CELL BIOLOGY. For the article ‘‘Gliding ghosts of Mycoplasmamobile,’’ by Atsuko Uenoyama and Makoto Miyata, whichappeared in issue 36, September 6, 2005, of Proc Natl Acad SciUSA (102:12754–12758; first published August 26, 2005;10.1073�pnas.0506114102), the authors note that on page 12754,right column, second full paragraph, the last sentence, ‘‘Thisstrain had a substitution at the 859th amino acid of the gli521gene, from serine to arginine, and was named gli521 mutant(S859R),’’ should instead read: ‘‘This strain had a substitution atthe 476th amino acid of the gli521 gene, from proline to arginine,and was named gli521 mutant (P476R).’’ Additionally, in thethird full paragraph, first sentence, the phrase ‘‘Cultured cells ofthe gli521 mutant (S859R) were collected’’ should instead read:‘‘Cultured cells of the gli521 mutant (P476R) were collected.’’These errors do not affect the conclusions of the article.

www.pnas.org�cgi�doi�10.1073�pnas.0708463104

DEVELOPMENTAL BIOLOGY. For the article ‘‘Lsh controls Hox genesilencing during development,’’ by Sichuan Xi, Heming Zhu,Hong Xu, Anja Schmidtmann, Theresa M. Geiman, and KathrinMuegge, which appeared in issue 36, September 4, 2007, of ProcNatl Acad Sci USA (104:14366–14371; first published August 28,2007; 10.1073�pnas.0703669104), the authors note that refs. 25and 26 were inadvertently omitted from the article. The refer-ences appear below. This error does not affect the conclusionsof the article.

25. Liu S, Dontu G, Mantle ID, Patel S, Ahn NS, Jackson KW, Suri P, Wicha MS(2006) Cancer Res 66:6063–6071.

26. van der Lugt NM, Domen J, Linders K, van Roon M, Robanus-Maandag E,te Riele H, van der Valk M, Deschamps J, Sofroniew M, van Lohuizen M, BernsA (1994) Genes Dev 8:757–769.

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Lsh controls Hox gene silencing during developmentSichuan Xi, Heming Zhu, Hong Xu, Anja Schmidtmann, Theresa M. Geiman, and Kathrin Muegge*

Laboratory of Cancer Prevention, SAIC-Frederick, National Cancer Institute, Frederick, MD 21702-1201

Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved July 26, 2007 (received for review April 20, 2007)

Polycomb-mediated repression and DNA methylation are impor-tant epigenetic mechanisms of gene silencing. Recent evidencesuggests a functional link between the polycomb repressive com-plex (PRC) and Dnmts in cancer cells. Here we provide evidence thatLsh, a regulator of DNA methylation, is also involved in normalcontrol of PRC-mediated silencing during embryogenesis. We dem-onstrate that Lsh, a SNF2 homolog, can associate with some Hoxgenes and regulates Dnmt3b binding, DNA methylation, and si-lencing of Hox genes during development. Moreover, Lsh canassociate with PRC1 components and influence PRC-mediated hi-stone modifications. Thus Lsh is part of a physiological feedbackloop that reinforces DNA methylation and silencing of PRC targets.

chromatin � DNA methylation � polycomb � Lsh

Recently, several connections have been suggested betweenDNA methylation (1, 2) and polycomb repressive complex

(PRC-mediated silencing (3) in cancer cells. First, it was dem-onstrated that genes that are CpG methylated in cancer cells arefrequently marked by PRC binding and histone 3 K27 methyl-ation early in development (4–6). In addition, it was reportedthat Ezh2 (a PRC2 component) controls Dnmt binding andDNA methylation at the Myt1 gene in cancer cell lines (7). Theseresults suggest that PRC may premark sites for de novo DNAmethylation at genes methylated in cancer, but this may be a rareand aberrant event in cells predisposed to cancer.

In this study, we tested whether PRC-mediated silencing andDNA methylation are normally linked during development,using the Lsh knockout model (8). Lsh, a member of the SNF2chromatin remodeling family (9, 10), is involved in the control ofDNA methylation patterns during embryonic development (11,12). Lsh-mediated DNA methylation is crucial for retroviralsilencing and repression of selected imprinted loci (13, 14). Lshmay be directly involved in the control of de novo methylation viaassociation with Dnmt3a and -3b in embryonic stem cells (15).Because Lsh�/� mice die at birth, we used Lsh�/� embryos toexamine a functional link between Lsh, DNA methylation, andsilencing of selected PRC targets such as Hox genes.

ResultsWe first examined Hox gene expression in various tissues fromLsh�/� mice. RT-PCR analysis shows HoxA5, HoxA6, andHoxA7 genes are silenced in wild-type murine embryonic fibro-blasts (MEFs) but reactivated in Lsh�/� MEFs (Fig. 1A). Incontrast, HoxA10 and HoxA11 genes are already expressed inwild-type MEFs and not significantly changed in the absence ofLsh, as revealed by conventional RT-PCR analysis as wellas real-time PCR analysis (Fig. 1). Similarly, liver, brain,and whole-embryo tissues showed derepression of HoxA6 andHoxA7 genes after Lsh depletion but unchanged HoxA10and HoxA11 gene expression levels (Fig. 1 A). In addition,HoxB4, HoxC6, HoxC8, and HoxD13 were derepressed inLsh�/� MEFs (Table 1), indicating that Lsh does not exclusivelyaffect silencing of the HoxA gene cluster. Thus, Lsh is animportant transcriptional regulator of selected PRC targetsduring normal development.

To address the molecular mechanism, we examined the DNAmethylation pattern at Hox genes. Using methylation-sensitivePCR, genomic DNA derived from Lsh�/� MEFs and Lsh�/�

liver samples was found sensitive to HpaII or AciI digestion incomparison with wild-type controls, indicating loss of methyl-ation at several sites located at the promoter region of HoxA6and HoxA7 genes. In addition, several sites in the gene body ofHoxA6 that were previously shown to bind PRC components(16) were hypomethylated in the absence of Lsh (Fig. 2). Toconfirm and quantify the methylation results bisulphite sequenc-ing was used, examining the same CpG regions of the HoxA6,HoxA7 genes as well as the control HoxA10 promoter region(Fig. 2 A). DNA methylation levels were reduced at two HoxA6sites, and a HoxA7 region comparing Lsh�/� MEFs to Lsh�/�

MEFs (58% vs. 18% and 81% vs. 54% at HoxA6 and 43% vs.16% at HoxA7) (Fig. 3). Moreover, Lsh�/� brain tissue revealedan even more pronounced loss of CpG methylation comparedwith wild-type controls (55% vs. 5%, 88% vs. 44% at HoxA6 and44% vs. 9% at HoxA7). In contrast, methylation levels at the

Author contributions: S.X., H.Z., H.X., A.S., T.M.G., and K.M. designed research; S.X., H.Z.,H.X., A.S., and T.M.G. performed research; S.X., H.Z., H.X., A.S., T.M.G., and K.M. analyzeddata; and S.X. and K.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Abbreviations: MEF, murine embryonic fibroblasts; PRC, polycomb repressive complex.

*To whom correspondence should be addressed. E-mail: muegge@ncifcrf.gov.

This article contains supporting information online at www.pnas.org/cgi/content/full/0703669104/DC1.

Fig. 1. Lsh deletion causes de-repression of Hox genes in various tissues. (A)RT-PCR analysis for detection of the indicated HoxA genes derived from Lsh�/�

and Lsh�/� MEFs, liver, brain, or whole embryo tissue (day 18 of gestation). (B)Real-time PCR analysis of HoxA gene expression comparing wild-type MEFswith Lsh�/� MEFs.

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HoxA10 promoter (that showed no change in gene expressionafter Lsh depletion) were unaltered in the absence of Lsh ineither MEFs or brain tissue (32% vs. 30% and 28% vs. 28%) (Fig.3C). Thus alterations in the DNA methylation pattern werecorrelated with transcriptional changes and controlled by Lsh.

To investigate whether Lsh is directly involved in Hox genesilencing, we initially examined the expression levels of knownPRC1 components to test whether Lsh deletion affects theirexpression level. However, there was no evidence that eitherPRC1 components such as Bmi1 (Pcgf4), M33 (Cbx2, mPc1),Mel18 (Pcgf2, Rnf110, and Zfp144), or PRC2 components suchas Ezh2 (PRC2) were differentially expressed when comparingnuclear extracts derived from Lsh�/� and Lsh�/� MEFs (Fig.

4A). Next we examined whether Lsh could directly associate withHoxA genes in P19 cells or MEFs (Fig. 4B). Using ChIPs withanti-Lsh antibodies followed by real-time PCR, we comparedgenes that were reactivated by Lsh deletion (HoxA6 and HoxA7)with those that were not affected (HoxA10). Whereas the HoxA6and HoxA7 genes that were affected by Lsh deletion revealedbinding of Lsh, the promoter regions of HoxA10 showed reducedassociation, and hardly any association was detected with inter-genic control regions. As expected, no significant binding for Lshwas detected for either HoxA sites in Lsh�/� MEFs. Thissuggested that Lsh may play a direct role in the methylation atsome HoxA sites. Next, we tested whether Lsh and Dnmts canassociate with PRC1 components. Using nuclear extracts derivedfrom P19 embryonal carcinoma cells (because these cells arehigh in de novo methyltransferase activity and Lsh proteinlevels), DNA methyltransferase activity was found to be associ-ated with immunoprecipitates after using specific antibodiesagainst Bmi1, M33, and Mel18 but was not detectable afterprecipitation using antibodies against Pol II as control (Fig. 4 Cand D). The activity was comparable to that found after pre-cipitation of Dnmt3b, the PRC2 subunit Ezh2 (7), or Lsh (15).In addition, immunoprecipitations of PRC1 components dem-onstrated a specific association between Bmi1, Mel18, or M33with Lsh (Fig. 4E) and vice versa, specific immunoprecipitationof Lsh or Dnmt3b demonstrated an interaction with PRC1components (Fig. 4E). The association between Dnmt3b andBmi1 was readily detectable in wild-type MEF nuclear extractsbut reduced in extracts derived from Lsh�/� MEFs (Fig. 4F).This suggested that the interaction of Dnmt3 with PRC at leastin part depends on the presence of Lsh, and that it may be Lshrather than DNA that performs a scaffolding-like function andpromotes this interaction. Taken together, these data suggest a

Table 1. Summary of the gene expression patterns at differentHox clusters in Lsh�/� or Lsh�/� tissues

MEF Brain Liver

Lsh�/� Lsh�/� Lsh�/� Lsh�/� Lsh�/� Lsh�/�

HoxA2 � � � � � �

HoxA5 � � � � � �

HoxA6 � � � � � �

HoxA7 � � � � � �

HoxA10 � � � � � �

HoxB3 � � � � � �

HoxB4 � � � � � �

HoxB6 � � � � � �

HoxC6 � � � � � �

HoxC8 � � � � � �

HoxC9 � � � � � �

HoxD10 � � � � � �

HoxD13 � � � � � �

Fig. 2. Decreased CpG methylation after Lsh deletion at selected Hox genesites. (A) Map of the HoxA6, HoxA7, and HoxA10 genes illustrating thelocation of primers (black triangles). The methylation-sensitive restrictionenzyme sites HpaII and AciI are shown, as well as the primers (designated F1/R1to F6/R6) used for methylation-sensitive PCR analysis. The ChIP primers aredesignated P1/2, P3/4, and P5/6. The regions analyzed for bisulphate sequenc-ing are indicated with a double arrow. (B) Methylation-sensitive PCR analysisusing genomic DNA derived from Lsh�/� and Lsh�/� MEFs or liver after diges-tion with HpaII or AciI. PCR analysis amplifying a region around an AciI siteafter HpaII digestion (or HpaII site after AciI digestion) served as a control forequal input of DNA. The primers F6/R6 amplify a region lacking methylationsensitive restriction enzyme sites.

Fig. 3. Lsh deletion reduces DNA methylation at HoxA6 and HoxA7 sites. (A)Genomic DNA derived from Lsh�/� and Lsh�/� MEFs or brain was subjected tobisulfite sequencing and examined at regions for two regions of the HoxA6gene, as indicated in the map of Fig. 2A. Methylated CpG are presented byblack circles and unmethylated sites by open circles. (B) Bisulfite sequencinganalysis for MEFs and brain at the HoxA7 gene. (C) Bisulfite sequencinganalysis for MEFs and brain at the HoxA10 gene.

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model in which Lsh plays a direct role in the control of Hox genesilencing.

To understand whether Lsh can affect association of Dnmt3bto target sites and whether the presence of Lsh can modulatePRC-associated activities, histone modifications were examinedby ChIP. Using quantitative PCR analysis, first histone acetyla-tion, a marker for transcriptional activation, was examinedcomparing genes whose expression levels were affected by theabsence of Lsh (such as HoxA6 and HoxA7) with HoxA10 thatwas unaffected by Lsh (Fig. 5A). H3 acetylation was enhanced inLsh�/� MEFs at HoxA6 and HoxA7 loci but not at the HoxA10gene and thus correlated well with transcriptional changes. Incontrast to histone acetylation, Dnmt3b binding was reduced atHoxA6 and HoxA7 genes (2- and 9-fold, respectively) whencomparing Lsh�/� MEFs to wild type (Fig. 5B). These datasupport the idea that Lsh at least in part promotes association ofDnmt3b to specific sites at Hox genes. To investigate whetherLsh and DNA methylation affect PRC-mediated histone mod-ifications, we first analyzed H2A ubiquitylation mediated byPRC1 (17) (Fig. 5C). Whereas HoxA6 and HoxA7 sites revealeda reduction of H2A-K116 ubiquitylation of �7-fold in Lsh�/�

samples compared with wild-type controls, HoxA10 sites wereunchanged. Lsh deletion resulted in reduced association of M33and Mel18 to HoxA6 and HoxA7 loci (ranging from 5- to

13-fold), in contrast to HoxA10 sites that were unaffected (Fig.4 D and E). Bmi1 binding, though, did not show a reduction,suggesting that the recruitment of Bmi1 is independent of PRC2activity and not sufficient to maintain silencing (Fig. 5F).However, decrease of M33 and Mel18 binding and reduced H2Aubiquitylation suggest that Lsh/DNA methylation was importantfor complete assembly and activity of the PRC1 complex.

Because PRC1 recruitment via M33 (mPc1) is thought todepend on PRC2-mediated histone methylation (18), we exam-ined Ezh2 binding and H3-K27 trimethylation levels. Both markswere decreased in Lsh�/� MEFs at HoxA6 and HoxA7 sites (2-to 4-fold) and unperturbed at HoxA10 sites compared withwild-type samples (Fig. 5 G and H). These data suggest thatPRC2 cannot fully assemble in the absence of Lsh and DNAmethylation.

DNA methylation has long been known to participate ingenomic imprinting, X inactivation, repression of repeats, andsilencing of tumor suppressor genes (1, 2). Here we provideevidence that Lsh can associate with some Hox genes, controlsDNA methylation levels at Hox genes, and is also crucial fornormal developmental regulation of Hox gene expression pat-tern. We further demonstrate that PRC1 and PRC2 activities aretightly correlated to DNA methylation, and that there may be afeedback loop between DNA methylation and PRC-mediated

Fig. 4. Lsh is associated with PRC1 components. (A) Western blot analysis for detection of Bmi1, M33, Mel18, and Ezh2 using nuclear extracts derived from Lsh�/�

and Lsh�/� MEFs. Detection of Pcna and Lsh served as controls. (B) ChIP assays were performed on chromatin extracts derived from P19 (gray bar), Lsh�/� (blackbar), and Lsh�/� (open bar) MEFs using anti-Lsh antibodies and control IgG to detect association to specific HoxA6, HoxA7, and HoxA10 sites. Primers used forreal-time PCR analysis are illustrated in Fig. 2A. In addition, two control primers were designed that are located within the HoxA cluster but �3,000 bp awayfrom the HoxA10 or HoxA11 genes. The percentage of input was calculated for each precipitate. The values for the IgG control were �0.1% of input. (C and D)Nuclear extracts of P19 cells were immunoprecipitated with the indicated specific antibodies and assayed for DNA methyltransferase activity. (E) Western blotanalysis for detection of Lsh after immunoprecipitation with anti-Bmi1, anti-Mel18, or anti-M33 antibodies using P19 nuclear extracts. Species-matched IgG oromission of antibody (Mock) served as controls. Western blot analysis for detection of Mel18, Bmi1, M33 after IP using anti-Lsh, or anti-Dnmt3b antibodies. (F)Western blot analysis for detection of Bmi1 after immunoprecipitation with anti-Dnmt3b comparing extracts derived from Lsh�/� or Lsh�/� MEFs.

14368 � www.pnas.org�cgi�doi�10.1073�pnas.0703669104 Xi et al.

histone modifications. This supports the idea of a complexnetwork of diverse epigenetic modifications rather than a simplesequential activation cascade during mammalian embryogenesis.

Based on Lsh homology with SNF2 family members, part ofits activity may depend on presumed nucleosomal remodelingactivity that may allow for better access of DNA-binding proteinsto their nucleosomal target sites (9, 10). In addition, Lsh may alsohave chromatin remodeling-independent or scaffold-like func-tions, for example in promoting Dnmt3 activity or stabilizing theinteractions of proteins. The observation that the association ofDnmt3b with Bmi1 is influenced by Lsh (Fig. 4F) would beconsistent with a role of Lsh in scaffolding function. Possibly, viaits association with PRC components, Lsh may bind to Hox lociand promote targeting of Dnmt. Alternatively, other not-yet-defined factors may recognize PRC-mediated histone modifica-tions and lead to Lsh and subsequent Dnmt3b recruitment.Increased DNA methylation may result in decreased histoneacetylation levels and a decline in transcription. Histone 3-K4methyltransferases coupled to Pol II may alter H3-K4 methyl-ation levels and may ultimately prevent spreading of the repres-sive H3-K27me mark (19). As another possibility, noncodingRNA transcripts that are prevalent in mammalian Hox clusters(20) may demarcate regions of gene silencing by regulatingPRC2 occupancy and H3-K27me levels (21). A rise in H3-K27memay enhance PRC1 targeting to Hox genes (18). Alternatively,

DNA methylation itself may affect PRC1 binding, as has beenshown for reduced Bmi1 localization to PcG bodies after Dnmt1depletion (22). More PRC1 binding may further promote DNAmethylation and subsequent H3-K27methylation reinforcing thesilencing marks in several feedback loops.

Although PRC silencing is highly conserved in differentspecies, Drosophila and Caenorhabditis elegans do not showsignificant levels of genomic DNA methylation and lack an Lshhomolog. Thus the involvement of Lsh-mediated DNA methyl-ation in PRC silencing shows a complexity that is unique tohigher organisms. We hypothesize that Lsh associates only witha subset of polycomb complexes, because PRC components canassemble into various functionally distinct complexes and, as wereport here, Lsh affects only some but not all examined Hoxgenes. More than 1,000 potential target sites have been reportedfor PRC components, and Lsh may also affect some of them (16,23, 24). Thus the biologic activities of Lsh may be partiallyoverlapping with PRC activities and may include effects on stemcell properties of breast epithelium, hematopoietic, and neuro-nal precursor potential and effects on skeletal development (3,25, 26).

Cancer cells are long known to show aberrant DNA methyl-ation patterns (1, 2) and recent evidence suggests that hyper-methylation at promoter regions is linked to PRC binding (3–7).This study suggests that PRC-mediated silencing and DNAmethylation are not aberrantly connected in cancer cells but partof an ordinary regulatory pathway involving Lsh. Whether thislink is unique for embryonic cells or is also present in adultdifferentiated cells and why these pathway are targeted in cancerto loci that are usually unmethylated remains unknown, but Lshis one possible candidate that could play a role in aberrantrecruitment of Dnmts. On the other hand, cancer is also asso-ciated with global DNA hypomethylation, which in turn mayderepress some Hox genes. Deregulation of Hox genes has beenimplied in hematopoietic malignancies and ectopic Hox geneexpression determines the phenotype in ovarian epithelial cellcancer (3, 27, 28). The suggested connection between the twoepigenetic pathways may shed new light on the molecularmechanisms involved in tumorigenesis and may prove helpful toimproving strategies for cancer treatment or prevention infuture.

MethodsWestern Blot Analysis and Immunoprecipitations. Samples wereseparated on 4–12% Tris-glycine SDS/PAGE gels and blottedonto Immobilon P membrane (Millipore, Bedford, MA). West-ern blotting was performed according to standard procedures byusing ECL detection reagents, according to the manufacturer’sinstructions (Amersham, Piscataway, NJ). Nuclear extracts wereprepared as described (29). For immunoprecipitations, the nu-clear extract buffer was adjusted to a final concentration of 50mM Tris (pH 7.5)/150 mM NaCl/1 mM EDTA/0.5% NonidetP-40. Nuclear extracts (200 �g) were precleared for 30 min withprotein G agarose (Invitrogen, Carlsbad, CA) and then incu-bated with 20 �l of antibodies for 2 h or overnight at 4°C.Washing was performed three times in 500 �l of buffer [50 mMTris (pH 7.5)/150 mM NaCl/1 mM EDTA/0.5% Nonidet P-40]at 4°C, 5 min each cycle on a rotator. Antibodies used forimmunoprecipitation or Western analysis were species-matchednormal IgG (Santa Cruz Biotechnology, Santa Cruz, CA), rabbitanti-Lsh recombinant protein affinity-purified antibody, anti-Dnmt3b (Alexis, San Diego, CA), anti-Bmi1 (Upstate Biotech-nology, Lake Placid, NY), anti-Ezh2 (Upstate Biotechnology),anti-Mel18 (Abcam, Cambridge, MA), anti-M33 antibody (BDTransduction Laboratories, Franklin Lanes, NJ), anti-Pcna(Santa Cruz Biotechnology), and anti-Pol II antibody (UpstateBiotechnology). The following secondary antibodies were used:goat anti-rabbit HRP-conjugated IgG, goat anti-mouse HRP-

Fig. 5. Lsh controls Dnmt3b recruitment and PRC-mediated histone modi-fications at Hox sites. ChIP assays were performed from chromatin extracts ofLsh�/� (open bar) and Lsh�/� (black bar) MEFs using the indicated antibodiesto detect specific histone modifications or association of specific proteins atHoxA genes. Primers used for real-time PCR analysis are illustrated in Fig. 2A.The percentage of input was calculated for each precipitate and the valuesexpressed as ratio of Lsh�/� samples over wild type. The following antibodieswere used: (A) Antiacetylation of H3. The asterix indicate the minimum ratioabove wild-type controls, because the actual values for Lsh�/� samples ex-ceeded the range of the standard curve. (B) Anti-Dnmt3b. (C) Anti-H2A-K116ubiquitylation. (D) Anti-M33. (E) Anti-Mel18. (F) Anti-Bmi1. (G) Anti-H3-K27trimethylation. (H) Anti-Ezh2.

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conjugated IgG, and rabbit anti-goat HRP-conjugated IgG(Santa Cruz Biotechnology).

In Vitro DNA Methyltransferase Activity Assay. After 150 �l ofnuclear extracts (29) derived from P19 cells was incubated withantibodies and protein G agarose for 2 h or overnight at 4°C withrotation, agarose beads were washed three times with nuclearextraction buffer and then again incubated with 150 �l of freshnuclear extracts and antibodies to improve the yield of DNAmethyltransferase activity. After a second round of incubation andwashes, the beads were rinsed with DNA methyltransferase assaybuffer (50 mM Tris, pH 7.8/1 mM EDTA/1 mM DTT/10%glycerol/1% Tween) and frozen at �80°C until future analysis.Assays were performed with immunoprecipitated material stillattached to agarose beads. DNA methyltransferase activity wasanalyzed by the standard glass fiber method using S-adenosyl-L-(3H-methyl)-methionine (Amersham–Amersham Pharmacia) asthe methyl donor and poly(d[I-C]) as the DNA substrate with anincubation time of 1.5 h. After washing, filters containing 3H wereplaced in scintillation fluid, and the level of radioactivity wascounted. Two immunoprecipitations were performed on indepen-dent nuclear extracts for each antibody and normal species IgGcontrols. The average of two immunoprecipitations using indepen-dent nuclear extracts was graphed with error bars representing thestandard deviation.

Methylation-Sensitive PCR. DNA was extracted by using theDNeasy kit (Qiagen, Valencia, CA). DNA was completelydigested with HpaII or AciI. To analyze the methylation statusof the genomic DNA, the following PCR primer pairs A6(F1/R1), A6(F2/R2), A6(F3/R3), A6(F4/R4), A7(F5/R5), andA10 (F6/R6) were used, as listed in supporting information (SI)Text.

PCRs were carried out as follows: 5 min at 94°C, 35 cycles of60 s at 94°C, 30 s at 60°C, and 60 s at 72°C, and finally 5 min at72°C. The PCR products were electrophoresed on 1% agarosegels, stained with ethidium bromide, and photographed.

RT-PCR. Total RNA was prepared from MEF cells, smashedembryos (day 18 of gestation), liver, and brain tissue (day 18 ofgestation) using TRIzol reagent (Invitrogen), according to themanufacturer’s instructions. Any genomic DNA present waseliminated with TURBO DNA-free Kit (Ambion, Austin, TX).Approximately 1 �g of total RNA was reverse-transcribed byusing iScript reverse transcriptase (Bio-Rad, Hercules, CA).Omission of reverse transcriptase served as a negative control.cDNA was amplified by using Platinum PCR SuperMix (Invitro-gen). PCR followed by agarose gel electrophoresis using Hoxprimers (17) was performed as follows: 5 min at 94°C, 35 cyclesof 60 s at 94°C, 60 s at 56–59°C, and 60 s at 72°C, followed by onecycle of 5 min at 72°C. For real-time PCR, the following primerswere used as listed in SI Text.

ChIP. For ChIP, cells were cross-linked with 1% formaldehyde,lysed, and sonicated on ice to generate DNA fragments with anaverage length of 200–800 bp. After preclearing, 1% of each

sample was saved as input fraction. Immunoprecipitation wasperformed by using specific antibodies against the indicatedproteins or IgG of different species used as control. Afterreversal of cross-linking, nucleic acids were prepared from theeluted complex, and PCR analysis was performed. Amplificationconditions were as follows: 94°C for 4 min; 94°C for 1 min; 55°Cfor 1 min; 72°C for 1 min (35 cycles) and 72°C for 7 min. Thefollowing antibodies were used for ChIPs: H3K27 triM, triMAcetyl-H3 (Lys-9/14), ubiquityl-histone H2A, Bmi1, Ezh2 anti-bodies (Upstate Biotechnology), anti-Lsh recombinant proteinaffinity-purified antibody, Dnmt3b antibody (Alexis), Mel18antibody (Abcam), and M33 antibody (BD Transduction Labo-ratories).

Real-time PCR primer pairs for ChIPs analysis are listed in SIText.

Real-Time PCR Analysis. For real-time PCR analysis, the MyiQSingle-Color Real-Time PCR machine (Bio-Rad) and PlatinumSYBR Green qPCR SuperMix UDG (Invitrogen) were used.The PCR for ChIPs was initiated with one cycle of 95°C for 3min, followed by 45 cycles of 95°C for 30 s, 55°C for 30 s, and 72°Cfor 30 s. PCR for the RT-PCR analysis was initiated with onecycle of 95°C for 3 min, followed by 45 cycles of 95°C for 30 s,59°C for 30 s, and 72°C for 30 s. The negative control withouttemplate was carried out for each PCR analysis. To quantify theamount of the template using real-time PCR data, standardtitration experiments for each template and each primer set wereperformed, and linear regression equation and the calculationfor DNA amounts were established by using Prism 3.0 software(GraphPad, San Diego, CA) and Microsoft (Redmond, WA)Excel. Every ChIP experiment includes species-specific IgGcontrols. The results have been calculated as percentage of Input(which lay usually between 5% and 20%) and then expressed asratio of Lsh�/� over wild type. For better comparison, thewild-type samples were set to one, and the values expressed asratio of Lsh�/� samples over wild type.

Bisulphite Sequencing. Genomic DNA from MEF cells and braintissue (day 18 of gestation) was subjected to bisulfite treatmentby using CpGenome DNA modification kit (Chemicon Interna-tional, Temecula, CA) according to the manufacturer’s instruc-tions. The PCR products were separated in agarose gels andpurified by using the QIAEX II gel extraction kit (Qiagen).Amplified fragments were subcloned into the pCR2.1-TOPOvector with the TOPO TA Cloning Kit (Invitrogen). Indepen-dent clones for each fragment were sequenced by using the M13F or M13 R and only sequences with individual fingerprintselected from analysis. The primers used are listed in SI Text.

We thank Rodney Wiles and Terry Stull for excellent technical assis-tance. We thank Nancy Colburn and Peter Johnson for helpful discussionof the manuscript. This project has been funded in whole or part withfederal funds from the National Cancer Institute, National Institutes ofHealth, under Contract No. N01-C0-12400. This research was supportedby the Intramural Research Program of the National Institutes of Health,National Cancer Institute, Center for Cancer Research.

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