Lsh Is Essential for Maintaining Global DNA Methylation Levels in … · 2016-01-26 ·...

Research ArticleLsh Is Essential for Maintaining Global DNA MethylationLevels in Amphibia and Fish and Interacts Directly with Dnmt1

Donncha S Dunican1 Sari Pennings2 and Richard R Meehan1

1MRC Human Genetics Unit MRC IGMM University of Edinburgh Western General Hospital Crewe RoadEdinburgh EH4 2XU UK2Centre for Cardiovascular Science Queenrsquos Medical Research Institute 47 Little France Crescent Edinburgh EH16 4TJ UK

Correspondence should be addressed to Donncha S Dunican donnchadunicanigmmedacuk andRichard R Meehan richardmeehanigmmedacuk

Received 29 May 2015 Revised 28 August 2015 Accepted 3 September 2015

Academic Editor Touati Benoukraf

Copyright copy 2015 Donncha S Dunican et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Eukaryotic genomes are methylated at cytosine bases in the context of CpG dinucleotides a pattern which is maintained throughcell division by the DNA methyltransferase Dnmt1 Dramatic methylation losses are observed in plant and mouse cells lackingLsh (lymphoid specific helicase) predominantly at repetitive sequences and gene promoters However the mechanism by whichLsh contributes to the maintenance of DNA methylation is unknown Here we show that DNA methylation is lost in Lsh depletedfrog and fish embryos both of which exhibit developmental delay Additionally we show that both Lsh and Dnmt1 are associatedwith chromatin and that Lsh knockdown leads to a decreased Dnmt1-chromatin association Coimmunoprecipitation experimentsreveal that Lsh and Dnmt1 are found in the same protein complex and pulldowns show this interaction is direct Our data indicatethat Lsh is usually diffuse in the nucleus but can be recruited to heterochromatin in a HP1120572-dependent mannerThese data together(a) show that the role of Lsh inDNAmethylation is conserved in plants amphibian fish andmice and (b) support amodel in whichLsh contributes to Dnmt1 binding to chromatin explaining how its loss can potentially lead to perturbations in DNA methylationmaintenance

1 Introduction

DNA methylation at the 51015840 cytosine position (5mC) in thecontext of CpG dinucleotides plays a central role in generepression in most eukaryotes and flowering plants 5mCrepresents 1of all nucleotides and 4of all cytosine residuesin mammalian genomes and is required for a wide rangeof biological processes including transcriptional silencing[1] Additionally 5mC is involved in allele-specific genomicimprinting X chromosome inactivation in female cells andsilencing of retrotransposons in germ cells and the soma [2]Moreover embryonic stem (ES) cells lacking DNA methyla-tion are capable of self-renewal but are unable to differentiate[3] Two classes of DNAmethyltransferases have been widelydescribed in animals Dnmt1 (maintenance methyltrans-ferase) and Dnmt3a3b (de novo methyltransferases) [4ndash8]The heritability of the 5mCmark is in part accounted for by

the ability of Dnmt1 to faithfully remethylate DNA daughterstrands during and after replication ensuring appropriatemethylation patterns in future progeny [9] Considerableefforts have led to the conclusion that the role of the de novomethyltransferases is to establish new methylation marksfollowing the postfertilisation wave of demethylation duringearly embryogenesis and germ cell development [10ndash12]

Chromatin structure influences transcriptional stateswithin the mouse genome and can be broadly consideredin two different flavours ldquoactiverdquo euchromatin which isenriched for histone marks associated with transcriptionalactivity (ie H3K4me3H3K27ac) and ldquoinactiverdquo heterochro-matin (ie H3K27me3 H3K9me3) [13ndash16] InterestinglyH3K9me3 acts as a ligand for the chromodomain proteinHP1120572 thus reinforcing silencing of heterochromatin [17]Another tier of chromatin conformation is controlled bynucleosome remodelling complexes including the SWISNF

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 740637 12 pageshttpdxdoiorg1011552015740637

2 BioMed Research International

family [18] The SNF domain has been shown to be anATPase-dependent protein domain capable of shifting nucle-osomes on chromatin templates an effect which can exposeor obscure transcription factor binding sites [19] Lymphoidspecific helicase (Lsh) (also known as Hells PASG andSMARCA6) is a putative member of the SWISNF family[20] in addition to a SNF domain it harbours a helicasemotif which can bendkinkDNAandRNAmolecules [21 22]Taking these two domains together implies that Lsh may beinvolved in chromatin remodellingThis hypothesis has beensupported by the decreased global DNA methylation levelsobserved at repeat elements and some single-copy genes inLsh deficient plants and mice [23ndash25]

Links between Lsh and DNA methylation have beeninvestigated extensively but the mechanism by which inter-ference of Lsh function contributes to global hypomethy-lation remains incomplete [26ndash28] Lsh was shown to bedispensable for the recruitment of Dnmt1 to normal replica-tion foci during late S phase [29] However support for anassociation between Lsh and the maintenance methyltrans-ferase Dnmt1 was suggested by the finding that transgenesilencing mediated via tethered Gal4-Lsh requires Dnmt1in cooperation with histone deacetylases and the de novomethyltransferase Dnmt3b [27] Evidence of a link betweenLsh and heterochromatin structure arose from studies thatdemonstrated that Lsh association with chromatin is lost incells treated with the histone deacetylase inhibitor tricho-statin A (TSA) which results in chromatin having a moreaccessible hyperacetylated signature [29] Additionally lossof Lsh leads to the accumulation of the ldquoactivatingrdquo markH3K4me2 globally [29] while the repressive mark H3K9me3is reduced [26] Lsh knockout mice die perinatally with grossrenal defects or shortly after birth with a spectrum of organdefects and a premature aging phenotype[30 31]

We present evidence that Lsh is essential for the com-pletion of a normal developmental program in amphibianand fish [32] In addition we report that Lsh and Dnmt1 caninteract directly in vivo and in vitro but rarely colocalise atheterochromatic foci in cells however this can be enhancedby the presence of HP1120572 Finally we show that both Lshand Dnmt1 are chromatin bound and that Lsh is requiredto recruit or facilitate the association between Dnmt1 andchromatin Taken together this study demonstrates that LshandDnmt1 are key protein partners and thismay underlie theloss of DNAmethylation and embryonic defects that occur inLsh depleted embryos

2 Materials and Methods

21 Embryos Morpholinos TNT Assay and TUNEL Xeno-pus laevis and zebrafish were maintained using standardprocedures All morpholinos were designed and obtainedfrom GeneTools LLC Xenopus laevis 2-cell embryos weremicroinjected into each blastomere (05ndash10 ng per cell) andallowed to develop Zebrafish stocks were maintained andembryo cultures were as described previously [32] Mor-pholinos were injected (5ndash10 ng per cell) at the 1-cell stage

Morpholino sequences xLMO(51015840-AGCTCTGTCCCACAG-GCATCTTATA-31015840 51015840-TTGGGTCATCATCAGATGGTT-CCAT-31015840) zLMO (51015840-GCTTGCTTTTTTCCATTGTGG-TCTC-31015840) control-MO (51015840-CCTCTTACCTCAGTTACA-ATTTATA-31015840) TNT assays were performed using a full-length cDNA xLsh clone as template and were labelled with35S-methionine Assays were performed in the presence orabsence of 200 nMmorpholino and products were separatedby PAGE TUNEL staining was carried out as described[33] TNT assays for GST-pulldowns were carried out byamplifying T7 tagged mLsh and mDnmt1 by PCR with linkerprimers PCR products were added to the TNT Quick T7for PCR DNA kit and translated in the presence of 35S-methionine Whole mount in situ hybridisation was carriedout as previously described [34]

22 Southern Blotting and DNADot Blotting Genomic DNAwas isolated from embryo batches (sim50ndash100) in SETN buffer1 SDS 1mM EDTA 10mM Tris pH8 and 150mM NaClLysates were RNaseA treated proteinase K treated phenol-extracted and precipitated yielding high-integrity genomicDNA For southern analysis 2ndash4120583g of DNA were digested tocompletion with 10U HpaII orMspI in a reaction volume of100 120583L for two hours at 37∘C followed by a further addition of5U enzyme overnight at 37∘C Southern blots were carriedout using established methods and probes for xSatI [35]and Dana [36] For dot blots DNA was dotted onto PVDF(Bio-Rad) and membranes were baked for 2 hours at 80∘Cunder vacuum and then probed with a monoclonal anti-5-methylcytosine antibody (Eurogentec)

23 Bisulfite Sequencing Genomic DNA (500 ng) was bisul-fite converted using EZ DNA Methylation-Lightning Kit(Zymo Research) PCR primers used were xSatI-Bis1 GTT-AATATTAATTTGAGGTTTAG xSatI-Bis2 GTTTGA-ATAGTTTAGTTGGTAG xSatI-Bis3 AAATACTAAATA-AAAAAACCC xSatI-Bis4 TTCAAACTAATACTAAAC-AAAC PCR products were cloned into pGEM-T Easy(Promega) and sequenced using BigDye 31 sequencingchemistry (Thermo Fisher Scientific) on an ABI Prism 3700DNA Analyzer (Applied Biosystems)

24 Cell Culture Mouse 3T3 and N2a cells and human293T and SW620 cells were grown in DMEM supplementedwith 10 serum and 1 penicillin-streptomycin p53minusminus andp53minusminusDnmt1minusminus mouse embryonic fibroblasts were grownas described [37]

25 GST Pulldowns and Immunoprecipitations GST andGST-fusions were produced in BL21 cells and crudely iso-lated using BugBuster (Novagen) followed by binding toglutathione beads (GE HealthSciences) For pulldowns fromextracts nuclear-enriched fractions were isolated using anestablished method [38] Briefly cells were washed in PBSand lysed using RSB-150 (10mM Tris pH 75 NaCl 150mM25mM MgCl

2 40 120583gmL digitonin and protease inhibitor

tablets (Roche)) on ice for 5 minutes followed by centrifu-gation at 2000 g for 8 minutes at 4∘C The cytoplasmic

BioMed Research International 3

supernatant was discarded and the pellet was resuspendedin RSB-150 supplemented with 05 Triton X-100 passedthrough a 40 120583M needle and harvested as before Nuclearextract supernatants were mixed with GST proteins for 1hour at 4∘C washed in RSB-150 three times resuspendedin Laemmli buffer and separated by PAGE Tagged proteinswere transfected into 293T cells using Lipofectamine-2000(Invitrogen) and nuclear extracts were prepared as aboveExtracts were precleared with protein G (AutoGen Bioclear)and supplemented with antibodies overnight at 4∘C Finallyfresh protein G was added for one hour at 4∘C and complexeswere washed three times with RSB-150 and separated byPAGE

26 Direct Fluorescence p53minusminus MEF cells were grown oncoverslips and transfected with the indicated plasmids usingLipofectamine-2000 (Invitrogen) for 24 hours Cells werewashed twice in PBS permeabilised in 01 Triton X-100PBS and stained in 01mgmL DAPI Coverslips weremounted inVectashield on slides andwere visualised using anAxioplan fluorescence microscope (Carl Zeiss Welwyn UK)fitted with a Chroma 84000 quadruple-band pass filter set(Chroma Technology Rockingham VT) Grayscale imageswere captured with an Orca AG CCD (Hamamatsu Photon-ics Welwyn Garden City Hertfordshire UK)

27 siRNA Sucrose Gradients and Micrococcal NucleaseAssays siRNA duplexes (sequences available on request)against human Lsh were obtained from Ambion (USA)and were introduced into 293T cells using Oligofectamine(Invitrogen) Knockdown efficiency was determined byimmunoblotting using a rabbit polyclonal Lsh antibody (giftfrom Kathrin Muegge) compared to endogenous PCNA(Abcam) levels Soluble chromatin was released from mouse3T3 nuclei using micrococcal nuclease (MNase) overnightand fractionated over 6ndash40 isokinetic sucrose gradients asdescribed [39] Fractions were precipitated using an equalvolume of 20 trichloroacetic acid and protein pellets werewashed in cold acetone and prepared for PAGE in Laemmlibuffer DNA was isolated from gradient fractions by ethanolprecipitation and separated on 1x TPE agarose gels MNaserelease of chromatin associated proteins was essentiallyperformed as described [40] Briefly nuclei were isolatedfrom p53minusminus cells using RSB-150 containing 05 Triton X-100 and 20120583g DNA equivalents of nuclei were equilibratedin 60120583L solution C (300mM sucrose 50mM Tris pH825mM KCl 4mM MgCl

2 and lmM CaCl

2) Aliquots were

either untreated or digested with various unit amounts ofMNase for 15mins at room temperature and reactions werestopped by supplementing with 20mM EDTA on ice for afurther 15mins Released proteins in the supernatant wereisolated by centrifugation at 13000 rpm for 10mins at 4∘Candboth soluble and pellet fractions were processed for proteinisolation The pellet fractions were also processed for DNAisolation to monitor the dynamics of chromatin digestion byMNase

28 Western Blotting Western blotting was carried out usingstandard methods In brief proteins were resolved on 4ndash12

precast gradient gels (Invitrogen) and transferred to PVDF(Bio-Rad) Blots were blocked in 5 marvel milk in PBSsupplemented with 01 Tween 20 and incubated with theappropriate antibody at 4∘C overnight Western blot signalswere detected using alkaline-phosphatase secondary anti-bodies (Bio-Rad) and exposed to film (GEHealthSciences)

3 Results and Discussion

31 Lsh Is Essential for Xenopus laevis and Danio rerioDevelopment Lsh orthologs are highly conserved from yeastto humans and both temporal and spatial analyses show thatxLsh is expressed largely ubiquitously throughout allXenopuslaevis embryonic stages (see S1-S2 in Supplementary Mate-rial available online at httpdxdoiorg1011552015740637)Studies in plants and mice have indicated that interferencewith endogenous Lsh function by gene targeting resultsin partial hypomethylation of the genome [23 24 26]To address whether this finding is conserved in amphibiaand fish we depleted Lsh in Xenopus laevis and Daniorerio embryos by microinjection with antisense morpholinosthat inhibit translation of the target mRNA [41] XenopusLsh (xLsh) morphants (xLMO) appeared normal throughthe midblastula transition (MBT) and neurulation In con-trast at early tailbud stages many xLMO embryos hadan aberrant phenotype in comparison with the controlmorpholino injected siblings (Figure 1(a) left) xLMO mid-tailbud embryos are axis-truncated and hyperventralisedand do not form proper head structures including theeye cement gland and brain structures (Figure 1(a) middlepanel) xLMO tadpole abnormalities are more pronounced(Figure 1(a) right) and by stages 44-45 (tadpole) manymutants have no tail structure and lack eyes mouth andhead structures (Figure 1(b)) Successful microinjection andmorpholino stability are verified by UV detection of themorpholino fluorescein tag (Figure 1(c)) indicating that themorpholino is stable in vivo for over 3 days In the absenceof a suitable antibody against xLsh we demonstrated xLMOknockdown efficacy by in vitro where translation of xLshmRNA was reduced reproducibly by 70 in the presenceof the morpholino (Figure 1(d)) To rule out nonspecificinhibition by xLMO we repeated the same experiment withrecombinant radiolabelled luciferase which was translatedefficiently (third lane in Figure 1(d) and data not shown)Finally we reproduced the similar axis-truncated late-stagephenotype with an xLMO design targeting a different regionof the xLsh mRNA (data not shown) xDnmt1- and xKaiso-depleted embryos both show general patterns of apoptosisthat is hallmark of their respective phenotypes [33 42] Incontrast the xLMOmorphants showed no significant TUNELpositive staining (Supplementary S3)

We also tested Lsh depletion by morpholino (zLMO) inthe model system Danio rerio Embryos were microinjectedand allowed to develop to 24 hours after fertilisation (hpf) Bytitrating the dose of morpholino injected (5ndash10 ngembryo)we observed a developmental phenotype compared to wildtype embryos (Figure 1(e) compare zLMO and control MO)Themorphant phenotype becomesmore pronounced the tailbecomes shorter somite numbers are reduced and head and


zLM

OC

ontro

l MO

zLMO dose

minus + minus xLMO

xLsh

Luciferase

(a)

(b)

(d)

(f)

(h)

(g)

(e)(c)

xSatI southern

xLMO

(kb)

(kb)

HpaII

MspI

minus minus minus +

minus minus + +

+ minus minus minus

1 2 3 4

10

5

1

3

zLMO

HpaII

MspI + minus minus minus

minus minus minus +

minus minus + +

1 2 3 4

Dana southern

10

5

1

3

025

24hpf

WT

zLMO

WT

xLMO

gDNA (ng)

gDNA (ng)

50 100

200

100

200

500

IB120572

-5m

eCIB

120572-5

meC

Tadpole (st 37ndash42)

Figure 1 Continued


WT (tadpole)xLMO (tadpole)

CpG number

100

1 2 3 4 5 6 70

Met

hyla

tion

()

(i)

Figure 1 Lsh is essential for both Xenopus laevis and Danio rerio development (andashc) Xenopus laevis embryos were injected with xLMO orcontrolmorpholinos and allowed to develop Each panel shows examples ofmorphant embryos and a control embryo (black arrows) xLMO isfluorescein labelled and successfully injected embryos can be visualised under UV light (c) Developmental stages are (a) 28 37-38 42 (b) 42ndash45 (c) 42ndash45 Scale bar = 1mm (d) In vitro inhibition of xLsh coupled transcription-translation (TNT) with xLMO 35S-Methionine labelledxLsh protein was prepared by TNT in the presence or absence of xLMO and products separated by PAGE xLsh production was inhibited byxLMO (compare left andmiddle lanes) Band on lower right is TNT luciferase protein (e)Danio rerio embryos were injected with zLMO andallowed to develop to the midsomite stage (24 hpf) Severity of phenotype is dose-dependent (compare panels left to right) UV light showingsuccessful microinjection of three doses of zLMO and severity of phenotype (top panel lateral view) Brightfield view of three doses of zLMO(middle panel lateral view) Two representative brightfield control morpholino injected embryos (lower panel lateral view) Scale bar =300 120583m (f) Southern blot analysis of genomic DNA isolated from control- and xLMO-injected tadpole embryos using a dispersed repeatxSatI probe DNA was digested with either HpaII (methylation-sensitive) or MspI (methylation-insensitive HpaII isoschizomer) resolvedand probed with radiolabelled xSatI Digestion with HpaII indicates that xLMO DNA from tadpoles is more frequently cut as indicated bythe lowmolecular weight banding pattern (black arrows) compared to control-injected genomic DNA (g) Southern blot analysis of genomicDNA isolated from control- and zLMO-injected 24 hpf embryos using a Danio rerio Dana probe A similar approach was taken as in (f)Compare the extent of HpaII digestion in lane 3 (control) and lane 4 (zLMO) Black bracket = wild type HpaII profile dashed red bracket =zLMO HpaII profile DNA sizes are indicated in kilobases to the left of each gel (h) Upper dot blot of Xenopus laevis genomic DNA probedwith 5-methylcytosine antibody Note the weaker binding of antibody to the xLMO DNA indicating global hypomethylation lower dot blotof Danio rerio genomic DNA probed with 5-methylcytosine antibody Note the reduced binding of antibody to the zLMO DNA indicatingglobal hypomethylation (i) Summary of bisulfite sequencing of xSat in wild type and xLMO tadpole embryos Vertical axis methylationhorizontal axis each CpG in xSat amplicon

brain structures are primitively formed or absent in a dose-dependent manner Control morpholino injected embryosare shown in Figure 1(e) bottom panel For more detailedinformation on embryo phenotypes and survival rates seeSupplementary Figures S4-S5

32 DNA Hypomethylation Is Conserved in Lsh DepletedEmbryos Interference with Lsh function in plants and miceleads to a global DNA methylation deficit in embryos andcultured cells [43] Loss of Arabidopsis thaliana repeat-associated DNAmethylation leads to increased rates of retro-transposition while loss of repetitive DNA methylation andsome single-copy genes occurs in Lshminusminus embryos Whetherthis is restricted to plants and mammals is unknown Previ-ously we have shown that cytosine methylation is reducedat an interspersed repeat sequence xSatI in xDnmt1-depletedXenopus embryos [42] Using a similar approach we tested ifDNA hypomethylation occurs at xSatI in xLMO morphantsby comparing the digestion profile of genomic DNA usingHpaII (methyl-sensitive) and MspI (methyl-insensitive) Inneurula staged embryos we detected no detectable changein methylation (data not shown) Upon probing with aradiolabelled xSat probe xLMO tadpole stage (coincidentwith the morphant phenotype) embryonic DNA is sensitive

to HpaII digestion compared to control embryonic DNA(Figure 1(f) compare low molecular weight smear in lanes3 and 4 ethidium gel in Supplementary S6) confirming lossof DNA methylation We note that this loss of methylation ispartial as HpaII does not digest to the same extent asMspI

To extend this analysis to fish we digested control andzLMO genomic DNA isolates from Danio rerio as aboveand probed with a radiolabeled short interspersed repeatelement sequence termed Dana [36 44] The range of themean size HpaII digested zLMO DNA is shifted comparedto the mean size of the control DNA but we did not observethe appearance of the low molecular weight band observedfor MspI digestion (Figure 1(g) compare lanes 3 and 4black bracket (wild type) red bracket (zLMO) ethidiumgel in Supplementary S6) This suggests like Lsh depletionin mouse and Xenopus that loss of DNA methylation inzLMO morphants is partial consistent with an incompleteknockdown To validate the observed restriction digestionDNA hypomethylation results we performed dot-blot anal-ysis using a 5-methylcytosine antibody Using this approachwe can distinguish between control DNA and xLMOzLMODNA which has approximately 50 less methylated DNAsignal compared to the control (Figure 1(h)) Blots werestained with methylene blue to show equal DNA loading


(Supplementary S6 [45]) Taken together these data implythat Lsh is essential for normal development in frogs andfish and that morphant embryos show partial losses in globalDNAmethylation levels Finally we used bisulfite sequencingto examine repeat methylation [34] in xLMO tadpole DNAcompared to wild type DNA showing loss of methylationfrom the xSat interspersed repeat in morphant DNA acrossseven CpG positions (Figure 1(i)) Taken together this sug-gests evolutionary conservation in Lsh function as a regulatorof DNAmethylation between plants fish frogs and rodents

33 Lsh and Dnmt1 Interact In Vivo and In Vitro Dnmt1 isthe major DNA cytosine methyltransferase in mammaliancells and has a prominent role in the faithful preservationof DNA methylation patterns in daughter cells after DNAreplication The most striking Lsh target sequences at whichDNA methylation is lost are repeat elements which areboth templates for the maintenance (Dnmt1) and de novo(Dnmt3a and 3b) methyltransferases in mice To explainthe losses of repeat sequence methylation in Lsh depletedcells we hypothesized that Lsh which lacks an obviousmethyltransferase domain may be a cofactor for Dnmt1 inmaintaining DNA methylation levels at repeat sequence loci(and perhaps genes) and directly participate in their silencing[20]

To test this hypothesis we carried out biochemical assaysto determine if Dnmt1 and Lsh can interact We first madeuse of a panel of GST-mLsh and GST-hDnmt1 fusionsproteins which we expressed purified (Supplementary S6)and used as bait for in vitro radiolabeled translated mLsh andmDnmt (Figure 2(a)) We observed GST pulldown signalsfrom the N-terminal and C-terminal domain mLsh GST-fusions for radiolabelled hDnmt1 (Figure 2(b) upper) Wenote that the N-terminal domain of Lsh contains two coiled-coil domains which are predicted to be protein-proteininteraction domains (PFAM httphmmerjaneliaorg) Sec-ondly the C-terminal domain of Lsh encompasses the heli-case domain which may imply coupling between Lsh andDnmt1 at unwinding chromatin The reciprocal experiment(radiolabeled mLsh and GST-Dnmt1 fusions) showed robustGST pulldown signals for all five hDnmt1 fusions withstrongest signals from GST-hDnmt1 (305ndash609) and GST-hDnmt1 (1000ndash1632) (Figure 2(b) lower) Next we testedwhether Lsh and Dnmt1 can interact in cellular contextsWe coexpressed full-length tagged Dnmt1 and Lsh fusionsin highly transfectable human 293T cells and performedcoimmunoprecipitations Both immunoprecipitated proteinswere capable of interacting with the partner tagged protein(Figure 2(c) right IP lanes) Finally we wanted to testwhether endogenous Lsh and Dnmt1 can interact Unrelatedexperiments showed that the human colorectal cancer cellline SW620 expresses high levels of both proteins (data notshown) and blotting of hDnmt1 immunoprecipitates fromthese cells gave a strong signal using a human Lsh antibody(Figure 2(d)) We also performed these experiments in thehigh salt conditions previously reported [27] and observedthe same interactions (data not shown) Collectively thesebiochemical experiments imply that Lsh and Dnmt1 interact

in vitro and in vivo and that this interaction can occur directlywithout additional nuclear protein partners

34 Bulk Lsh Is Predominantly Nuclear Diffuse Cell biologyapproaches in cultured murine cells suggest that Dnmt1 ispredominantly associated with pericentric heterochromaticnuclear foci at S-phase however this localisation may fluc-tuate during the cell cycle and can be lost in cancer cells[46ndash49] Others have suggested that Lsh protein expressionis essentially nuclear and that this overlaps with Dnmt1and PCNA at replication foci in late S-phase but not ininterphase nuclei [29] To further explore these findingswe took advantage of a p53minusminus MEF cell line [37] which isresistant to overexpression induced cell death to determineLsh localisationWe observed mouse Lsh (cherry red tagged)to be nuclear diffuse in the majority of nuclei and in somecases present at subtle nuclear foci which overlap in part withpericentric heterochromatin (Figure 2(e) compare upperand lower panels) which implies that the majority of Lshprotein is not associated with pericentric heterochromatin inMEFs In addition we tested a T7-tagged xLsh fusion andGFP mLsh in additional mouse cells largely showing diffusenuclear staining in gt90 of cells (Supplementary S7) Wedetected a similar nuclear diffuse pattern with the previouslypublished GFP-tagged mLsh fusion [29] (Figure 2(f) toppanel) In contrast we observed both GFP-xDnmt1 and GFP-hDnmt1 colocalise with DAPI bright pericentric heterochro-matin in up to 50 cells otherwise these Dnmt1 fusionswere nuclear diffuse in the remaining cells (SupplementaryS7) Efforts to recruit exogenous Lsh from diffuse nuclearstaining to heterochromatic foci in the presence of exogenousDnmt1 were unsuccessful in p53minusminus MEFs however weobserve widespread nuclear diffuse colocalisation implyingthat these proteins overlap at nonheterochromatic regions inthe nucleus (Supplementary S7) In summary the bulk of Lshis diffusely stained across nuclei from a variety of cells typeswith a minor fraction localising with heterochromatic foci

35 HP1120572 Can Recruit Lsh to Heterochromatin A role forLsh in regulation of histone methylation and the formationof normal heterochromatin was proposed in experimentswhich demonstrated that H3K4me2 levels were increasedin Lshminusminus cells and this could be recapitulated by treatingcells with 51015840-azacytidine [50] This suggests a pathway whereloss of DNA methylation precedes the gain of activatinghistone marks at normally silent loci in Lshminusminus cells Lshcan colocalise with and precipitate HP1120572 after cross-linkingsuggesting a close (if not direct) association of Lsh with HP1120572on heterochromatic nucleosomes [29] Thus it is possiblethat HP1120572 facilitates Lsh localisation to heterochromatinTo investigate this we explored the localisation of HP1120572together with Lsh in p53minusminus MEFs As expected GFP-HP1120572localises almost exclusively to heterochromatic DAPI brightspots (Figure 2(f) bottom panel) In the presence of HP1120572weobserved a higher proportion of cells (gt30) exhibiting Lshaccumulation at heterochromatin (Figure 2(g)) comparedto expression of Lsh alone (Figure 2(e)) implying that anexogenous pool of active HP1120572 is sufficient to drive Lsh to


GSTmLsh

GSTmDnmt1

MTaseNLS PCNA Zn-finger Bromodomain

Snf2 HelicaseNLSCC

1125ndash1620740ndash1115613ndash740305ndash6091ndash343

560ndash822211ndash5601ndash208

(a)

mLsh (aa)

mDnmt1 (aa)

205

95

(kDa)

GST

GSTInpu

t(1

0)

Inpu

t(1

0)

(kDa)

35S-mDnmt1

35S-mLsh

1125

ndash1620

740

ndash1115

613

ndash740

305

ndash609

1ndash3

43

560

ndash822

211

ndash560

1ndash208

(b)

IgGIn Lsh

Dnmt1IgGIn

205

95

(kDa)

120572Dnmt1

120572Lsh

(c)

Dnmt1IgGIn

95

(kDa)

120572Lsh

(d)

85

15

DAPI mLsh Merge

(e)

DAPI mLsh Merge

(f)

DAPI MergehHP1120572

(g)

DAPImLsh

30

XhHP1120572

(h)

100DAPImLsh +

hHP1120572V21M

(i)

100DAPIxLsh +

hHP1120572A129R

(j)

Figure 2 Lsh and Dnmt1 proteins interact in vitro and in vivo and Lsh is predominantly excluded from pericentric heterochromatin (a)Cartoon of Lsh and Dnmt1 GST-fusions used Individual fusions are indicated by numbering under each protein (b) Direct interactionbetween Lsh and Dnmt1 Top mLsh GST-fusions 1ndash208 and 560ndash822 pulldown radiolabelled full-length mDnmt1 Bottom mDnmt1 GSTpulldown radiolabelled full-length mLsh All assays performed in the presence of 50 120583gmL ethidium bromide (c) Full-length taggedDnmt1 and Lsh can interact in vivo in cultured cells Tagged proteins (GFP-xDnmt1 and T7-xLsh) were transfected into 293T cells andimmunoprecipitated under high salt conditions (250mMNaCl) Both proteins coimmunoprecipitate reciprocally (see IP lanes right of eachpanel) (d) Endogenous immunoprecipitation of human Lsh and Dnmt1 in SW620 cells (e) Lsh is predominantly nuclear diffuse Expressionof tagged (cherry red) mLsh in p53minusminus MEF White arrows indicate less frequent colocalisation with pericentric heterochromatin 119899 = 100(f) Expression of previously published [29] GFP-tagged mLsh is nuclear diffuse in contrast expression of GFP-tagged HP1120572 overlaps withpericentric heterochromatin foci (white arrows) 119899 = 100 (g) Coexpression of Lsh and HP1120572 drives Lsh to heterochromatin 119899 = 80 (h-i)HP1120572mutants (V21M-chromodomain and A129R-chromoshadow domain) do not redirect Lsh to heterochromatin 119899 = 90


heterochromatin Coexpression of HP1120572 mutants with Lsh(HP1120572V21M chromodomain mutant HP1120572A129R chromoshadow domain mutant) abrogates Lsh presence at hete-rochromatic foci implying that wild type HP1120572 is sufficientand necessary to recruit Lsh to heterochromatin (Figures2(h)ndash2(j)) Interestingly as we have found for Lsh HP1 familymembers are known to interact directly with Dnmt1 andmediate its activity [8]

36 Lsh Can Recruit Dnmt1 to Chromatin and Can Repressa Nonmethylated Reporter Gene Previous studies have high-lighted that Lsh is chromatin associated by showing its pres-ence in the detergent insoluble chromatin fraction derivedfrom mouse nuclei [29] To test this orthogonally we exam-ined the coupling of Lsh to chromatin by treating 293Tnuclei with micrococcal nuclease (MNase) and assayingfor the presence of Lsh in the supernatant (soluble andfree) or pellet (insoluble and chromatin bound) [40] Asshown in Figure 3(a) endogenous Lsh is absent from thesupernatants of untreated nuclei in contrast to the high levelspresent in the soluble fraction ofMNase treated nuclei whichdemonstrates that Lsh is tightly coupled to chromatin Asimilar finding was seen for endogenous Dnmt1 using thesame assay (Figure 3(a)) An alternative method of assay-ing for chromatin bound proteins is fractionating solublechromatin by sedimentation across sucrose gradients [39]followed by immunoblotting for the protein of interest Wefractionated mouse 3T3 soluble chromatin across isokinetic6ndash40 sucrose gradients and precipitated the protein fromeach fraction and blotted for endogenous Lsh (sedimen-tation of open and compacted chromatin was confirmedby gel electrophoresis (Figure 3(b))) Three Lsh peaks wereobserved across the gradient (Figure 3(b) lanes 2ndash6 lanes12ndash19 lanes 21ndash25) implying that Lsh exists in mouse cells inboth monomeric (top of gradient open chromatin) and inoligomeric nucleosomal fractions (middle (bulk chromatin)and bottom of gradient (compact chromatin))

Taking the Lsh-chromatin association and LshDnmt1interaction data we tested the hypothesis that Lsh recruitsDnmt1 to chromatin by combining Lsh siRNA knockdownwithMNase dependent Dnmt1-chromatin release [40]Threedifferent siLsh duplexes were transfected into 293T cells(Figure 3(c)) where siLsh3 achieved highest knockdown ofendogenous Lsh levels In non-siRNA treated cells Dnmt1 isreleased after MNase treatment in contrast Dnmt1 is foundin the supernatant of non-MNase treated Lsh knockdownp53minusminus cells (Figure 3(d) compare untreated lanes of both toppanel western blots) Emerin was used as a control proteinwhich is not chromatin bound under the conditions usedDensitometry of the western blots was used to calculatea Dnmt1-emerin ratio which illustrates the shift of Dnmt1from ldquoboundrdquo (no siLsh) to enrichment in the ldquounboundrdquo(siLsh3) fraction These findings suggest the association ofDnmt1 with chromatin can be Lsh dependent

4 Conclusions

A series of investigations have implicated Lsh as a globalDNAmethylation accessory factor alongside other polypeptides

including Dnmt1 Dnmt3a and Dnmt3b [27 28] This rolefor Lsh was initiated by experiments in DDM1minusminus plants(DDM1 is the Arabidopsis Lsh orthologue) showing globalhypomethylation in these mutants at repeat sequences [24]This hypothesis was supported when Lsh was knocked out inmice (by two similar strategies) [30 31] leading to postnatallethality with concomitant losses in DNA methylation inrepeat sequences and more recently at the HoxA gene cluster[51] This wholesale hypomethylator phenotype in mice wasexplained byZhu and colleagueswith the finding that Lsh andthe de novo methyltransferases (Dnmt3a and Dnmt3b) caninteract and contribute to the silencing of an episomal trans-gene independent of DNA replication [28] We contribute tothe current view of Lsh function by reporting that (a) Lsh isessential for frog and fish embryonic development (b) Lshand Dnmt1 can associate in vivo and interact directly in vitro(c) Lsh recruitment to heterochromatin can be augmentedby HP1120572 (d) the association of Dnmt1 with chromatin ismediated by Lsh

Interestingly the phenotype of frog and fishmorphants isrelatively late-onset (subsequent to themidblastula transition(MBT) and in most cases after neurulation) which is in con-trast to phenotypes associated with knockdown experimentsof other proteins linked toDNAmethylation such as xDnmt1xKaiso and xMBD3 [33 34 52] One possibility is thatabundant stores of maternal xLsh protein are not depleted byxLMOuntil later developmental stage (ie neurula onwards)An alternative is that Lsh is not essential in early Xenopusembryonic genomic silencing Moreover we do not see anychanges in global DNAmethylation until long after the MBTat the tailbud and tadpole stages The phenotypic effect ofLsh depletion in frogs and zebrafish is not associated withloss of any particular germ layer or organ which dovetailswith the range of phenotypes observed in DDM1minusminus andantisense MET1 plants [53 54] Similar to what we haveestablished for frogs and fish in relation to the mouse Lshphenotype early development is relatively normal afterwhichmice die either perinatally [30] or a few weeks after birth [31]These studies report that although embryonic developmentis overall normal knockout embryos fail after birth due toa range of defects including renal dysfunction respiratoryproblems (lung defects) growth retardation and an agingphenotype

In terms of DNA methylation in frog embryo mor-phants we observed losses at the high-copy interspersedrepeat sequence xSatI We previously demonstrated that thisrepeat is heavily methylated in all developmental stagesbut that this CpG methylation is lost in severely xDnmt1-depleted genomic DNA [42] The kinetics and extent ofxSatI hypomethylation between Lsh and Dnmt1 morphantsare different with partial losses of methylation observedin Lsh tadpole morphants (compared to complete loss atMBT for in Dnmt1 antisense RNA injected mutants) Itis possible that Lsh is not involved in maintaining DNAmethylation at this repeat in early development but has amore prominent role at late stages Dnmt1 is highly abundantin early Xenopus development and may be sufficient tomediate early repression [34] but as development proceeds


MNase (U)

Unt

reat

ed

Sup

95

205

32

(kDa)120572Lsh

120572Dnmt1

120572Emerin

(a)

6 40

10

5

1

10

30

5

25 25

Genomic DNA gel

Fraction1 25

(kb)(kb) Openchromatin

Origin

Compactchromatin

120572Lsh

6 40

Core histones

Input(i) (ii) (iii)Fraction 2 6 12 19 21 25

95(kDa)

13ndash17

(b)

95

30

(kDa)siLsh

1

siLsh

2

siLsh

3

siLsh

1+2+3

No

siRN

A

120572Lsh

120572Pcna

(c)

siLsh3 minus minus minus minusminus + + + ++

Sup

MNase (U) MNase (U)

MNase (U)MNase (U)

2

1

2

1

Unbound Bound Unbound

Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Bound

20532

(kDa)120572Dnmt1

120572Emerin

Dnm

t1 e

mer

in

Dnm

t1 e

mer

in

(d)

Figure 3 Lsh is associated with chromatin and is required for Dnmt1-chromatin association (a) MNase treatment of 293T nuclei indicatesthat endogenous Lsh andDnmt1 are chromatin bound (see untreated lanes) (b) Endogenous Lsh is associatedwith soluble chromatin Sucrosegradient sedimentation was used to fractionate 3T3 soluble chromatin and both protein and genomic DNA were isolated from each fractionFractionation of chromatin was validated by DNA gel electrophoresis of all gradient fractions Western blotting of fractions shows that mLsh(free) is enriched at the top of the gradient (open chromatin) and also cosediments with bulk chromatin (chromatin bound) in themiddle andend of the gradient (compact chromatin) (c) siRNAs against human Lsh were tested in knockdown experiments in 293T cells and siLsh3gives sim70 knockdown (d) Lsh is required for the Dnmt1-chromatin association Comparison of wild type and siRNA treated 293T cellsby MNase treatment of nuclei shows that Dnmt1chromatin association is decreased in knockdown cells (comparison of amounts of Dnmt1released into the supernatant showhigher levels released in knockdown cells) Densitometry of thewestern blots shows thatDnmt1 is enrichedin the chromatin bound fraction (left panel) knockdown of Lsh shifts Dnmt1 into the unbound fraction Emerin was used as a control for aprotein which is unaffected by MNase treatment

its levels are titrated out after multiple cell divisions perhapspermitting Lsh to have a more prominent role in specifyingrepression at discrete loci

Here we show a novel direct in vivo interaction betweenLsh and Dnmt1 Existing data has implied that Lsh interacts

predominantly with the de novo methyltransferases Dnmt3aandDnmt3b inMEFs while this interaction occurs bymeansof HDAC1 andHDAC2 in transformed cancer cells (HCT116)[27] Similar to work from Yan et al [29] we propose thatLsh and Dnmt1 colocalisation in somatic cells is a rare event


(lt15) Although this interaction is rare it is likely to bephysiologically relevant as our in vitro experiments showa direct interaction between Lsh and Dnmt1 biochemicallyunder physiological salt (sim150mM) conditions and the morestringent conditions (400mM) employed previously by [27]implying that the interaction is robust even in the presenceof ethidium bromide (an inhibitor of DNAprotein interac-tions) Furthermore we are able to show immunoprecipita-tion between Lsh andDnmt1 in SW620 colorectal cancer cellsindicating the proteins are partners in vivoThis demonstratesfor the first time that while Lsh and Dnmt1 can associatethe in vivo protein association may be transient and or cell-cycle regulated It is a possibility that Lsh cooperates withde novo methylation activities in early embryonic cells [2855] and that the Lsh and Dnmt1 association is crucial fordifferentiated and fate-determined soma [27] FurthermoreXenopus Dnmt3 may not be a de novomethylation candidatepartner for Lsh as sequence database searches revealed onlyone Dnmt3 orthologue in the Xenopus tropicalis genome thatis most similar to murine Dmnt3a2 a truncated form ofDnmt3a lacking the N-terminal 219 amino acids involved inthe repression of euchromatic loci [56]The same homologueis the only Dnmt3-like protein present in the Xenopus laevisEST database Expression analysis of the Xenopus laevistranscript indicates that it is only present in later stagesof development (Supplementary S8) which argues againstXenopus Lsh and Dnmt3a2 having a role in maintainingglobal DNA methylation during early embryogenesis

Nuclear protein localization studies give useful indica-tions of protein function This is further assisted by theclear staining of blocks of silent pericentric heterochromatinby DAPI (410158406-diamidino-2-phenylindole) in murine cellswhich is composed of tandem repeats of satellite sequencesInvolvement of Lsh in heterochromatin structure has beenreported in mouse Lshminusminus cells which accumulate the acti-vating H3K4me2 mark and by its localisation to DAPI brightspots In unsynchronised somatic cells (MEFs3T3N2a) werarely (lt15) observe Lsh that is coincident with pericentricheterochromatic foci Replication of the mammalian genomeis organised into early mid and late replicating loci withregions containing high gene density early interspersedrepeats later and condensed heterochromatin at the lateststages of S-phase Diffuse Lsh staining in gt85 of cells maybe indicative of localisation at euchromatic gene regions andinterspersed repeat sequences We propose a model whereLsh can cooperate with Dnmt1 at condensed pericentricheterochromatin during late S-phase but these protein part-ners may also have a role in repressing gene expression(ie Hox genes [51]) and nonheterochromatic interspersedrepeat elements and this is facilitated by HP1120572 (see model inFigure 4)

Evidence for a model where Lsh can recruit Dnmt1 tochromatin is strengthened by ourMNase release assays whichhave also been used to demonstrate the association betweenMeCP2 and chromatin [40] We show that both Dnmt1 andLsh are tightly coupled to chromatin in human 293T cellsUsing an siRNA strategy to deplete endogenous Lsh we showthat the Dnmt1-chromatin association requires normal levelsof Lsh These data are consistent with the idea that Lsh can

H3K9trime

HP1

LshDnmt1

MeCpGCpG

Methylated and ldquosilentrdquo

(a)

MeCpGCpG

H3K9trime

HP1

LshDnmt1

H3K4dime H3K4dime

Methylation losses and permissive

(b)

Figure 4 Model for Lsh and Dnmt1 cooperation in silencing(a) Model for LshDnmt1 mediated repression In wild type cellsthe H3K9trime mark acts as a ligand in HP1120572 recruitment tosilent regions of the genome Taking together our data and that ofothers both Dnmt1 and Lsh can be associated with HP1120572 (perhapsrequiring HDACs 1 and 2) thereby allowing the parallel dockingof DNA methyltransferase and chromatin remodelling activitiesto silent loci (b) In Lsh depleted cells (and knockout plants andanimals) targeting of Dnmt1 is diminished leading to reduced DNAmethylation maintenance and partial genomic hypomethylationThe accumulation of the activating H3K4me2 mark in Lshminusminus cellsmay be a downstream effect of DNA hypomethylation

recruit and modify local nucleosome positioning or act asa cofactor for Dnmt1 binding to chromatin which wouldexplain the hypomethylation phenotype in Lsh mutantsInterestingly van Heeringen and colleagues [57] have shownthat specific nonmethylated Xenopus tropicalis sequences aregenetically instructive for H3K27me3 deposition a findingwhich supports the opposing paradigm that heterochromatinis epigenetically regulated through recruitment of Dnmt1 tothese repetitive genomic regions Moreover the action ofHDACs may be critical for this process as Lsh-mediatedrepression of a reporter is alleviated in part by treatmentwith TSA (data not shown) and the observation that Dnmt1and Lsh may signal through HDACs [27] (see model in


Figure 4) To definitively test these possibilities sequentialChIP-Seq with antisera against Lsh and Dnmt1 (and Lsh andDnmt3a3b)will reveal the genetic targets of these complexes

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Hazel Cruickshanks and members of theChromosomes and Gene Expression Section at the HGUMRC IGMM for helpful comments and corrections duringpaper preparation and Nick Hastie for advice and generalsupport They thank Alexey Ruzov for assistance in Xenopusmicroinjections This study was supported by an MRC grantto Richard R Meehan (MC PC U127574433) Sari Penningsacknowledges BBSRC funding They thank Nick Gilbert forongoing technical discussions and assistance with sucrosegradient sedimentation experiments They also thank thefollowing for plasmid reagents GSThDnmt1 (Sara Nakielny)GSTmDnmt1 (Francois Fuks) FlaghHP1120572 (Frank RauscherIII) GFP-mLsh (Kathrin Muegge) GFPhDnmt1 (WilliamNelson)

References

[1] M G Goll and T H Bestor ldquoEukaryotic cytosine methyltrans-ferasesrdquo Annual Review of Biochemistry vol 74 pp 481ndash5142005

[2] WReik ldquoStability and flexibility of epigenetic gene regulation inmammalian developmentrdquo Nature vol 447 no 7143 pp 425ndash432 2007

[3] A Tsumura T Hayakawa Y Kumaki et al ldquoMaintenanceof self-renewal ability of mouse embryonic stem cells inthe absence of DNA methyltransferases Dnmt1 Dnmt3a andDnmt3brdquo Genes to Cells vol 11 no 7 pp 805ndash814 2006

[4] S K T Ooi and T H Bestor ldquoCytosine methylation remainingfaithfulrdquo Current Biology vol 18 no 4 pp R174ndashR176 2008

[5] S K TOoi andTH Bestor ldquoThe colorful history of activeDNAdemethylationrdquo Cell vol 133 no 7 pp 1145ndash1148 2008

[6] P-O EsteveHGChinA Smallwood et al ldquoDirect interactionbetween DNMT1 and G9a coordinates DNA and histonemethylation during replicationrdquo Genes and Development vol20 no 22 pp 3089ndash3103 2006

[7] J Sharif M Muto S-I Takebayashi et al ldquoThe SRA proteinNp95 mediates epigenetic inheritance by recruiting Dnmt1 tomethylated DNArdquo Nature vol 450 no 7171 pp 908ndash912 2007

[8] A Smallwood P-O Esteve S Pradhan and M Carey ldquoFunc-tional cooperation between HP1 and DNMT1 mediates genesilencingrdquoGenes and Development vol 21 no 10 pp 1169ndash11782007

[9] G Liang M F Chan Y Tomigahara et al ldquoCooperativitybetween DNAmethyltransferases in the maintenance methyla-tion of repetitive elementsrdquoMolecular and Cellular Biology vol22 no 2 pp 480ndash491 2002

[10] Y Kato M Kaneda K Hata et al ldquoRole of the Dnmt3 familyin de novo methylation of imprinted and repetitive sequences

during male germ cell development in the mouserdquo HumanMolecular Genetics vol 16 no 19 pp 2272ndash2280 2007

[11] H D Morgan F Santos K Green W Dean and W ReikldquoEpigenetic reprogramming in mammalsrdquo Human MolecularGenetics vol 14 no 1 pp R47ndashR58 2005

[12] M Okano D W Bell D A Haber and E Li ldquoDNA methyl-transferases Dnmt3a and Dnmt3b are essential for de novomethylation and mammalian developmentrdquo Cell vol 99 no 3pp 247ndash257 1999

[13] S Khorasanizadeh ldquoThe nucleosome from genomic organiza-tion to genomic regulationrdquo Cell vol 116 no 2 pp 259ndash2722004

[14] A J Ruthenburg H Li D J Patel and C David AllisldquoMultivalent engagement of chromatin modifications by linkedbinding modulesrdquo Nature Reviews Molecular Cell Biology vol8 no 12 pp 983ndash994 2007

[15] S L Schreiber and B E Bernstein ldquoSignaling network modelof chromatinrdquo Cell vol 111 no 6 pp 771ndash778 2002

[16] G G Wang C D Allis and P Chi ldquoChromatin remodelingand cancer part I covalent histone modificationsrdquo Trends inMolecular Medicine vol 13 no 9 pp 363ndash372 2007

[17] R R Meehan C-F Kao and S Pennings ldquoHP1 binding tonative chromatin in vitro is determined by the hinge region andnot by the chromodomainrdquo The EMBO Journal vol 22 no 12pp 3164ndash3174 2003

[18] C S Kwon andDWagner ldquoUnwinding chromatin for develop-ment and growth a few genes at a timerdquo Trends in Genetics vol23 no 8 pp 403ndash412 2007

[19] P B Becker and W Horz ldquoAtp-dependent nucleosome remod-elingrdquoAnnual Review of Biochemistry vol 71 pp 247ndash273 2002

[20] R R Meehan S Pennings and I Stancheva ldquoLashings ofDNA methylation forkfuls of chromatin remodelingrdquo Genesand Development vol 15 no 24 pp 3231ndash3236 2001

[21] C D Jarvis T GeimanM P Vila-Storm et al ldquoA novel putativehelicase produced in early murine lymphocytesrdquoGene vol 169no 2 pp 203ndash207 1996

[22] T M Geiman S K Durum and K Muegge ldquoCharacterizationof gene expression genomic structure and chromosomal local-ization of Hells (Lsh)rdquo Genomics vol 54 no 3 pp 477ndash4831998

[23] K Dennis T Fan T Geiman Q Yan and K Muegge ldquoLsha member of the SNF2 family is required for genome-widemethylationrdquo Genes and Development vol 15 no 22 pp 2940ndash2944 2001

[24] A Vongs T Kakutani R A Martienssen and E J RichardsldquoArabidopsis thaliana DNA methylation mutantsrdquo Science vol260 no 5116 pp 1926ndash1928 1993

[25] W Yu C McIntosh R Lister et al ldquoGenome-wide DNAmethylation patterns in LSH mutant reveals de-repression ofrepeat elements and redundant epigenetic silencing pathwaysrdquoGenome Research vol 24 no 10 pp 1613ndash1623 2014

[26] D S Dunican H A Cruickshanks M Suzuki et al ldquoLshregulates LTR retrotransposon repression independently ofDnmt3b functionrdquo Genome Biology vol 14 article R146 2013

[27] K Myant and I Stancheva ldquoLSH cooperates with DNAmethyl-transferases to repress transcriptionrdquo Molecular and CellularBiology vol 28 no 1 pp 215ndash226 2008

[28] H Zhu T M Geiman S Xi et al ldquoLsh is involved in de novomethylation ofDNArdquoTheEMBO Journal vol 25 no 2 pp 335ndash345 2006


[29] Q Yan E Cho S Lockett and K Muegge ldquoAssociation ofLsh a regulator of DNA methylation with pericentromericheterochromatin is dependent on intact heterochromatinrdquoMolecular and Cellular Biology vol 23 no 23 pp 8416ndash84282003

[30] TM Geiman L Tessarollo M R Anver J B Kopp J MWardand K Muegge ldquoLsh a SNF2 family member is required fornormal murine developmentrdquo Biochimica et Biophysica Actavol 1526 no 2 pp 211ndash220 2001

[31] L-Q Sun D W Lee Q Zhang et al ldquoGrowth retardation andpremature aging phenotypes in mice with disruption of theSNF2-like gene PASGrdquo Genes and Development vol 18 no 9pp 1035ndash1046 2004

[32] A Ruzov E Savitskaya J AHackett et al ldquoThenon-methylatedDNA-binding function of Kaiso is not required in earlyXenopuslaevis developmentrdquo Development vol 136 no 5 pp 729ndash7382009

[33] A Ruzov D S Dunican A Prokhortchouk et al ldquoKaiso isa genome-wide repressor of transcription that is essential foramphibian developmentrdquo Development vol 131 no 24 pp6185ndash6194 2004

[34] D S Dunican A Ruzov J A Hackett and R R MeehanldquoxDnmt1 regulates transcriptional silencing in pre-MBT Xeno-pus embryos independently of its catalytic functionrdquo Develop-ment vol 135 no 7 pp 1295ndash1302 2008

[35] H Lei S P Oh M Okano et al ldquoDe novo DNA cytosinemethyltransferase activities in mouse embryonic stem cellsrdquoDevelopment vol 122 no 10 pp 3195ndash3205 1996

[36] D Macleod V H Clark and A Bird ldquoAbsence of genome-wide changes in DNA methylation during development of thezebrafishrdquo Nature Genetics vol 23 no 2 pp 139ndash140 1999

[37] L Lande-Diner J Zhang I Ben-Porath et al ldquoRole of DNAmethylation in stable gene repressionrdquo Journal of BiologicalChemistry vol 282 no 16 pp 12194ndash12200 2007

[38] S Pinol-Roma Y D Choi M J Matunis and G DreyfussldquoImmunopurification of heterogeneous nuclear ribonucleopro-tein particles reveals an assortment of RNA-binding proteinsrdquoGenes amp Development vol 2 no 2 pp 215ndash227 1988

[39] NGilbert S BoyleH Fiegler KWoodfineN P Carter andWA Bickmore ldquoChromatin architecture of the human genomegene-rich domains are enriched in open chromatin fibersrdquo Cellvol 118 no 5 pp 555ndash566 2004

[40] R R Meehan J D Lewis and A P Bird ldquoCharacterizationof MeCP2 a vertebrate DNA binding protein with affinity formethylated DNArdquo Nucleic Acids Research vol 20 no 19 pp5085ndash5092 1992

[41] L J N Brent and P Drapeau ldquoTargeted ldquoknockdownrdquo ofchannel expression in vivo with an antisense morpholinooligonucleotiderdquoNeuroscience vol 114 no 2 pp 275ndash278 2002

[42] I Stancheva C Hensey and R R Meehan ldquoLoss of themaintenance methyltransferase xDnmt1 induces apoptosis inXenopus embryosrdquoThe EMBO Journal vol 20 no 8 pp 1963ndash1973 2001

[43] K Muegge ldquoLsh a guardian of heterochromatin at repeatelementsrdquo Biochemistry and Cell Biology vol 83 no 4 pp 548ndash554 2005

[44] Z Izsvak Z Ivics D Garcia-Estefania S C Fahrenkrug andP B Hackett ldquoDANA elements a family of composite tRNA-derived short interspersedDNAelements associatedwithmuta-tional activities in zebrafish (Danio rerio)rdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 93 no 3 pp 1077ndash1081 1996

[45] M A Pereira W Wang P M Kramer and L Tao ldquoDNAhypomethylation induced by non-genotoxic carcinogens inmouse and rat colonrdquo Cancer Letters vol 212 no 2 pp 145ndash1512004

[46] A T Agoston P Argani A M De Marzo J L Hicks andW G Nelson ldquoRetinoblastoma pathway dysregulation causesDNA methyltransferase 1 overexpression in cancer via MAD2-mediated inhibition of the anaphase-promoting complexrdquo TheAmerican Journal of Pathology vol 170 no 5 pp 1585ndash15932007

[47] H P Easwaran L Schermelleh H Leonhardt and M C Car-doso ldquoReplication-independent chromatin loading of Dnmt1duringG2 andMphasesrdquo EMBOReports vol 5 no 12 pp 1181ndash1186 2004

[48] J B Margot M Cristina Cardoso and H Leonhardt ldquoMam-malian DNA methyltransferases show different subnucleardistributionsrdquo Journal of Cellular Biochemistry vol 83 no 3 pp373ndash379 2001

[49] L Schermelleh A Haemmer F Spada et al ldquoDynamics ofDnmt1 interaction with the replication machinery and its rolein postreplicative maintenance of DNA methylationrdquo NucleicAcids Research vol 35 no 13 pp 4301ndash4312 2007

[50] Q Yan J Huang T Fan H Zhu and K Muegge ldquoLsh amodulator of CpG methylation is crucial for normal histonemethylationrdquoThe EMBO Journal vol 22 no 19 pp 5154ndash51622003

[51] S Xi H Zhu H Xu A Schmidtmann T M Geiman and KMuegge ldquoLsh controlsHox gene silencing during developmentrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 104 no 36 pp 14366ndash14371 2007

[52] H Iwano M Nakamura and S Tajima ldquoXenopus MBD3 playsa crucial role in an early stage of developmentrdquo DevelopmentalBiology vol 268 no 2 pp 416ndash428 2004

[53] E J Finnegan W J Peacock and E S Dennis ldquoReduced DNAmethylation in Arabidopsis thaliana results in abnormal plantdevelopmentrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 93 no 16 pp 8449ndash84541996

[54] T Kakutani J A Jeddeloh S K Flowers K Munakata andE J Richards ldquoDevelopmental abnormalities and epimutationsassociated with DNA hypomethylation mutationsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 22 pp 12406ndash12411 1996

[55] J Ren V Briones S Barbour et al ldquoThe ATP binding site of thechromatin remodeling homolog Lsh is required for nucleosomedensity and de novo DNA methylation at repeat sequencesrdquoNucleic Acids Research vol 43 no 3 pp 1444ndash1455 2015

[56] T Chen Y Ueda S Xie and E Li ldquoA novel Dnmt3aisoform produced from an alternative promoter localizes toeuchromatin and its expression correlates with Active de novomethylationrdquo Journal of Biological Chemistry vol 277 no 41pp 38746ndash38754 2002

[57] S J van Heeringen R C Akkers I van Kruijsbergen etal ldquoPrinciples of nucleation of H3k27 methylation duringembryonic developmentrdquo Genome Research vol 24 no 3 pp401ndash410 2014


family [18] The SNF domain has been shown to be anATPase-dependent protein domain capable of shifting nucle-osomes on chromatin templates an effect which can exposeor obscure transcription factor binding sites [19] Lymphoidspecific helicase (Lsh) (also known as Hells PASG andSMARCA6) is a putative member of the SWISNF family[20] in addition to a SNF domain it harbours a helicasemotif which can bendkinkDNAandRNAmolecules [21 22]Taking these two domains together implies that Lsh may beinvolved in chromatin remodellingThis hypothesis has beensupported by the decreased global DNA methylation levelsobserved at repeat elements and some single-copy genes inLsh deficient plants and mice [23ndash25]

Links between Lsh and DNA methylation have beeninvestigated extensively but the mechanism by which inter-ference of Lsh function contributes to global hypomethy-lation remains incomplete [26ndash28] Lsh was shown to bedispensable for the recruitment of Dnmt1 to normal replica-tion foci during late S phase [29] However support for anassociation between Lsh and the maintenance methyltrans-ferase Dnmt1 was suggested by the finding that transgenesilencing mediated via tethered Gal4-Lsh requires Dnmt1in cooperation with histone deacetylases and the de novomethyltransferase Dnmt3b [27] Evidence of a link betweenLsh and heterochromatin structure arose from studies thatdemonstrated that Lsh association with chromatin is lost incells treated with the histone deacetylase inhibitor tricho-statin A (TSA) which results in chromatin having a moreaccessible hyperacetylated signature [29] Additionally lossof Lsh leads to the accumulation of the ldquoactivatingrdquo markH3K4me2 globally [29] while the repressive mark H3K9me3is reduced [26] Lsh knockout mice die perinatally with grossrenal defects or shortly after birth with a spectrum of organdefects and a premature aging phenotype[30 31]

We present evidence that Lsh is essential for the com-pletion of a normal developmental program in amphibianand fish [32] In addition we report that Lsh and Dnmt1 caninteract directly in vivo and in vitro but rarely colocalise atheterochromatic foci in cells however this can be enhancedby the presence of HP1120572 Finally we show that both Lshand Dnmt1 are chromatin bound and that Lsh is requiredto recruit or facilitate the association between Dnmt1 andchromatin Taken together this study demonstrates that LshandDnmt1 are key protein partners and thismay underlie theloss of DNAmethylation and embryonic defects that occur inLsh depleted embryos

2 Materials and Methods

21 Embryos Morpholinos TNT Assay and TUNEL Xeno-pus laevis and zebrafish were maintained using standardprocedures All morpholinos were designed and obtainedfrom GeneTools LLC Xenopus laevis 2-cell embryos weremicroinjected into each blastomere (05ndash10 ng per cell) andallowed to develop Zebrafish stocks were maintained andembryo cultures were as described previously [32] Mor-pholinos were injected (5ndash10 ng per cell) at the 1-cell stage

Morpholino sequences xLMO(51015840-AGCTCTGTCCCACAG-GCATCTTATA-31015840 51015840-TTGGGTCATCATCAGATGGTT-CCAT-31015840) zLMO (51015840-GCTTGCTTTTTTCCATTGTGG-TCTC-31015840) control-MO (51015840-CCTCTTACCTCAGTTACA-ATTTATA-31015840) TNT assays were performed using a full-length cDNA xLsh clone as template and were labelled with35S-methionine Assays were performed in the presence orabsence of 200 nMmorpholino and products were separatedby PAGE TUNEL staining was carried out as described[33] TNT assays for GST-pulldowns were carried out byamplifying T7 tagged mLsh and mDnmt1 by PCR with linkerprimers PCR products were added to the TNT Quick T7for PCR DNA kit and translated in the presence of 35S-methionine Whole mount in situ hybridisation was carriedout as previously described [34]

22 Southern Blotting and DNADot Blotting Genomic DNAwas isolated from embryo batches (sim50ndash100) in SETN buffer1 SDS 1mM EDTA 10mM Tris pH8 and 150mM NaClLysates were RNaseA treated proteinase K treated phenol-extracted and precipitated yielding high-integrity genomicDNA For southern analysis 2ndash4120583g of DNA were digested tocompletion with 10U HpaII orMspI in a reaction volume of100 120583L for two hours at 37∘C followed by a further addition of5U enzyme overnight at 37∘C Southern blots were carriedout using established methods and probes for xSatI [35]and Dana [36] For dot blots DNA was dotted onto PVDF(Bio-Rad) and membranes were baked for 2 hours at 80∘Cunder vacuum and then probed with a monoclonal anti-5-methylcytosine antibody (Eurogentec)

23 Bisulfite Sequencing Genomic DNA (500 ng) was bisul-fite converted using EZ DNA Methylation-Lightning Kit(Zymo Research) PCR primers used were xSatI-Bis1 GTT-AATATTAATTTGAGGTTTAG xSatI-Bis2 GTTTGA-ATAGTTTAGTTGGTAG xSatI-Bis3 AAATACTAAATA-AAAAAACCC xSatI-Bis4 TTCAAACTAATACTAAAC-AAAC PCR products were cloned into pGEM-T Easy(Promega) and sequenced using BigDye 31 sequencingchemistry (Thermo Fisher Scientific) on an ABI Prism 3700DNA Analyzer (Applied Biosystems)

24 Cell Culture Mouse 3T3 and N2a cells and human293T and SW620 cells were grown in DMEM supplementedwith 10 serum and 1 penicillin-streptomycin p53minusminus andp53minusminusDnmt1minusminus mouse embryonic fibroblasts were grownas described [37]

25 GST Pulldowns and Immunoprecipitations GST andGST-fusions were produced in BL21 cells and crudely iso-lated using BugBuster (Novagen) followed by binding toglutathione beads (GE HealthSciences) For pulldowns fromextracts nuclear-enriched fractions were isolated using anestablished method [38] Briefly cells were washed in PBSand lysed using RSB-150 (10mM Tris pH 75 NaCl 150mM25mM MgCl

2 40 120583gmL digitonin and protease inhibitor

tablets (Roche)) on ice for 5 minutes followed by centrifu-gation at 2000 g for 8 minutes at 4∘C The cytoplasmic





2 and lmM CaCl

2) Aliquots were








zLM

OC

ontro

l MO

zLMO dose

minus + minus xLMO

xLsh

Luciferase

(a)

(b)

(d)

(f)

(h)

(g)

(e)(c)

xSatI southern

xLMO

(kb)

(kb)

HpaII

MspI

minus minus minus +

minus minus + +

+ minus minus minus

1 2 3 4

10

5

1

3

zLMO

HpaII


minus minus minus +

minus minus + +

1 2 3 4

Dana southern

10

5

1

3

025

24hpf

WT

zLMO

WT

xLMO

gDNA (ng)

gDNA (ng)

50 100

200

100

200

500

IB120572

-5m

eCIB

120572-5

meC


Figure 1 Continued



CpG number

100

1 2 3 4 5 6 70

Met

hyla

tion

()

(i)














GSTmLsh

GSTmDnmt1


Snf2 HelicaseNLSCC



(a)

mLsh (aa)

mDnmt1 (aa)

205

95

(kDa)

GST

GSTInpu

t(1

0)

Inpu

t(1

0)

(kDa)

35S-mDnmt1

35S-mLsh

1125

ndash1620

740

ndash1115

613

ndash740

305

ndash609

1ndash3

43

560

ndash822

211

ndash560

1ndash208

(b)

IgGIn Lsh

Dnmt1IgGIn

205

95

(kDa)

120572Dnmt1

120572Lsh

(c)

Dnmt1IgGIn

95

(kDa)

120572Lsh

(d)

85

15

DAPI mLsh Merge

(e)

DAPI mLsh Merge

(f)


(g)

DAPImLsh

30

XhHP1120572

(h)

100DAPImLsh +

hHP1120572V21M

(i)

100DAPIxLsh +

hHP1120572A129R

(j)






4 Conclusions






MNase (U)

Unt

reat

ed

Sup

95

205

32

(kDa)120572Lsh

120572Dnmt1

120572Emerin

(a)

6 40

10

5

1

10

30

5

25 25

Genomic DNA gel

Fraction1 25


Origin

Compactchromatin

120572Lsh

6 40

Core histones


95(kDa)

13ndash17

(b)

95

30

(kDa)siLsh

1

siLsh

2

siLsh

3

siLsh

1+2+3

No

siRN

A

120572Lsh

120572Pcna

(c)


Sup

MNase (U) MNase (U)

MNase (U)MNase (U)

2

1

2

1


Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Bound

20532

(kDa)120572Dnmt1

120572Emerin

Dnm

t1 e

mer

in

Dnm

t1 e

mer

in

(d)









H3K9trime

HP1

LshDnmt1

MeCpGCpG


(a)

MeCpGCpG

H3K9trime

HP1

LshDnmt1

H3K4dime H3K4dime


(b)







Acknowledgments


References
































































2 and lmM CaCl

2) Aliquots were








zLM

OC

ontro

l MO

zLMO dose

minus + minus xLMO

xLsh

Luciferase

(a)

(b)

(d)

(f)

(h)

(g)

(e)(c)

xSatI southern

xLMO

(kb)

(kb)

HpaII

MspI

minus minus minus +

minus minus + +

+ minus minus minus

1 2 3 4

10

5

1

3

zLMO

HpaII


minus minus minus +

minus minus + +

1 2 3 4

Dana southern

10

5

1

3

025

24hpf

WT

zLMO

WT

xLMO

gDNA (ng)

gDNA (ng)

50 100

200

100

200

500

IB120572

-5m

eCIB

120572-5

meC


Figure 1 Continued



CpG number

100

1 2 3 4 5 6 70

Met

hyla

tion

()

(i)














GSTmLsh

GSTmDnmt1


Snf2 HelicaseNLSCC



(a)

mLsh (aa)

mDnmt1 (aa)

205

95

(kDa)

GST

GSTInpu

t(1

0)

Inpu

t(1

0)

(kDa)

35S-mDnmt1

35S-mLsh

1125

ndash1620

740

ndash1115

613

ndash740

305

ndash609

1ndash3

43

560

ndash822

211

ndash560

1ndash208

(b)

IgGIn Lsh

Dnmt1IgGIn

205

95

(kDa)

120572Dnmt1

120572Lsh

(c)

Dnmt1IgGIn

95

(kDa)

120572Lsh

(d)

85

15

DAPI mLsh Merge

(e)

DAPI mLsh Merge

(f)


(g)

DAPImLsh

30

XhHP1120572

(h)

100DAPImLsh +

hHP1120572V21M

(i)

100DAPIxLsh +

hHP1120572A129R

(j)






4 Conclusions






MNase (U)

Unt

reat

ed

Sup

95

205

32

(kDa)120572Lsh

120572Dnmt1

120572Emerin

(a)

6 40

10

5

1

10

30

5

25 25

Genomic DNA gel

Fraction1 25


Origin

Compactchromatin

120572Lsh

6 40

Core histones


95(kDa)

13ndash17

(b)

95

30

(kDa)siLsh

1

siLsh

2

siLsh

3

siLsh

1+2+3

No

siRN

A

120572Lsh

120572Pcna

(c)


Sup

MNase (U) MNase (U)

MNase (U)MNase (U)

2

1

2

1


Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Bound

20532

(kDa)120572Dnmt1

120572Emerin

Dnm

t1 e

mer

in

Dnm

t1 e

mer

in

(d)









H3K9trime

HP1

LshDnmt1

MeCpGCpG


(a)

MeCpGCpG

H3K9trime

HP1

LshDnmt1

H3K4dime H3K4dime


(b)







Acknowledgments


References





























































zLM

OC

ontro

l MO

zLMO dose

minus + minus xLMO

xLsh

Luciferase

(a)

(b)

(d)

(f)

(h)

(g)

(e)(c)

xSatI southern

xLMO

(kb)

(kb)

HpaII

MspI

minus minus minus +

minus minus + +

+ minus minus minus

1 2 3 4

10

5

1

3

zLMO

HpaII


minus minus minus +

minus minus + +

1 2 3 4

Dana southern

10

5

1

3

025

24hpf

WT

zLMO

WT

xLMO

gDNA (ng)

gDNA (ng)

50 100

200

100

200

500

IB120572

-5m

eCIB

120572-5

meC


Figure 1 Continued



CpG number

100

1 2 3 4 5 6 70

Met

hyla

tion

()

(i)














GSTmLsh

GSTmDnmt1


Snf2 HelicaseNLSCC



(a)

mLsh (aa)

mDnmt1 (aa)

205

95

(kDa)

GST

GSTInpu

t(1

0)

Inpu

t(1

0)

(kDa)

35S-mDnmt1

35S-mLsh

1125

ndash1620

740

ndash1115

613

ndash740

305

ndash609

1ndash3

43

560

ndash822

211

ndash560

1ndash208

(b)

IgGIn Lsh

Dnmt1IgGIn

205

95

(kDa)

120572Dnmt1

120572Lsh

(c)

Dnmt1IgGIn

95

(kDa)

120572Lsh

(d)

85

15

DAPI mLsh Merge

(e)

DAPI mLsh Merge

(f)


(g)

DAPImLsh

30

XhHP1120572

(h)

100DAPImLsh +

hHP1120572V21M

(i)

100DAPIxLsh +

hHP1120572A129R

(j)






4 Conclusions






MNase (U)

Unt

reat

ed

Sup

95

205

32

(kDa)120572Lsh

120572Dnmt1

120572Emerin

(a)

6 40

10

5

1

10

30

5

25 25

Genomic DNA gel

Fraction1 25


Origin

Compactchromatin

120572Lsh

6 40

Core histones


95(kDa)

13ndash17

(b)

95

30

(kDa)siLsh

1

siLsh

2

siLsh

3

siLsh

1+2+3

No

siRN

A

120572Lsh

120572Pcna

(c)


Sup

MNase (U) MNase (U)

MNase (U)MNase (U)

2

1

2

1


Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Bound

20532

(kDa)120572Dnmt1

120572Emerin

Dnm

t1 e

mer

in

Dnm

t1 e

mer

in

(d)









H3K9trime

HP1

LshDnmt1

MeCpGCpG


(a)

MeCpGCpG

H3K9trime

HP1

LshDnmt1

H3K4dime H3K4dime


(b)







Acknowledgments


References






























































CpG number

100

1 2 3 4 5 6 70

Met

hyla

tion

()

(i)














GSTmLsh

GSTmDnmt1


Snf2 HelicaseNLSCC



(a)

mLsh (aa)

mDnmt1 (aa)

205

95

(kDa)

GST

GSTInpu

t(1

0)

Inpu

t(1

0)

(kDa)

35S-mDnmt1

35S-mLsh

1125

ndash1620

740

ndash1115

613

ndash740

305

ndash609

1ndash3

43

560

ndash822

211

ndash560

1ndash208

(b)

IgGIn Lsh

Dnmt1IgGIn

205

95

(kDa)

120572Dnmt1

120572Lsh

(c)

Dnmt1IgGIn

95

(kDa)

120572Lsh

(d)

85

15

DAPI mLsh Merge

(e)

DAPI mLsh Merge

(f)


(g)

DAPImLsh

30

XhHP1120572

(h)

100DAPImLsh +

hHP1120572V21M

(i)

100DAPIxLsh +

hHP1120572A129R

(j)






4 Conclusions






MNase (U)

Unt

reat

ed

Sup

95

205

32

(kDa)120572Lsh

120572Dnmt1

120572Emerin

(a)

6 40

10

5

1

10

30

5

25 25

Genomic DNA gel

Fraction1 25


Origin

Compactchromatin

120572Lsh

6 40

Core histones


95(kDa)

13ndash17

(b)

95

30

(kDa)siLsh

1

siLsh

2

siLsh

3

siLsh

1+2+3

No

siRN

A

120572Lsh

120572Pcna

(c)


Sup

MNase (U) MNase (U)

MNase (U)MNase (U)

2

1

2

1


Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Bound

20532

(kDa)120572Dnmt1

120572Emerin

Dnm

t1 e

mer

in

Dnm

t1 e

mer

in

(d)









H3K9trime

HP1

LshDnmt1

MeCpGCpG


(a)

MeCpGCpG

H3K9trime

HP1

LshDnmt1

H3K4dime H3K4dime


(b)







Acknowledgments


References




































































GSTmLsh

GSTmDnmt1


Snf2 HelicaseNLSCC



(a)

mLsh (aa)

mDnmt1 (aa)

205

95

(kDa)

GST

GSTInpu

t(1

0)

Inpu

t(1

0)

(kDa)

35S-mDnmt1

35S-mLsh

1125

ndash1620

740

ndash1115

613

ndash740

305

ndash609

1ndash3

43

560

ndash822

211

ndash560

1ndash208

(b)

IgGIn Lsh

Dnmt1IgGIn

205

95

(kDa)

120572Dnmt1

120572Lsh

(c)

Dnmt1IgGIn

95

(kDa)

120572Lsh

(d)

85

15

DAPI mLsh Merge

(e)

DAPI mLsh Merge

(f)


(g)

DAPImLsh

30

XhHP1120572

(h)

100DAPImLsh +

hHP1120572V21M

(i)

100DAPIxLsh +

hHP1120572A129R

(j)






4 Conclusions






MNase (U)

Unt

reat

ed

Sup

95

205

32

(kDa)120572Lsh

120572Dnmt1

120572Emerin

(a)

6 40

10

5

1

10

30

5

25 25

Genomic DNA gel

Fraction1 25


Origin

Compactchromatin

120572Lsh

6 40

Core histones


95(kDa)

13ndash17

(b)

95

30

(kDa)siLsh

1

siLsh

2

siLsh

3

siLsh

1+2+3

No

siRN

A

120572Lsh

120572Pcna

(c)


Sup

MNase (U) MNase (U)

MNase (U)MNase (U)

2

1

2

1


Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Bound

20532

(kDa)120572Dnmt1

120572Emerin

Dnm

t1 e

mer

in

Dnm

t1 e

mer

in

(d)









H3K9trime

HP1

LshDnmt1

MeCpGCpG


(a)

MeCpGCpG

H3K9trime

HP1

LshDnmt1

H3K4dime H3K4dime


(b)







Acknowledgments


References





























































GSTmLsh

GSTmDnmt1


Snf2 HelicaseNLSCC



(a)

mLsh (aa)

mDnmt1 (aa)

205

95

(kDa)

GST

GSTInpu

t(1

0)

Inpu

t(1

0)

(kDa)

35S-mDnmt1

35S-mLsh

1125

ndash1620

740

ndash1115

613

ndash740

305

ndash609

1ndash3

43

560

ndash822

211

ndash560

1ndash208

(b)

IgGIn Lsh

Dnmt1IgGIn

205

95

(kDa)

120572Dnmt1

120572Lsh

(c)

Dnmt1IgGIn

95

(kDa)

120572Lsh

(d)

85

15

DAPI mLsh Merge

(e)

DAPI mLsh Merge

(f)


(g)

DAPImLsh

30

XhHP1120572

(h)

100DAPImLsh +

hHP1120572V21M

(i)

100DAPIxLsh +

hHP1120572A129R

(j)






4 Conclusions






MNase (U)

Unt

reat

ed

Sup

95

205

32

(kDa)120572Lsh

120572Dnmt1

120572Emerin

(a)

6 40

10

5

1

10

30

5

25 25

Genomic DNA gel

Fraction1 25


Origin

Compactchromatin

120572Lsh

6 40

Core histones


95(kDa)

13ndash17

(b)

95

30

(kDa)siLsh

1

siLsh

2

siLsh

3

siLsh

1+2+3

No

siRN

A

120572Lsh

120572Pcna

(c)


Sup

MNase (U) MNase (U)

MNase (U)MNase (U)

2

1

2

1


Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Bound

20532

(kDa)120572Dnmt1

120572Emerin

Dnm

t1 e

mer

in

Dnm

t1 e

mer

in

(d)









H3K9trime

HP1

LshDnmt1

MeCpGCpG


(a)

MeCpGCpG

H3K9trime

HP1

LshDnmt1

H3K4dime H3K4dime


(b)







Acknowledgments


References
































































4 Conclusions






MNase (U)

Unt

reat

ed

Sup

95

205

32

(kDa)120572Lsh

120572Dnmt1

120572Emerin

(a)

6 40

10

5

1

10

30

5

25 25

Genomic DNA gel

Fraction1 25


Origin

Compactchromatin

120572Lsh

6 40

Core histones


95(kDa)

13ndash17

(b)

95

30

(kDa)siLsh

1

siLsh

2

siLsh

3

siLsh

1+2+3

No

siRN

A

120572Lsh

120572Pcna

(c)


Sup

MNase (U) MNase (U)

MNase (U)MNase (U)

2

1

2

1


Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Bound

20532

(kDa)120572Dnmt1

120572Emerin

Dnm

t1 e

mer

in

Dnm

t1 e

mer

in

(d)









H3K9trime

HP1

LshDnmt1

MeCpGCpG


(a)

MeCpGCpG

H3K9trime

HP1

LshDnmt1

H3K4dime H3K4dime


(b)







Acknowledgments


References





























































MNase (U)

Unt

reat

ed

Sup

95

205

32

(kDa)120572Lsh

120572Dnmt1

120572Emerin

(a)

6 40

10

5

1

10

30

5

25 25

Genomic DNA gel

Fraction1 25


Origin

Compactchromatin

120572Lsh

6 40

Core histones


95(kDa)

13ndash17

(b)

95

30

(kDa)siLsh

1

siLsh

2

siLsh

3

siLsh

1+2+3

No

siRN

A

120572Lsh

120572Pcna

(c)


Sup

MNase (U) MNase (U)

MNase (U)MNase (U)

2

1

2

1


Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Unt

reat

ed

Bound

20532

(kDa)120572Dnmt1

120572Emerin

Dnm

t1 e

mer

in

Dnm

t1 e

mer

in

(d)









H3K9trime

HP1

LshDnmt1

MeCpGCpG


(a)

MeCpGCpG

H3K9trime

HP1

LshDnmt1

H3K4dime H3K4dime


(b)







Acknowledgments


References
































































H3K9trime

HP1

LshDnmt1

MeCpGCpG


(a)

MeCpGCpG

H3K9trime

HP1

LshDnmt1

H3K4dime H3K4dime


(b)







Acknowledgments


References
































































Acknowledgments


References


























































































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