-localizes tightly MBD4

Accepted Article( region. intervening its via UHRF1 with interacts MBD4 B4 opee wt UR1 n S7 ( USP7. and UHRF1 with complexes MBD4 C B . ) or below ( below or ) E i

B & ). Co- ). ) Distribution of FLAG-UHRF1 in CMT93 cells. ( cells. CMT93 FLAG-UHRF1ofin Distribution ) ii ), the cells exhibit an increase in cell size and marked large-scale reorganization of reorganization large-scale marked and size cell in increase an exhibit cells the ), precipitated proteins were detected via western blot. Input (3% of 500μg GFP 500μg of (3% Input blot. western via detected were proteins precipitated nd 500μg of each MBD4 protein mutant was measured for the subsequent subsequent the for measured was mutant protein MBD4 each of 500μg nd D C . control) and ( UHRF1 zd ih AI Sae as 10μ bars, Scale DAPI. with ized . i to , scale). The vertical gray shadow indicates the domain region of USP7 of region domain the indicates shadow gray vertical The scale). ii – , AEadCoasebu tiig rti ad o HF and UHRF1 of bands Protein staining. blue Coomassie and PAGE m. ) correspond to magnifications of the areas indicated by the two the by indicated areas the of magnifications to correspond iii) ii )

corresponding peptide sequence ( sequence peptide corresponding E a . 6HIS IP), and 3% of 500μg MBD4 protein mutants in mutants protein MBD4 500μg of 3% and IP), 6HIS . to USP7 cDNA fragment that iinteracts with MBD4 in a Y2H a in MBD4 with iinteracts that fragment cDNA USP7 scale) and the representative scheme of generating the four four the generating of scheme representative the and scale) B ) The schematic representation of the fusion proteinsof fusion the of representation schematic The ) B C

E & ). The cells were fixed 48 hours later and analyzed and later hours 48 fixed were cells The ). ). C

C ) Flow chart of the reciprocal co-IP procedure. ( procedure. co-IP reciprocal the of chart Flow) ) Asynchronously growing mouse CMT93 cells cells CMT93 mouse growing Asynchronously ) A ) UHRF1 and USP7 are identified as new new as identified are USP7 and UHRF1 ) . ( m. B ,o c-rnfce y GFP-MBD4 by co-transfected or ), A C A Dsrbto o GPMD in GFP-MBD4 of Distribution ) ) GFP-MBD4 and FLAG-UHRF1 GFP-MBD4FLAG-UHRF1 and) ) Schematic representation of the of representation Schematic ) ), schematic representation of representation schematic iii), A ), FLAG-UHRF1( ), B ) Immunoprecipitation of Immunoprecipitation ) ) The representative The A) B

& a square on the the on square C D ). Nuclear Nuclear ). B . control) . ), or co- or ), D and 15 no D -

chromocenters in all the cells co-transfected cells the all in chromocenters ( 3. Figure bars, 10μ supplement to represented are examples Additional ( spots. of pattern and GFP-Mlh1 FLAG-MBD4 transfection of distribution nucleus Diffused (i) which 4). grown in & 3 MLH1 were (Figure in cells as analyzed and CMT93 stained transfected, Mouse chromatins. condensed heterochromatic ( with foci. heterochromatic USP7 and MBD4 1. Figure Supplementary of colocalization indicate arrows black while diminishing ordisappearing DAPI bright spots. spots, bright DAPI and USP7 MBD4, of co-localization triple indicate arrows White line. white parallel two the by indicated heterochromatin marked exhibit cells the co- C, cells in CMT93 images the immunostaining all ( right the on insets The remodeling. the in all chromocenters In to transfected. exclusively MCherry-USP7 recruits GFP-MBD4 ( DAPI. with visualized was counterstaining ( MCherry-USP7 and B m. Accepted Article co-transfected. cells the all in chromocenters at UHRF1 colocalizes tightly MBD4 )

evl sand AI pt wr ecue fo te L1 itiuin (ii) distribution. MLH1 the from excluded were spots DAPI stained heavily C A ). The cells were fixed 48 hours later and analyzed directly by IF. Nuclear Nuclear IF. by directly analyzed and later hours 48 fixed were cells The ). ) MBD4 recruits Mlh1 to bright DAPI spots that are tightly associated with associated tightly are that spots DAPI bright to Mlh1 recruits MBD4 )

Co -localization of MBD4 of -localization B ) or below ( below or ) . More cells are represented to supplement Figure 4. Scale Scale 4. Figure supplement to represented are cells More . resulted resulted B itiuino Cer-S7i M9 el. ( cells. CMT93 in MCherry-USP7 of Distribution ) Scale bars,Scale 10μ C . i with Mlh1, UHRF1, and recruitment of USP7 at USP7 of recruitment and UHRF1, Mlh1, with , in the recruitment of GFP-MLH1 to DAPIGFP-MLH1 bright to of in the recruitment ii , ) correspond to magnifications of the areas the of magnifications to correspond iii) m. m. C MD rcut UP to USP7 recruits MBD4 ) snhoosy then asynchronously, Co C 16 ) -

Figure 1 Accepted Article

Figure 2 Accepted Article

Figure 3 Accepted Article

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