10.1128/MCB.23.23.8416-8428.2003.

2003, 23(23):8416. DOI:Mol. Cell. Biol. MueggeQingsheng Yan, Edward Cho, Stephen Lockett and Kathrin HeterochromatinHeterochromatin Is Dependent on IntactMethylation, with Pericentromeric Association of Lsh, a Regulator of DNA

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MOLECULAR AND CELLULAR BIOLOGY, Dec. 2003, p. 8416–8428 Vol. 23, No. 230270-7306/03/$08.00�0 DOI: 10.1128/MCB.23.23.8416–8428.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Association of Lsh, a Regulator of DNA Methylation, withPericentromeric Heterochromatin Is Dependent on

Intact HeterochromatinQingsheng Yan,1 Edward Cho,2 Stephen Lockett,2 and Kathrin Muegge1*

Laboratory of Molecular Immunoregulation, Basic Research Program,1 and Image Analysis Laboratory,2

SAIC-Frederick, National Cancer Institute, Frederick, Maryland 21702

Received 19 June 2003/Returned for modification 30 June 2003/Accepted 15 August 2003

The eukaryotic genome is packaged into distinct domains of transcriptionally active euchromatin and silentheterochromatin. A hallmark of mammalian heterochromatin is CpG methylation. Lsh, a member of the SNF2family, is a major regulator of DNA methylation in mice and thus crucial for normal heterochromatinformation. In order to define the molecular function of Lsh, we examined its cellular localization and itsassociation with chromatin. Our studies demonstrate that Lsh is an exclusively nuclear protein, and we definea nuclear localization domain within the N-terminal portion of Lsh. Lsh strongly associates with chromatinand requires the internal and C-terminal regions for this interaction. Lsh accumulates at pericentromericheterochromatin, suggesting a direct role for Lsh in the methylation of centromeric DNA sequences and theformation of heterochromatin. In search of a signal that is responsible for Lsh recruitment to pericentromericheterochromatin, we found that histone tail modifications were critical. Prolonged treatment with histonedeacetylase inhibitors has been reported to disrupt higher-order heterochromatin organization, and this wasaccompanied by dissociation of Lsh from pericentromeric heterochromatin. These results are consistent witha model in which Lsh is recruited by intact heterochromatin structure and then assists in maintainingheterochromatin organization by establishing CpG methylation patterns.

The chromatin of eukaryotic cells is organized into two ma-jor types: euchromatin and heterochromatin (6, 39). Euchro-matin contains the majority of single-copy genes, replicatesduring early S phase, and contains acetylated histones. Het-erochromatin is composed of long stretches of DNA repeats,usually replicates in late S phase, and contains underacetylatedhistones. A specific methylation mark in heterochromatin inhistone 3 at lysine 9 serves as a recognition signal for bindingof the heterochromatin protein 1 (HP1) (26). Heterochroma-tin regulates many diverse nuclear functions, including genesilencing, normal centromere function and nuclear organiza-tion.

Another hallmark of mammalian heterochromatin is DNAmethylation (5–7, 9, 34, 39). The addition of methyl groups tocytosines within the CpG dinucleotide by DNA methyltrans-ferases is involved in regulating transcription, maintaining ge-nome stability, imprinting, and X chromosome inactivation. Inmammalian cells, DNA methylation is catalyzed by two impor-tant classes of DNA methyltransferases (5). DNA methyltrans-ferase 1 (Dnmt1) resides at the replication fork and methylatesCpG dinucleotides in the newly synthesized daughter strand.The function of Dnmt1 is thought to be essential for maintain-ing DNA methylation patterns in proliferating cells (30). Thereare two members of the second class of methyltransferases,Dnmt3a and Dnmt3b, which are required for the initiation ofde novo methylation during embryonic development (37).

Although DNA methylation and heterochromatin are

closely associated, their precise relationship has not been elu-cidated. Recently, several members of the SNF2 family ofchromatin remodeling enzymes have been found to regulateDNA methylation, suggesting that chromatin structure influ-ences DNA methylation pattern (34).

SNF2 family members are marked by the presence of sevenconserved “helicase” motifs involved in ATP binding and hy-drolysis (13, 14). Many ATP-dependent chromatin remodelingcomplexes have been identified, such as SWI/SNF/BRM,ISWI, or Mi-2/NuRD, and each chromatin remodeling com-plex contains at least one member of the SNF2 superfamily.Chromatin remodeling complexes disrupt histone-DNA inter-actions and allows for the physical movement, or sliding, ofnucleosomes on the DNA utilizing the energy derived fromATP hydrolysis (3). Thus, they alter the accessibility of chro-matin to various proteins that regulate DNA-based processessuch as DNA replication, transcription, and repair. DDM1,ATRX, and Lsh all belong to the SNF2 superfamily and havebeen demonstrated to regulate DNA methylation (12, 20, 25).Recombinant DDM1 has been recently demonstrated to in-duce nucleosome repositioning on a short DNA fragment, thusconfirming its membership in the SNF2 family of chromatinremodeling proteins (8). However, neither DDM1, ATR-X,nor Lsh have been reported as yet to interact with large proteincomplexes, a characteristic of other SNF2 family members.

The DDM1 gene (named for decrease in DNA methylation)deficiency causes a 70% reduction of genomic DNA methyl-ation in the plant Arabidopsis thaliana (27). The loss of DNAmethylation is most pronounced at repetitive sequences, fol-lowed by hypomethylation at single-copy genes. ATRX defi-ciency gives rise to changes in the pattern of methylation ofseveral highly repeated sequences, including ribosomal DNA

* Corresponding author. Mailing address: LMI, SAIC, NationalCancer Institute, Bldg. 469, Rm. 243, Frederick, MD 21702-1201.Phone: (301) 846-1386. Fax: (301) 846-7077. E-mail: muegge@ncifcrf.gov.

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arrays, a Y-specific satellite sequence, and subtelomeric re-peats. Mutations in the human ATRX gene result in the inher-ited disease �-thalassemia (19, 20).

Lymphoid-specific helicase (Lsh) is another member of theSNF2 family of chromatin remodeling proteins (17, 24, 28).Lsh is a nonredundant member of the SNF2 family and isessential for normal murine development and survival. Tar-geted deletion of Lsh results in perinatal lethality, with abnor-mal lymphoid development and renal defects (16, 18). DNAderived from Lsh�/� tissue shows a substantial loss of CpGmethylaton (12). This suggests that Lsh is an important factorin determining DNA methylation and thus crucial for hetero-chromatin organization in mammals. However, it is not knownwhether Lsh controls methylation directly or indirectly, forexample, via induction of other enzymes involved in DNAmethylation. We have previously reported that Lsh does notinfluence methyltransferase activity or Dnmt1, Dnmt3a, orDnmt3b protein levels, suggesting a more direct role of Lsh inestablishing or maintaining methylation (12). Here we soughtto examine the cellular localization of Lsh and its associationwith chromatin. We report a close association of Lsh withpericentromeric heterochromatin, suggesting that Lsh via CpGmethylation directly participates in the organization of hetero-chromatin. Disruption of higher-order heterochromatin struc-ture by prolonged exposure to the histone acetylase inhibitortrichostatin A (TSA) is followed by delocalization of Lsh.These results are consistent with a model in which the histonetail modifications that shape higher-order heterochromatinstructure provide a signal for Lsh recruitment that in turn iscrucial for establishing methylation patterns and the formationof heterochromatin.

MATERIALS AND METHODS

Tissue culture. For analysis of Lsh expression fetal murine thymus was used(C57BL/6J strain; Jackson Laboratory). PD31, a pre-B-cell line, and EL4, aT-cell line (American Type Culture Collection), were cultured in RPMI medium1640 (Gibco) supplemented with 2 mM glutamine, 100 U of penicillin/ml, and100 �g of streptomycin/ml (all from Gibco) and 10% fetal calf serum (HyClone).Embryos from crosses between Lsh�/� mice were removed at day 13.5 of ges-tation and genotyped by PCR as described before (18). Preparation of primarymouse embryonal fibroblast (MEF) cells are described elsewhere (15), andMEFs were grown in Dulbecco modified Eagle medium (Gibco) supplementedwith 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, and antibiotics(Gibco). NIH 3T3 cells (American Type Culture Collection) were maintained inDulbecco modified Eagle medium with 10% bovine calf serum, and Lsh-express-ing GeneSwitch-3T3 cells (Invitrogen) were cultured in the same medium sup-plemented with 50 �g of hygromycin/ml to select for hygromycin resistance. Cellsin exponential growth were treated in the presence of TSA (Sigma) as describedpreviously (31, 42); the drug and medium were renewed regularly. NIH 3T3 cellswere incubated at 75 ng of TSA/ml.

Vector construction. Based on the mouse Lsh cDNA (U25691) sequence,three primers were designed. The first, a 5� primer designated LSHN01 (5�-AGAGATGGCCGAACAAACGGAG-3�), encoded the N-terminal of LSH, includ-ing the initiation codon. The second, a 3� primer designated LSHFC1 (5�-CCGGCTAGCTCACTTGTCATCGTCGTCCTTGTAGTCAAATAAACATTCAGCACTGG-3�), encoded the C-terminal sequence of the Lsh peptide, followed bya DNA sequence encoding the eight-amino-acid FLAG tag (DYKDDDDK) anda stop codon. The third was another 3� primer, LSHFC2 (5�-CCTTGTCATCGTCGTCCTTGAGTCAAATAAACATTCAGCACTGG-3�), that contained a C-terminal sequence for the Lsh protein followed by a DNA sequence encoding theFlag tag without a stop codon. The complete open reading frame of the full-length mouse Lsh cDNA was amplified by reverse transcription-PCR with totalRNA purified from PD31 cells as a template and the primer pair LSHN01 andLSHFC1. A 2.5-kb portion of PCR-amplified product was subcloned directly intopCRBLUNT (Invitrogen), forming the vector pCRLSHF1. By using the

pCRLSHF1 as a template and two primers, LSHN01 and LSHFC2, the PCR-amplified product was subcloned into pCRBLUNT (Invitrogen), and the newvector was termed pCRLSHF2. The plasmid pCRLSHF1 was cut with EcoRI,and the LSH DNA fragment was subcloned into the EcoRI site of pGene/V5-his(Invitrogen), designating the vector pGeneLSHF. The same EcoRI DNA frag-ment was subcloned into the EcoRI site of pEGFP-C1 (Clontech), generating thevector pEGFPLSH that encoded the EGFP-tagged Lsh at the C terminus. ThepEGFPLSH was partially digested with EcoRI, MunI, and BamHI, respectively.The appropriate DNA fragments were recollected and self-ligated. The serialdeletion mutants of Lsh were generated as follows. After EcoRI digestion, thedeletion mutant, �1-176, did not contain the DNA sequence encoding the 1 to176 of amino acids of Lsh. The vector generated after MunI digestion had theDNA fragment deleted that encoded amino acids 244 to 469 of Lsh and was thusdesignated �244-469. The vector generated after BamHI digestion did not en-code amino acids 475 to 768 of Lsh and was therefore designated �475-768. Theplasmid pCRLSHF2 was digested with EcoRI and cloned into the EcoRI site ofpEGFP-N1 (Clontech), producing the vector pLSHEGFP that encoded theenhanced green fluorescent protein (EGFP)-tagged Lsh at the N terminus. ThepLSHEGFP vector was digested with XhoI, and the appropriate DNA fragmentswere collected and self-ligated. The deletion vector, termed �1-31, generated byXhoI digestion did not encode amino acids 1 to 31 of Lsh.

Stable and transient transfections. The GeneSwitch system (Invitrogen), amifepristone (Invitrogen)-regulated expression system for mammalian cells, waschosen to produce a cell line with inducible expression of the Lsh gene. TheGeneSwitch 3T3 cells express the GeneSwitch regulatory protein from thepSwitch vector. GeneSwitch 3T3 cells were seeded at a concentration of 106 in10-cm dishes overnight before transfection. Cells were transfected with 2.5 �g ofLsh-inducible vector pGeneLSHF or empty vector pGene/V5-his as a control, byusing FuGENE 6 transfection reagent (Roche) according to the manufacturer’srecommendations. Stably transfected cells were selected with 500 to 1,000 �g ofZeocin (Invitrogen)/ml. Zeocin-resistant cells were induced with 100 pM mife-pristone for induction of the Lsh protein. For EGFP expression NIH 3T3 cellswere seeded at a concentration of 1 � 105 to 2 � 105 cells per well in a six-wellplate (Costar cluster multiple-well plates) for 15 h before transfection. Cells weretransfected with 2 to 4 �g of EGFP-tagged Lsh vector or control vector by usingSuperFect transfection reagent (Qiagen) or FuGENE 6 transfection reagent(Roche) according to manufacturers’ recommendations. Transiently transfectedcells were cultured overnight, and Lsh-GFP expression was observed by fluores-cence microcopy or Lsh expression was examined by Western analysis.

Cell cycle analysis. For cell cycle synchronization, EL4 cells were seeded at ca.105/ml in a total volume of 10 ml per 25-cm2 flask, with L-mimosine (400 �M;Sigma), aphidicolin (250 ng/ml; Sigma), and nocodazole (400 ng/ml; Sigma). Thetreated cells were incubated for 24 h in the presence of the blocking reagents,and the drugs were removed for flow cytometric analysis or Western analysis. Forcell cycle synchronization, NIH 3T3 cells were incubated at 3 �g of aphidico-lin/ml for 18 h and then released into aphidicolin-free medium (10). For cell cycleanalysis, cell suspensions were washed twice in phosphate-buffered saline (PBS)and fixed with 70% of ethanol at �20°C for at least 2 h. Cells were washed oncewith PBS and then incubated in 25 �g of propidium iodide (Sigma)/ml with 100�g of RNase A/ml in PBS at 37°C for 30 min. Flow cytometric analysis wasperformed on a Coulter EPICS XL-MCL cytometer. The data were collectedand analyzed by using the Coulter System II software provided by the manufac-turer.

Indirect immunofluorescence studies. MEFs were seeded on glass coverslipsto 50 to 60% confluency. Cells were fixed in 2% paraformaldehyde solution andpermeabilized with 0.5% Triton X-100 (Triton X) solution. Coverslips wereblocked with 5% goat serum for 30 min at room temperature and then incubatedwith primary antibodies in a humidifying chamber at room temperature for 1 hor at 4°C overnight. After the coverslips were washed five times, they wereincubated with Cy5 and fluorescein isothiocyanate-conjugated secondary anti-bodies (Jackson Immunoresearch) in PBS with 1.0 mM MgCl2 at room temper-ature for 1 h. After an extensive washing, coverslips were mounted onto glassmicroscope slides by using mounting reagent (Molecular Probes). To immuno-stain newly synthesized DNA and related proteins, cell staining was performed aspreviously described (29, 40). The following primary antibodies were used.Dnmt1 was kindly provided by T. Bestor (Department of Genetics and Devel-opment, Columbia University, New York, N.Y.) as published before (29), PCNA(PC10; 05-347) was purchased from Upstate Biotechnology, and M2 (anti-Flag)was purchased from Sigma, monoclonal bromodeoxyuridine (BrdU) antibodywas from Pharmingen (catalog no. 347580) or Roche (catalog no. 1296736);HP1� (1H5; MAb3584) was purchased from Chemicon, and branched anti-histone 3 lysine 9 methylation antiserum was kindly provided by T. Jenuwein(Research Institute of Molecular Pathology, Vienna, Austria). The cells were

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observed under a fluorescence microscope (Zeiss) or by three-dimensional con-focal microscopy (LSM 510 confocal microscope; Zeiss).

Western analysis and protein fractionation. Protein extracts were separatedon NuPAGE Bis-Tris Pre-Cast gels (for low- to proteins), or NuPAGETris-Acetate gels (for high-molecular-weight proteins) by electrophoresis andtransferred onto nitrocellulose. Each lane contained 10 �g of protein ex-tracts, with the exception of the cell cycle experiments or fractionation ex-periments, in which proteins were extracted from equal amounts of cells (i.e.,105). After a blocking step, the membrane was incubated with primary anti-bodies to distinct proteins, followed by incubation with secondary horseradishperoxidase-coupled antibodies (Amersham Pharmacia). Detection of horse-radish peroxidase was performed by using the enhanced chemiluminescenceWestern blotting detection reagents according to the manufacturer’s instruc-tions, followed by visualization on Hyperfilm ECL chemiluminescent film.The following antibodies were used for detection of proteins as the primaryantibodies. Lsh antiserum was used as described previously (18). Brg-1 (sc-8749), HDAC1 (sc-7872), MBD1 (sc-10751), CBP (sc-369), and p300 (sc-584)were purchased from Santa Cruz Biotechnology. M2 (anti-Flag; F3165) andVimentin (LN-6; V2258) were purchased from Sigma. MeCP2 (07-013),PCNA (PC10; 05-347), histone H3 (05-499), acetylated histone 3 (06-599),histone4 (07-108), and acetylated histone 4 (06-598), and dimethyl-histoneH3 lysine 9 (MeK9; 07-212) were purchased from Upstate Biotechnology.HP1� (2G9; MAB3446) was purchased from Chemicon, EFGP (JL-8; 8873)was from Clontech, and DNMT3a (64B1446; IMG-268) was from Imgenex.Cell extraction with Triton X, chromatin fractionation, and nuclear matrixisolation were modified as described previously (22, 38). After two washes inPBS, cells were incubated and extracted for 3 min at 4°C in cytoskeletonbuffer (CSK; 10 mM PIPES [pH 6.8], 100 mM NaCl, 300 mM sucrose, 3 mMMgCl2, 1 mM EGTA) supplemented with leupeptin, aprotinin, and pepstatin(1 �g/ml each), 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol,and 0.5% (vol/vol) Triton X. The nuclear skeleton that was resistant to TritonX was separated from soluble proteins by centrifugation at 5,000 � g for 3min. Chromatin proteins were solubilized by DNA digestion with 1 mg ofRNase-free DNase I/ml in CSK buffer (with adjusted 50 mM NaCl) in thepresence of proteinase inhibitors for 15 min at room temperature, and thesupernatant, containing chromatin proteins, was collected by centrifugation.The pellet was washed with 0.25 M ammonium sulfate for 5 min at 4°C andpelleted again. The second pellet was washed with 2 M NaCl in CSK bufferfor 5 min at 4°C and then centrifuged. The nuclear matrix was then solubi-lized in 8 M urea buffer.

RESULTS

Nuclear localization domain of Lsh. Since Lsh belongs tothe SNF2 family of chromatin remodeling proteins and Lsh hasbeen demonstrated to be a major modulator of CpG methyl-ation, Lsh is predicted to have a nuclear localization. By com-paring nuclear and cytoplasmic extracts derived from differenttissues by Western analysis, we determined that Lsh was onlydetectable in the nuclear fraction and not in the cytoplasmicpreparation (Fig. 1A). Lsh was found to be highly expressed incells of the lymphoid lineage such as the pre-B-cell line PD31and embryonic thymocytes but minimally expressed by MEFs(Fig. 1A) and barely detectable in NIH 3T3 fibroblasts (datanot shown). As expected, MEFs derived from mice with atargeted deletion of Lsh showed no detectable signal whenprobed with a specific antiserum raised against the C terminusof Lsh (Fig. 1A). The predominantly nuclear expression of Lshwas further confirmed in 3T3 fibroblasts, when Lsh was taggedwith the Flag peptide and the expression was under the controlof a mifepristone response element. In this system Lsh wasexpressed �10-fold above endogenous Lsh levels (Fig. 1B).Furthermore, Lsh localized to the nucleus throughout allstages of the cell cycle (Fig. 1C) even during the S phase, whenendogenous Lsh expression was highest. Thus, the nuclearexpression profile of Lsh in all cell types examined corre-sponded well with Lsh’s presumed chromatin remodeling ac-tivity.

In order to confirm the exclusive nuclear localization and todefine the motif within the Lsh protein that is responsible fortargeting of Lsh to the nucleus, 3T3 cells were transientlytransfected with Lsh expression vectors. A series of LSH de-letion mutants were constructed and fused to GFP (Fig. 2A).As shown by Western analysis in Fig. 2B, all constructs resultedin expression of the Lsh protein with the expected molecular

FIG. 1. Nuclear localization of Lsh. (A) Lsh is localized in nuclear extracts. For analysis of Lsh expression the pre-B-cell line PD31, fetal thymusand embryonal fibroblasts (MEFs) were examined by Western analysis and probed with a specific antibody raised against the C-terminal portionof Lsh. Lanes: C, cytoplasmic extract; N, nuclear extract (equal loading of protein extracts). (B) Lsh is localized in overexpressed nuclear extracts.Lsh was induced in 3T3 cells in the GeneSwitch system and examined by Western analysis. Lanes: C, cytoplasmic extract; N, nuclear extract (equalloading of protein extracts). (C) Lsh is preferentially expressed during S phase. The T-cell line EL4 was synchronized with mimosine (M),aphidicolin (A), or nocodazole (N) or left untreated (Ctrl) and then examined by Western analysis for the presence of Lsh or -actin. Lanes: C,cytoplasmic extract; N, nuclear extract (extracts were prepared from equal number of cells). The lower panel shows the result of the cell cycleanalysis and the distribution of cells at different stages of the cell cycle.

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size. Expression of wild-type Lsh (amino acids 1 to 821) re-sulted in an exclusive nuclear expression pattern as shown byfluorescence microscopy, a finding which supported the previ-ous Western analysis of nuclear extracts (Fig. 2C). The posi-tion of GFP toward Lsh, either N terminal or C terminal, didnot influence the proper nuclear localization (data not shown).In contrast, expression of the GFP protein alone without Lshrevealed a diffuse cytoplasmic distribution (Fig. 2C). In orderto determine the nuclear localization domain, mutant Lsh vec-tors were transiently transfected. Fluorescence analysis re-vealed that the C-terminal portion of Lsh (amino acids 574 to768), as well as the internal deletion mutant (amino acids 244to 469), were dispensable for nuclear localization of Lsh. Incontrast the N-terminal deletion (amino acids 1 to 176) com-pletely abolished targeting of Lsh to the nucleus. Since theregion from amino acids 1 to 31 was not critical for the nuclearlocalization of Lsh, these results suggest that the nuclear lo-calization domain of murine Lsh is located between aminoacids 31 and 176, a region containing several lysine-richstretches.

Association of Lsh with Triton X-resistant fractions. In or-der to determine the nuclear function of Lsh, we further in-

vestigated the nuclear compartment of Lsh localization. Wefirst examined whether Lsh was present in a soluble and/orextractable form (as many DNA-binding factors are) orwhether it was closely bound to nuclear components, as hasbeen reported for other SNF2 family members (4, 36, 38, 46).Lsh was induced as a Flag-tagged fusion protein in 3T3 cells(Fig. 1B), and the nuclei were then washed with high concen-trations of Triton X detergent (Fig. 3A). Western analysis offlushed material and the Triton X-resistant fraction demon-strated that as much as about half of the induced Lsh wasfound to be Triton resistant, indicating a close association ofLsh with chromatin or the nuclear matrix. By comparison, mosttranscription factors or nuclear proteins, such as MECP2 andPCNA, can be efficiently extracted by Triton X washes,whereas chromatin-associated proteins such as BRG1 andhBRM, are fairly Triton X resistant (38, 46) (Fig. 3A). In orderto determine whether overexpression of Lsh “saturates” po-tential chromatin-binding sites and thus is responsible for theextractable form of Lsh, we performed a time kinetic experi-ment to induce low amounts of Lsh. However, even at 0 and 3 hof induction, when low levels of Lsh were detectable, the re-lationship of free and bound Lsh was unaltered, and the ratio

FIG. 2. Nuclear localization domain of Lsh. (A) Construction map of Lsh deletional mutants. (B) Expression of deletion mutants. Nuclearextracts were examined 24 h after transfection by Western analysis with specific antiserum raised against the GFP peptide (equal loading of proteinextracts). (C) The nuclear localization domain is N terminal. At 24 h after transfection with indicated plasmids, 3T3 cells were examined undera fluorescence microscope.

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of bound to free Lsh remained at ca. 50:50 (Fig. 3B). Thissuggested that both forms may play a physiological role and arenot simply a by-product of overexpression. Since Lsh expres-sion peaked during S phase (Fig. 1C), we tested whether theresistance of Lsh to Triton X washes is correlated with thereplication phase. At confluency, when 3T3 cells are arrestedat G1, Lsh was found to be predominantly Triton X extract-able, whereas during the growth phase the ratio of free tobound Lsh shifted back to 50:50 (Fig. 3C). As a control, Vi-mentin, a nuclear scaffolding protein, was examined that wascontained within the Triton X-resistant fraction throughoutthe cell cycle. In contrast, PCNA (proliferation cellular nuclearantigen) shifted slightly within the Triton X-resistant fractionfrom chromatin association at 25% confluency to dissociationat 100% confluency. The shift in Lsh’s Triton X resistanceduring cell cycle progression suggests that the ability of Lsh tobind to chromatin is best in proliferating cells.

In order to define which domains of the Lsh protein areresponsible for Triton X resistance, we transfected GFP-tagged Lsh mutants into 3T3 cells and extracted them with

Triton X. A deletion of 31 amino acids at the N-terminalportion of Lsh did not affect the ratio of free to bound Lsh andwas similar to that of wild-type Lsh (Fig. 3D). However, dele-tion of the internal region comprising amino acids 244 to 469,as well as deletion of the C-terminal located portion (aminoacids 574 to 768), shifted the distribution of Lsh toward thefree form. This suggests that the entire Lsh protein down-stream of amino acid 244 is critical for efficient chromatinbinding. In addition, these domains may contain protein inter-action or protein folding functions.

Lsh localizes to pericentromeric heterochromatin. In orderto examine to which nuclear structure Lsh is recruited, theTriton X-resistant fractions were further eluted by digestionwith DNase I and high-salt washes. Under these conditionschromatin-bound fractions are separated from the nuclear ma-trix (22). Vimentin, for example, a component of the nuclearmatrix scaffold, resists elution of high-salt concentrations (Fig.4A). DNA-binding proteins (MecP2 and MBD1) or transcrip-tional regulators (p300) or the replication factor PCNA areonly loosely associated with the nuclear components and are

FIG. 3. Lsh is associated with chromatin. (A) Lsh is present in the Triton X-resistant fraction. After Lsh induction in 3T3 cells that weretransfected with the Lsh expression vector (Lsh) or transfected with the empty control vector (Ctrl) cells were extracted with Triton X-100, andthe resistant and soluble fractions were examined by Western analysis for the presence of Lsh with antibodies against the C-terminal portion ofLsh (Lsh), the Flag epitope (Flag), Brg-1, MeCP2, and PCNA (extracts were prepared from equal number of cells). (B) Kinetics of Lsh expression.At different time points after Lsh induction 3T3 cells were extracted with Triton X, and the different fractions were examined by Western analysisfor the presence of Lsh, PCNA, or Vimentin (extracts were prepared from equal number of cells). (C) Association with Triton X-resistant fractionis dependent on cell cycle. 3T3 cells at different stages of confluency were extracted with Triton X, and the different fractions were examined byWestern analysis for the presence of Lsh or PCNA and Vimentin serving as controls (extracts were prepared from equal number of cells).(D) TheC-terminal and internal domain are required for Triton X resistance. After transfection with the indicated deletion mutants, Triton X-resistant(lanes R) and soluble (Triton flush out [lanes F]) fractions were examined by Western analysis for the presence of GFP, Vimentin, and PCNA(extracts were prepared from equal number of cells).

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readily eluted by detergent. In contrast, about half of Lshresists Triton X washes, and most of this fraction is detectablein the “chromatin” fraction which also contains histones, HP1�and Dnmt3a (Fig. 4A) (10). This suggests that the bound Lshprotein primarily associates with chromatin, as opposed tobinding to the nuclear scaffold (for comparison, see lanes 2 and5 in Fig. 4A). However, a small portion of induced Lsh appearsin the scaffolding fraction and, at present, it cannot be deter-mined whether this is of physiologic relevance or an artifactdue to overexpression of Lsh.

To determine whether Lsh binds to euchromatin or hetero-chromatin, we transiently expressed Lsh as a GFP-tagged fu-sion protein. As revealed by confocal immunofluorescenceanalysis, Lsh colocalized with DAPI (4�,6�-diamidino-2-phe-nylindole) spots that are indicative for pericentromeric hetero-chromatin in murine cells (Fig. 4B). Pericentromeric hetero-chromatin DNA consists primarily of minor and major satellitesequences and large blocks of repetitive sequences and pre-sents the bulk of heterochromatin visualized by immunefluo-rescence analysis. Pericentromeric heterochromatin is thoughtto contribute to normal centromere functions during mitosissuch as spindle formation and chromosome segregation. Thecolocalization of Lsh with DAPI was confirmed in 3T3 cellsthat overexpressed Flag-tagged Lsh (data not shown). Further-more, Lsh colocalized with HP1�, a major component of peri-centromeric heterochromatin (Fig. 4C). In addition, we havedemonstrated that chromatin immunoprecipitated with anti-Lsh/Flag antibodies in 3T3 cells showed a close association ofLsh with minor and major satellite sequences that are the maincomponents of pericentromeric DNA (Q. Yan and K. Muegge,unpublished data). Thus, our data show that Lsh is localized atpericentromeric heterochromatin, and previous work indicated

that Lsh is crucial for CpG methylation at pericentromericDNA (12). Taken together, these results support the idea thatLsh plays a direct role in the formation of pericentromericheterochromatin.

Lsh is not required for targeting of Dnmt1 to replicationfoci. Lsh-deficient cells show a global methylation defect that issimilar to the one reported in Dnmt1�/� embryos. Thus, wehypothysed that Lsh and its presumed chromatin remodelingactivity maybe important for targeting Dnmt1 to sites of meth-ylation. Dnmt1 has a significant preference for hemimethyl-ated DNA relative to unmethylated DNA in vitro. Dnmt1associates with sites of DNA synthesis, the replication foci,throughout the S phase but shows a diffuse nucleoplasmicdistribution pattern during other phases of the cell cycle. Thedirect interaction with PCNA allows Dnmt1 to track along thenewly synthesized strands and to ensure proper copying of themethylation “code.” Thus, Dnmt1 is thought to play a role inthe maintenance of CpG methylation (5). Based on Lsh’s nu-clear expression pattern, we tested whether Lsh could directlyparticipate in the maintenance of genomic methylation. There-fore, we first determined whether Lsh could associate withreplication foci, the site of newly synthesized DNA. Lsh-in-duced 3T3 fibroblasts were synchronized with a high concen-tration of the cell cycle inhibitor aphidicolin. Cells were thenreleased into normal cell cycle and at different time pointspulsed with BrdU in order to detect newly synthesized DNA.Costaining for detection of incorporated BrdU, as well as Lsh,allowed the identification of different stages of the cell cycle.As shown in Fig. 5A, Lsh associated with replication foci onlyduring late S phase, when few, large foci are visible by BrdUincorporation. Lsh did not colocalize with replication foci atearly S phase, characterized by a fine punctate staining pattern,

FIG. 4. Lsh localizes to pericentromeric heterochromatin. (A) Lsh predominantly associates with chromatin. 3T3 cells were extracted withTriton X, and different fractions examined by Western analysis with the indicated antibodies: lane 1, flushed fraction; lane 2, Triton X-resistantfraction after DNase digestion; lane 3, ammonium sulfate wash; lane 4, 2 M NaCl wash; lane 5, solubilized pellet. (B) Lsh colocalizes with DAPI.At 24 h after transfection, GFP-tagged Lsh was examined by fluorescence microscopy. (C) Lsh colocalizes with HP1�. At 24 h after transfection,GFP-tagged Lsh was immunostained for detection of HP1�.

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or mid-phase with a staining pattern of BrdU at the peripheryof the nucleus and perinucleolar sites (29). Lsh also colocalizedwith Dnmt1 in late S phase (Fig. 5B). Thus, Lsh potentiallymay facilitate maintenance of methylation during the latephase of replication.

In order to test the possibility that Lsh is crucial for recruit-

ment of Dnmt1 to late replication foci, MEFs derived fromLsh-deficient mice or wild-type controls were stained for BrdUincorporation and probed for PCNA or Dnmt1 to detect rep-lication foci. As shown in Fig. 6A and B, PCNA, Dnmt1, andBrdU colocalized with each other in wild-type cultures, indi-cating the formation of normal replication foci. The same

FIG. 5. Lsh colocalizes with late replication foci. (A) Lsh colocalizes with late replication foci. Lsh was induced in 3T3 cells and immunostainedwith anti-Flag tag antibody and with anti-BrdU antibody for detection of incorporated BrdU. (B) Lsh colocalizes with Dnmt1. Lsh-induced 3T3cells were immunostained with anti-Flag tag antibody and immunostained for detection of Dnmt1.

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FIG. 6. Lsh is not required for targeting of Dnmt1 to replication foci. Lsh�/� (A) or Lsh�/� (B) MEFs were immunostained for detection ofPCNA, Dnmt1, and incorporated BrdU.

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colocalization was detectable in Lsh�/� MEFs, suggesting thatLsh is dispensable for proper localization of Dnmt1 during lateS phase. Thus, Lsh, although present at replication foci, doesnot alter targeting of Dnmt1 to replication foci.

Delocalization of Lsh follows disruption of higher-order het-erochromatin organization. Next, we sought to determine thesignal that is required to target Lsh to pericentromeric hetero-chromatin. Targeted deletion of Lsh in MEFs leads to hypo-methylation at pericentromeric minor and major satellite se-quences (12). Since Lsh is crucial for methylation atcentromeric DNA, it may also require CpG methylation as asignal for targeting, and thus Lsh could participate in a self-reinforcing loop of DNA methylation. To test this idea, wesought to compare the ability of Lsh to associate with hetero-chromatin in Lsh wild-type and Lsh-deficient MEFs. When

GFP-tagged Lsh was transiently expressed in Lsh�/� hypo-methylated MEFs, Lsh still retained the ability to colocalizewith DAPI (Fig. 7A). This suggests that Lsh does not requireDNA methylation for heterochromatic localization.

Many SNF2 family members have been reported to partic-ipate in large protein complexes, a hallmark of SNF/SWI chro-matin remodeling complexes but, to date, no direct interac-tions of Lsh with other proteins have been found. Duringchromatin immunoprecipitation, Lsh can precipitate HP1� af-ter cross-linking (Yan and Muegge, unpublished), suggesting aclose association of Lsh with HP1 on heterochromatic nucleo-somes. However, Lsh is not directly interacting with HP1�,since Flag-tagged Lsh cannot directly precipitate HP1� with-out cross-linking. In order to understand better the signal thatrecruits Lsh to pericentromeric heterochromatin, we examined

FIG. 7. Lsh dissociates from chromatin after TSA treatment. (A) Lsh associates with DAPI in hypomethylated MEFs. Lsh wild-type and Lsh�/�

MEFs were transfected with a GFP-tagged Lsh expression vector and examined after 24 h. (B) TSA induces hyperacteylation. 3T3 cells weretreated in the presence of 75 ng of TSA/ml, and extracts were examined by Western analysis with antibodies raised against histone 3 (H3),acetylated H3 (AcH3), or acetylated H4 (AcH4; equal loading of protein extracts). (C) Lsh dissociates from chromatin after TSA treatment andreassociates after recovery. After TSA treatment or after an additional 24 h of recovery, 3T3 cells were extracted with Triton X, and the differentfractions were examined by Western analysis for the presence of induced Lsh; Vimentin served as a control. Triton X-resistant (lanes R) andsoluble (Triton flush out [lanes F]) (extracts were prepared from equal number of cells) fractions are shown.

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whether Lsh requires higher-order heterochromatin organiza-tion for its localization. The pericentromeric heterochromatinstructure depends on the presence of underacetylated histones.Prolonged treatment with the histone deacetylase inhibitorTSA results in disruption of pericentromric heterochromatinstructure and centromere function (42). HP1�, which is rec-ognized by methylated lysine 9 residue of histone 3, is nolonger retained at centromeric heterochromatin. Also, thebinding of the H3-K9me branched peptide antibody, recogniz-ing a close configuration of methylated lysine residues athistone 3 tails, is disturbed (31, 42). To test whether Lsh’spericentric localization was dependent on higher-order hetero-chromatin organization, we treated 3T3 cells with TSA to in-crease global levels for histone 3 and 4 acetylation (Fig. 7B).After induction of hyperacetylation, 3T3 cells displayed analtered DAPI staining pattern with relocation of centromericregions toward the nuclear periphery (Fig. 8), as has been

previously reported (42). Similarly, staining with the branchedanti-H3-K9 methylation antibody was disturbed, indicating asuccessful disruption of the heterochromatin structure (31)(Fig. 8B). Using anti-Flag antibody as a probe, indirect immu-nofluorescence staining revealed a diffuse staining pattern forLsh and a failure of Lsh to associate with DAPI. This indicatedthat Lsh localization requires a higher-order heterochromatinstructure (Fig. 8A). In order to confirm that Lsh binding topericentromeric heterochromatin depends on a higher-orderstructure, we examined the Triton X resistance of the Lshprotein. As shown in Fig. 7C, almost all Lsh was extractablewith Triton X, indicating a loss of the ability to bind to chro-matin. Under the same conditions HP1� was flushed by TritonX and no longer retained by heterochromatin. To demonstratethat cells did not simply succumb to histone hyperacteylationafter TSA treatment, cells were washed with PBS and culturedfor 24 to 48 h to allow for recovery. The association of either

FIG. 8. Lsh requires higher-order heterochromatin structure for localization. (A) Lsh dissociates from DAPI after TSA treatment andreassociates after recovery. After TSA treatment of 3T3 cells, Lsh was induced and cells were immunostained with anti-Flag antibody. (B) Dis-sociation of branched anti-H3-K9me from DAPI after TSA treatment and reassociation after recovery. The cells were also immunostained withthe antibody raised against the branched H3-K9 methylated peptide.

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Lsh (Fig. 7C) or HP1 (data not shown) with heterochromatinwas reversible and returned to levels of untreated cells withina day, as judged by Western analysis. The colocalization of Lshwith DAPI could also be restored within a day of recovery (Fig.8A). Similarly, the higher-order structure as indicated by stain-ing with the branched H3-K9me antibody reversed after 1 dayof recovery (Fig. 8B). Therefore, the signal that is required forrecruitment of Lsh was not erased even after TSA treatment,and removal of the drug resulted in the relocalization of Lsh.These results suggest that binding of Lsh to pericentromericheterochromatin is dependent on histone modifications andclosely associated with normal higher-order structure of het-erochromatin.

In order to define whether Lsh plays a role in maintenanceversus initiation of pericentromeric heterochromatin, we ex-amined the time course of HP1 association in the absence ofLsh (Table 1). We have recently found that HP1� associationwith pericentromeric heterochromatin is not altered in theabsence of Lsh, despite changes in histone methylation levels(45). In order to determine whether HP1 reassembly is delayedin the absence of Lsh, we disrupted HP1� association withpericentromeric chromatin by TSA treatment in wild-type andLsh-deficient cells. Table 1 demonstrates that the dissociationof HP1 with DAPI occurs in ca. 77 to 85% of cells in bothcultures. The recovery rate of HP1 localization in Lsh-deficientcells after 3, 6, or 12 h resembles that of wild-type cells, furthersupporting the idea that Lsh plays a role in maintaining ratherthan initiating heterochromatin formation, as further discussedbelow.

DISCUSSION

CpG methylation is a major hallmark in mammalian hetero-chromatin (5–7, 9, 34, 39). It plays an important role in genesuppression and is thought to control chromatin structure, forexample, by modulating histone tail modifications. We havepreviously reported that Lsh is a crucial regulator of CpGmethylation in mice (12). Furthermore, we have recently ob-served abnormal H3-K4 methylation patterns at heterochro-matin in the absence of Lsh and reactivation of retroviral geneexpression (45). Thus, Lsh modifies some (although not all, seebelow) characteristics of heterochromatin in mice. In thepresent study, we investigated the possibility that Lsh partici-pates directly in the configuration of heterochromatin as op-posed to indirectly inducing other protein factors. We demon-strated here a nuclear localization of Lsh, a strong association

with chromatin, and a specific accumulation of Lsh at pericen-tromeric heterochromatin. These results provide an importantlink with previous results that demonstrated an effect of Lsh onheterochromatin structure and support a model in which Lsh isa direct participant with pericentromeric heterochromatin andplays an important role in normal heterochromatin formation.

Lsh deletion strongly affects DNA methylation at manyother repetitive sequences that are not pericentromeric. Theeffect of DNA hypomethylation on single-copy genes is lesspronounced (only five of nine genes show hypomethylation bySouthern analysis [see also reference 12]), a phenomen alsoreported in DDM1 mutants. These pronounced Lsh effects onrepetitive sequences are consistent with a model in which Lshprimarily guards heterochromatin at repetitive sites, and alter-ations of chromatin structure after Lsh deletion may thenspread into single-copy genes.

Since Lsh-deficient cells have residual CpG methylation,functional homologues for Lsh may exist that can also localizeto heterochromatin. ATRX, another member of the SNF2chromatin remodeling family, has been reported to localize topericentromeric heterochromatin (4, 33), and ATRX muta-tions cause DNA methylation defects in patients; however,these defects do not occur at pericentromeric DNA sequencesbut at other repetitive elements (20). Possibly, ATRX and Lshmay have redundant roles with respect to DNA methylation atheterochromatic sites such that Lsh substitutes for ATRXfunction. Another SNF2 family member, SNF2H, is associatedwith heterochromatin during late S phase of the cell cycle (11).SNF2H is thought to play a role at heterochromatin by en-abling DNA replication through highly condensed regions dur-ing S phase. An effect on CpG methylation has not yet beendemonstrated. DNA methyltransferases also localize to het-erochromatic regions (1, 10, 32). Dnmt1, thought to be themajor “maintenance” methyltransferase, colocalizes to hetero-chromatin during the late S phase of replication (29, 40). Sincethe global DNA methylation deficiency in Lsh�/� mutants issimilar to the one reported for Dnmt1�/� mutants (12, 30), wehypothesized cooperative effects of Lsh with Dnmt1. Thus, wetested for the role of Lsh in targeting of Dnmt1 to replicationfoci. Although we did not see any difference in the compositionof replication foci with or without Lsh, this does not exclude apossible role for Lsh in promoting the efficiency of Dnmt1methylation. However, these questions have to be resolved inan in vitro system utilizing recombinant Lsh.

Heterochromatin has various properties, and it is a currentchallenge to understand the relationship and sequential re-quirements of distinct chromatin modifications and chromatincomponents (39). A major concern has been the interplay ofhistone modifications and CpG methylation. Several lines ofevidence suggest that histone modifications provide a key sig-nal for DNA methylation in lower organisms. For example,mutants of H3-K9 methyltransferase (such as dim-5 or KYP)lower cytosine methylation in Neurospora crassa or Arabidopsisthaliana (23, 43). Hyperacetylation after treatment with thehistone deacetylase inhibitor TSA affects CpG methylation inNeurospora, a phenomenon not reported for mammalian spe-cies (42). On the other hand, there have been reports support-ing the reverse relationship in which DNA methylation affectshistone modifications. For example, mutants of met1 (aDnmt1-like DNA methyltransferase in Arabidopsis) can reduce

TABLE 1. HP1 recovery after TSA treatmenta

Time (h)% HP1 recovery

LSH�/� cells LSH�/� cells

0 25.2 33.03 58.5 62.56 71.9 74.5

12 78.9 86.9

a MEFs derived from Lsh�/� or Lsh�/� embryos were treated for 5 days withTSA (75 ng/ml), washed, and cultured for the indicated time periods. Cells werethen stained with HP1� antibody and DAPI in order to monitor the recovery ofhigher-order chromatin structure in the absence of Lsh. The percentage indicatesthe percentage of cells in which HP1 colocalized with DAPI.

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H3-K9 methylation (41). Although treatment with the demeth-ylation agent 5-azacytidine does not alter HP1 binding or stain-ing with the branched H3-K9me antibody (42, 45), it does alterH3-K4 methylation levels at heterochromatin (45). In humancancer cells, 5-azacytidine treatment or deletion of DNMT1and DNMT3b can alter the H3-K9 methylation status at thespecific promoter region of tumor suppressor genes, thus link-ing CpG methylation to chromatin modifications in mammals(2, 35). In the present study we observed that the reverserelationship also exists: disruption of higher-order heterochro-matin structure by histone hyperacteylation correlated withdissociation of Lsh from pericentromeric heterochromatin.This suggests a model in which heterochromatin organizationconstitutes the signal for Lsh localization (and CpG methyl-ation), and Lsh (and CpG methylation) plays a role in perpet-uating heterochromatin formation rather than initiating it (7).

Pericentromeric heterochromatin plays an important role inspindle fiber attachment and sister chromatid cohesion duringmitosis. The association of Lsh at pericentric regions and theeffect on the DNA methylation of pericentric satellite se-quences suggest a role in mitosis. We have recently found asevere growth defect in MEF derived from Lsh�/� embryoswith signs of abnormal mitosis, including centrosome hyper-amplification, abnormal spindle formation, and micronucleiformation (15). That treatment with the DNA-demethylatingagent azacytidine mimicked the absence of Lsh (15) suggeststhat Lsh localization to pericentromeric heterochromatin iscrucial to ensuring normal DNA methylation patterns that arerequired for normal mitosis.

At present, we do not know the precise heterochromaticsignal that is required for Lsh localization. A three-dimen-sional configuration may be recognized by Lsh either directlyor indirectly through other heterochromatic components.H3-K9 methylation or HP1 may contribute to Lsh recruitment,a hypothesis awaiting further testing. Recently, short RNAmolecules have been proposed to be involved in the formationof heterochromatin structure, since deletion of RNA interfer-ence machinery inhibits heterochromatin structure in fissionyeast (44). RNA interference leads to short double-strandedRNA that inhibits the accumulation of homologous transcripts.The process appears to be involved in the regulation of H3-K9methylation and heterochromatic gene silencing (44). DNArepeats that allow bidirectional transcription may lead to theformation of double-stranded RNA and have been suggestedas a nucleation center for heterochromatin (21). The pairing ofRNA molecules with corresponding sequences may serve astarget for Suv39h histone methyltransferases (26). After suc-cessful methylation of histone 3 at lysine 9, HP1 is recruitedand contributes to establishment of a higher-order heterochro-matin structure. This in turn may signal Lsh recruitment, whichthen facilitates CpG methylation. DNA methylation, occurringat repetitive sequences and at centromeric sites may then for-tify heterochromatin structure, thus granting its stable propa-gation through multiple rounds of cell division. Thus, our re-sults are consistent with a model in which Lsh, althoughimportant for heterochromatin formation, serves as a link inmaintaining rather than initiating heterochromatin structure.

ACKNOWLEDGMENTS

We are grateful to Joost Oppenheim, Zack Howard, and DougFerris for critical review of the manuscript. We thank Timothy Bestorand Thomas Jenuwein for the generous gifts of the Dnmt1 and thebranched H3-K9me antiserum.

The content of this publication does not necessarily reflect the viewsor policies of the Department of Health and Human Services, nor doesthe mention of trade names, commercial products, or organizationsimply endorsement by the U.S. government.

This project was funded in whole or in part with federal funds fromthe National Cancer Institute and the Alternative Medicine Program,National Institutes of Health, under contract no. NO1-CO-12400.

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