CELL BIOLOGY. For the article ‘‘Gliding ghosts of Mycoplasma DEVELOPMENTAL BIOLOGY. For the article ‘‘Lsh controls Hox mobile,’’ by Atsuko Uenoyama and Makoto Miyata, which silencing during development,’’ by Sichuan Xi, Heming Zhu, appeared in issue 36, September 6, 2005, of Proc Natl Acad Sci Hong Xu, Anja Schmidtmann, Theresa M. Geiman, and Kathrin USA (102:12754–12758; first published August 26, 2005; Muegge, which appeared in issue 36, September 4, 2007, of Proc 10.1073͞pnas.0506114102), the authors note that on page 12754, Natl Acad Sci USA (104:14366–14371; first published August 28, right column, second full paragraph, the last sentence, ‘‘This 2007; 10.1073͞pnas.0703669104), the authors note that refs. 25 strain had a substitution at the 859th amino acid of the gli521 and 26 were inadvertently omitted from the article. The refer- gene, from serine to , and was named gli521 mutant ences appear below. This error does not affect the conclusions (S859R),’’ should instead read: ‘‘This strain had a substitution at of the article. the 476th amino acid of the gli521 gene, from proline to arginine, and was named gli521 mutant (P476R).’’ Additionally, in the third full paragraph, first sentence, the phrase ‘‘Cultured cells of 25. Liu S, Dontu G, Mantle ID, Patel S, Ahn NS, Jackson KW, Suri P, Wicha MS the gli521 mutant (S859R) were collected’’ should instead read: (2006) Cancer Res 66:6063–6071. ‘‘Cultured cells of the gli521 mutant (P476R) were collected.’’ 26. van der Lugt NM, Domen J, Linders K, van Roon M, Robanus-Maandag E, These errors do not affect the conclusions of the article. te Riele H, van der Valk M, Deschamps J, Sofroniew M, van Lohuizen M, Berns A (1994) Dev 8:757–769.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708463104 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708754104 CORRECTION

PNAS ͉ October 9, 2007 ͉ vol. 104 ͉ no. 41 ͉ 16389 Downloaded by guest on September 28, 2021 Lsh controls Hox gene silencing during development

Sichuan 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 evidence suggests a functional link between the polycomb repressive com- plex (PRC) and Dnmts in cancer cells. Here we provide evidence that Lsh, a regulator of DNA methylation, is also involved in normal control of PRC-mediated silencing during embryogenesis. We dem- onstrate that Lsh, a SNF2 homolog, can associate with some Hox genes and regulates Dnmt3b binding, DNA methylation, and si- lencing of Hox genes during development. Moreover, Lsh can associate with PRC1 components and influence PRC-mediated hi- stone modifications. Thus Lsh is part of a physiological feedback loop that reinforces DNA methylation and silencing of PRC targets. chromatin ͉ DNA methylation ͉ polycomb ͉ Lsh

ecently, several connections have been suggested between RDNA 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 are frequently marked by PRC binding and histone 3 K27 methyl- ation early in development (4–6). In addition, it was reported that Ezh2 (a PRC2 component) controls Dnmt binding and DNA methylation at the Myt1 gene in cancer cell lines (7). These results suggest that PRC may premark sites for de novo DNA methylation at genes methylated in cancer, but this may be a rare and aberrant event in cells predisposed to cancer. 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 LshϪ/Ϫ In this study, we tested whether PRC-mediated silencing and ϩ ϩ and Lsh / MEFs, liver, brain, or whole embryo tissue (day 18 of gestation). (B) DNA methylation are normally linked during development, Real-time PCR analysis of HoxA comparing wild-type MEFs using the Lsh knockout model (8). Lsh, a member of the SNF2 with LshϪ/Ϫ MEFs. chromatin remodeling family (9, 10), is involved in the control of DNA methylation patterns during embryonic development (11, 12). Lsh-mediated DNA methylation is crucial for retroviral liver samples was found sensitive to HpaII or AciI digestion in silencing and repression of selected imprinted loci (13, 14). Lsh comparison with wild-type controls, indicating loss of methyl- may be directly involved in the control of de novo methylation via ation at several sites located at the promoter region of HoxA6 association with Dnmt3a and -3b in embryonic stem cells (15). and HoxA7 genes. In addition, several sites in the gene body of Because LshϪ/Ϫ mice die at birth, we used LshϪ/Ϫ embryos to HoxA6 that were previously shown to bind PRC components examine a functional link between Lsh, DNA methylation, and (16) were hypomethylated in the absence of Lsh (Fig. 2). To silencing of selected PRC targets such as Hox genes. confirm and quantify the methylation results bisulphite sequenc- ing was used, examining the same CpG regions of the HoxA6, Results HoxA7 genes as well as the control HoxA10 promoter region We first examined Hox gene expression in various tissues from (Fig. 2A). DNA methylation levels were reduced at two HoxA6 Ϫ/Ϫ Lsh mice. RT-PCR analysis shows HoxA5, HoxA6, and sites, and a HoxA7 region comparing Lshϩ/ϩ MEFs to LshϪ/Ϫ HoxA7 genes are silenced in wild-type murine embryonic fibro- MEFs (58% vs. 18% and 81% vs. 54% at HoxA6 and 43% vs. Ϫ/Ϫ blasts (MEFs) but reactivated in Lsh MEFs (Fig. 1A). In 16% at HoxA7) (Fig. 3). Moreover, LshϪ/Ϫ brain tissue revealed contrast, HoxA10 and HoxA11 genes are already expressed in an even more pronounced loss of CpG methylation compared wild-type MEFs and not significantly changed in the absence of with wild-type controls (55% vs. 5%, 88% vs. 44% at HoxA6 and Lsh, as revealed by conventional RT-PCR analysis as well 44% vs. 9% at HoxA7). In contrast, methylation levels at the as real-time PCR analysis (Fig. 1). Similarly, liver, brain, and whole-embryo tissues showed derepression of HoxA6 and HoxA7 genes after Lsh depletion but unchanged HoxA10 Author contributions: S.X., H.Z., H.X., A.S., T.M.G., and K.M. designed research; S.X., H.Z., and HoxA11 gene expression levels (Fig. 1A). In addition, H.X., A.S., and T.M.G. performed research; S.X., H.Z., H.X., A.S., T.M.G., and K.M. analyzed HoxB4, HoxC6, HoxC8, and HoxD13 were derepressed in data; and S.X. and K.M. wrote the paper. LshϪ/Ϫ MEFs (Table 1), indicating that Lsh does not exclusively The authors declare no conflict of interest. affect silencing of the HoxA gene cluster. Thus, Lsh is an This article is a PNAS Direct Submission. important transcriptional regulator of selected PRC targets Freely available online through the PNAS open access option. during normal development. Abbreviations: MEF, murine embryonic fibroblasts; PRC, polycomb repressive complex. To address the molecular mechanism, we examined the DNA *To whom correspondence should be addressed. E-mail: [email protected]. methylation pattern at Hox genes. Using methylation-sensitive This article contains supporting information online at www.pnas.org/cgi/content/full/ Ϫ Ϫ Ϫ Ϫ PCR, genomic DNA derived from Lsh / MEFs and Lsh / 0703669104/DC1.

14366–14371 ͉ PNAS ͉ September 4, 2007 ͉ vol. 104 ͉ no. 36 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703669104 Table 1. Summary of the gene expression patterns at different Hox clusters in Lsh؊/؊ or Lsh؉/؉ tissues MEF Brain Liver

Lshϩ/ϩ LshϪ/Ϫ Lshϩ/ϩ LshϪ/Ϫ Lshϩ/ϩ LshϪ/Ϫ

HoxA2 ϩϩϪϩϩϩ HoxA5 ϪϩϩϩϪϪ HoxA6 ϪϩϪϩϪϩ HoxA7 ϪϩϪϩϪϩ HoxA10 ϩϩϩϩϩϩ HoxB3 ϩϩϪϪϪϩ HoxB4 ϪϩϪϩϪϩ HoxB6 ϩϩϩϩϩϩ HoxC6 ϪϩϪϩϩϩ HoxC8 ϪϩϪϪϪϪ HoxC9 ϩϩϩϩϩϩ HoxD10 ϩϩϩϩϩϩ HoxD13 ϪϩϪϪϪϪ

HoxA10 promoter (that showed no change in gene expression after Lsh depletion) were unaltered in the absence of Lsh in either MEFs or brain tissue (32% vs. 30% and 28% vs. 28%) (Fig. 3C). Thus alterations in the DNA methylation pattern were correlated with transcriptional changes and controlled by Lsh. To investigate whether Lsh is directly involved in Hox gene silencing, we initially examined the expression levels of known Fig. 3. Lsh deletion reduces DNA methylation at HoxA6 and HoxA7 sites. (A) Genomic DNA derived from LshϪ/Ϫ and Lshϩ/ϩ MEFs or brain was subjected to PRC1 components to test whether Lsh deletion affects their bisulfite sequencing and examined at regions for two regions of the HoxA6 expression level. However, there was no evidence that either gene, as indicated in the map of Fig. 2A. Methylated CpG are presented by PRC1 components such as Bmi1 (Pcgf4), M33 (Cbx2, mPc1), black circles and unmethylated sites by open circles. (B) Bisulfite sequencing Mel18 (Pcgf2, Rnf110, and Zfp144), or PRC2 components such analysis for MEFs and brain at the HoxA7 gene. (C) Bisulfite sequencing as Ezh2 (PRC2) were differentially expressed when comparing analysis for MEFs and brain at the HoxA10 gene. nuclear extracts derived from LshϪ/Ϫ and Lshϩ/ϩ MEFs (Fig. 4A). Next we examined whether Lsh could directly associate with HoxA genes in P19 cells or MEFs (Fig. 4B). Using ChIPs with anti-Lsh antibodies followed by real-time PCR, we compared genes that were reactivated by Lsh deletion (HoxA6 and HoxA7) with those that were not affected (HoxA10). Whereas the HoxA6 and HoxA7 genes that were affected by Lsh deletion revealed binding of Lsh, the promoter regions of HoxA10 showed reduced association, and hardly any association was detected with inter- genic control regions. As expected, no significant binding for Lsh was detected for either HoxA sites in LshϪ/Ϫ MEFs. This suggested that Lsh may play a direct role in the methylation at some HoxA sites. Next, we tested whether Lsh and Dnmts can associate with PRC1 components. Using nuclear extracts derived from P19 embryonal carcinoma cells (because these cells are

high in de novo activity and Lsh protein BIOLOGY

levels), DNA methyltransferase activity was found to be associ- DEVELOPMENTAL ated with immunoprecipitates after using specific antibodies against Bmi1, M33, and Mel18 but was not detectable after precipitation using antibodies against Pol II as control (Fig. 4 C and 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- Fig. 2. Decreased CpG methylation after Lsh deletion at selected Hox gene sites. (A) Map of the HoxA6, HoxA7, and HoxA10 genes illustrating the onstrated a specific association between Bmi1, Mel18, or M33 location of primers (black triangles). The methylation-sensitive restriction with Lsh (Fig. 4E) and vice versa, specific immunoprecipitation sites HpaII and AciI are shown, as well as the primers (designated F1/R1 of Lsh or Dnmt3b demonstrated an interaction with PRC1 to F6/R6) used for methylation-sensitive PCR analysis. The ChIP primers are components (Fig. 4E). The association between Dnmt3b and designated P1/2, P3/4, and P5/6. The regions analyzed for bisulphate sequenc- Bmi1 was readily detectable in wild-type MEF nuclear extracts ing are indicated with a double arrow. (B) Methylation-sensitive PCR analysis Ϫ/Ϫ Ϫ/Ϫ ϩ/ϩ but reduced in extracts derived from Lsh MEFs (Fig. 4F). using genomic DNA derived from Lsh and Lsh MEFs or liver after diges- This suggested that the interaction of Dnmt3 with PRC at least tion with HpaII or AciI. PCR analysis amplifying a region around an AciI site after HpaII digestion (or HpaII site after AciI digestion) served as a control for in part depends on the presence of Lsh, and that it may be Lsh equal input of DNA. The primers F6/R6 amplify a region lacking methylation rather than DNA that performs a scaffolding-like function and sensitive restriction enzyme sites. promotes this interaction. Taken together, these data suggest a

Xi et al. PNAS ͉ September 4, 2007 ͉ vol. 104 ͉ no. 36 ͉ 14367 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 LshϪ/Ϫ and Lshϩ/ϩ MEFs. Detection of Pcna and Lsh served as controls. (B) ChIP assays were performed on chromatin extracts derived from P19 (gray bar), Lshϩ/ϩ (black bar), and LshϪ/Ϫ (open bar) MEFs using anti-Lsh antibodies and control IgG to detect association to specific HoxA6, HoxA7, and HoxA10 sites. Primers used for real-time PCR analysis are illustrated in Fig. 2A. In addition, two control primers were designed that are located within the HoxA cluster but Ͼ3,000 bp away from the HoxA10 or HoxA11 genes. The percentage of input was calculated for each precipitate. The values for the IgG control were Ͻ0.1% 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 blot analysis for detection of Lsh after immunoprecipitation with anti-Bmi1, anti-Mel18, or anti-M33 antibodies using P19 nuclear extracts. Species-matched IgG or omission 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 Lshϩ/ϩ or LshϪ/Ϫ MEFs. model in which Lsh plays a direct role in the control of Hox gene 13-fold), in contrast to HoxA10 sites that were unaffected (Fig. silencing. 4 D and E). Bmi1 binding, though, did not show a reduction, To understand whether Lsh can affect association of Dnmt3b suggesting that the recruitment of Bmi1 is independent of PRC2 to target sites and whether the presence of Lsh can modulate activity and not sufficient to maintain silencing (Fig. 5F). PRC-associated activities, histone modifications were examined However, decrease of M33 and Mel18 binding and reduced H2A by ChIP. Using quantitative PCR analysis, first histone acetyla- ubiquitylation suggest that Lsh/DNA methylation was important tion, a marker for transcriptional activation, was examined for complete assembly and activity of the PRC1 complex. comparing genes whose expression levels were affected by the Because PRC1 recruitment via M33 (mPc1) is thought to absence of Lsh (such as HoxA6 and HoxA7) with HoxA10 that depend on PRC2-mediated histone methylation (18), we exam- was unaffected by Lsh (Fig. 5A). H3 acetylation was enhanced in ined Ezh2 binding and H3-K27 trimethylation levels. Both marks LshϪ/Ϫ MEFs at HoxA6 and HoxA7 loci but not at the HoxA10 were decreased in LshϪ/Ϫ MEFs at HoxA6 and HoxA7 sites (2- gene and thus correlated well with transcriptional changes. In to 4-fold) and unperturbed at HoxA10 sites compared with contrast to histone acetylation, Dnmt3b binding was reduced at wild-type samples (Fig. 5 G and H). These data suggest that HoxA6 and HoxA7 genes (2- and 9-fold, respectively) when PRC2 cannot fully assemble in the absence of Lsh and DNA comparing LshϪ/Ϫ MEFs to wild type (Fig. 5B). These data methylation. support the idea that Lsh at least in part promotes association of DNA methylation has long been known to participate in Dnmt3b to specific sites at Hox genes. To investigate whether , X inactivation, repression of repeats, and Lsh and DNA methylation affect PRC-mediated histone mod- silencing of tumor suppressor genes (1, 2). Here we provide ifications, we first analyzed H2A ubiquitylation mediated by evidence that Lsh can associate with some Hox genes, controls PRC1 (17) (Fig. 5C). Whereas HoxA6 and HoxA7 sites revealed DNA methylation levels at Hox genes, and is also crucial for a reduction of H2A-K116 ubiquitylation of Ϸ7-fold in LshϪ/Ϫ normal developmental regulation of Hox gene expression pat- samples compared with wild-type controls, HoxA10 sites were tern. We further demonstrate that PRC1 and PRC2 activities are unchanged. Lsh deletion resulted in reduced association of M33 tightly correlated to DNA methylation, and that there may be a and Mel18 to HoxA6 and HoxA7 loci (ranging from 5- to feedback loop between DNA methylation and PRC-mediated

14368 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703669104 Xi et al. DNA methylation itself may affect PRC1 binding, as has been shown for reduced Bmi1 localization to PcG bodies after Dnmt1 depletion (22). More PRC1 binding may further promote DNA methylation and subsequent H3-K27methylation reinforcing the silencing marks in several feedback loops. Although PRC silencing is highly conserved in different species, Drosophila and Caenorhabditis elegans do not show significant levels of genomic DNA methylation and lack an Lsh homolog. Thus the involvement of Lsh-mediated DNA methyl- ation in PRC silencing shows a complexity that is unique to higher organisms. We hypothesize that Lsh associates only with a subset of polycomb complexes, because PRC components can assemble into various functionally distinct complexes and, as we report here, Lsh affects only some but not all examined Hox genes. More than 1,000 potential target sites have been reported for PRC components, and Lsh may also affect some of them (16, 23, 24). Thus the biologic activities of Lsh may be partially overlapping with PRC activities and may include effects on stem cell 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 DNA methylation are not aberrantly connected in cancer cells but part of an ordinary regulatory pathway involving Lsh. Whether this link is unique for embryonic cells or is also present in adult differentiated cells and why these pathway are targeted in cancer Fig. 5. Lsh controls Dnmt3b recruitment and PRC-mediated histone modi- to loci that are usually unmethylated remains unknown, but Lsh fications at Hox sites. ChIP assays were performed from chromatin extracts of is one possible candidate that could play a role in aberrant LshϪ/Ϫ (open bar) and Lshϩ/ϩ (black bar) MEFs using the indicated antibodies recruitment of Dnmts. On the other hand, cancer is also asso- to detect specific histone modifications or association of specific proteins at ciated with global DNA hypomethylation, which in turn may HoxA genes. Primers used for real-time PCR analysis are illustrated in Fig. 2A. derepress some Hox genes. Deregulation of Hox genes has been The percentage of input was calculated for each precipitate and the values implied in hematopoietic malignancies and ectopic Hox gene Ϫ Ϫ expressed as ratio of Lsh / samples over wild type. The following antibodies expression determines the phenotype in ovarian epithelial cell were used: (A) Antiacetylation of H3. The asterix indicate the minimum ratio Ϫ/Ϫ cancer (3, 27, 28). The suggested connection between the two above wild-type controls, because the actual values for Lsh samples ex- epigenetic pathways may shed new light on the molecular ceeded the range of the standard curve. (B) Anti-Dnmt3b. (C) Anti-H2A-K116 ubiquitylation. (D) Anti-M33. (E) Anti-Mel18. (F) Anti-Bmi1. (G) Anti-H3-K27 mechanisms involved in tumorigenesis and may prove helpful to trimethylation. (H) Anti-Ezh2. improving strategies for cancer treatment or prevention in future. histone modifications. This supports the idea of a complex Methods network of diverse epigenetic modifications rather than a simple Western Blot Analysis and Immunoprecipitations. Samples were sequential activation cascade during mammalian embryogenesis. separated on 4–12% Tris-glycine SDS/PAGE gels and blotted Based on Lsh homology with SNF2 family members, part of onto Immobilon P membrane (Millipore, Bedford, MA). West- its activity may depend on presumed nucleosomal remodeling ern blotting was performed according to standard procedures by activity that may allow for better access of DNA-binding proteins using ECL detection reagents, according to the manufacturer’s to their nucleosomal target sites (9, 10). In addition, Lsh may also instructions (Amersham, Piscataway, NJ). Nuclear extracts were prepared as described (29). For immunoprecipitations, the nu- have chromatin remodeling-independent or scaffold-like func- BIOLOGY clear extract buffer was adjusted to a final concentration of 50

tions, for example in promoting Dnmt3 activity or stabilizing the DEVELOPMENTAL interactions of proteins. The observation that the association of mM Tris (pH 7.5)/150 mM NaCl/1 mM EDTA/0.5% Nonidet P-40. Nuclear extracts (200 ␮g) were precleared for 30 min with Dnmt3b with Bmi1 is influenced by Lsh (Fig. 4F) would be protein G agarose (Invitrogen, Carlsbad, CA) and then incu- consistent with a role of Lsh in scaffolding function. Possibly, via bated with 20 ␮l of antibodies for2horovernight at 4°C. its association with PRC components, Lsh may bind to Hox loci Washing was performed three times in 500 ␮l of buffer [50 mM and promote targeting of Dnmt. Alternatively, other not-yet- Tris (pH 7.5)/150 mM NaCl/1 mM EDTA/0.5% Nonidet P-40] defined factors may recognize PRC-mediated histone modifica- at 4°C, 5 min each cycle on a rotator. Antibodies used for tions and lead to Lsh and subsequent Dnmt3b recruitment. immunoprecipitation or Western analysis were species-matched Increased DNA methylation may result in decreased histone normal IgG (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit acetylation levels and a decline in transcription. Histone 3-K4 anti-Lsh recombinant protein affinity-purified antibody, anti- coupled to Pol II may alter H3-K4 methyl- Dnmt3b (Alexis, San Diego, CA), anti-Bmi1 (Upstate Biotech- ation levels and may ultimately prevent spreading of the repres- nology, Lake Placid, NY), anti-Ezh2 (Upstate Biotechnology), sive H3-K27me mark (19). As another possibility, noncoding anti-Mel18 (Abcam, Cambridge, MA), anti-M33 antibody (BD RNA transcripts that are prevalent in mammalian Hox clusters Transduction Laboratories, Franklin Lanes, NJ), anti-Pcna (20) may demarcate regions of gene silencing by regulating (Santa Cruz Biotechnology), and anti-Pol II antibody (Upstate PRC2 occupancy and H3-K27me levels (21). A rise in H3-K27me Biotechnology). The following secondary antibodies were used: may enhance PRC1 targeting to Hox genes (18). Alternatively, goat anti-rabbit HRP-conjugated IgG, goat anti-mouse HRP-

Xi et al. PNAS ͉ September 4, 2007 ͉ vol. 104 ͉ no. 36 ͉ 14369 conjugated IgG, and rabbit anti-goat HRP-conjugated IgG sample was saved as input fraction. Immunoprecipitation was (Santa Cruz Biotechnology). performed by using specific antibodies against the indicated proteins or IgG of different species used as control. After In Vitro DNA Methyltransferase Activity Assay. After 150 ␮lof reversal of cross-linking, nucleic acids were prepared from the nuclear extracts (29) derived from P19 cells was incubated with eluted complex, and PCR analysis was performed. Amplification antibodies and protein G agarose for2horovernight at 4°C with conditions were as follows: 94°C for 4 min; 94°C for 1 min; 55°C rotation, agarose beads were washed three times with nuclear for 1 min; 72°C for 1 min (35 cycles) and 72°C for 7 min. The extraction buffer and then again incubated with 150 ␮loffresh following antibodies were used for ChIPs: H3K27 triM, triM nuclear extracts and antibodies to improve the yield of DNA Acetyl-H3 (Lys-9/14), ubiquityl-histone H2A, Bmi1, Ezh2 anti- methyltransferase activity. After a second round of incubation and bodies (Upstate Biotechnology), anti-Lsh recombinant protein washes, the beads were rinsed with DNA methyltransferase assay affinity-purified antibody, Dnmt3b antibody (Alexis), Mel18 buffer (50 mM Tris, pH 7.8/1 mM EDTA/1 mM DTT/10% antibody (Abcam), and M33 antibody (BD Transduction Labo- glycerol/1% Tween) and frozen at Ϫ80°C until future analysis. ratories). Assays were performed with immunoprecipitated material still Real-time PCR primer pairs for ChIPs analysis are listed in SI attached to agarose beads. DNA methyltransferase activity was Text. analyzed by the standard glass fiber method using S-adenosyl-L- 3 ( H-methyl)-methionine (Amersham–Amersham Pharmacia) as Real-Time PCR Analysis. For real-time PCR analysis, the MyiQ the methyl donor and poly(d[I-C]) as the DNA substrate with an Single-Color Real-Time PCR machine (Bio-Rad) and Platinum incubation time of 1.5 h. After washing, filters containing 3H were SYBR Green qPCR SuperMix UDG (Invitrogen) were used. placed in scintillation fluid, and the level of radioactivity was The PCR for ChIPs was initiated with one cycle of 95°C for 3 counted. Two immunoprecipitations were performed on indepen- min, followed by 45 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C dent nuclear extracts for each antibody and normal species IgG for 30 s. PCR for the RT-PCR analysis was initiated with one controls. The average of two immunoprecipitations using indepen- cycle of 95°C for 3 min, followed by 45 cycles of 95°C for 30 s, dent nuclear extracts was graphed with error bars representing the 59°C for 30 s, and 72°C for 30 s. The negative control without standard deviation. template was carried out for each PCR analysis. To quantify the amount of the template using real-time PCR data, standard Methylation-Sensitive PCR. DNA was extracted by using the titration experiments for each template and each primer set were DNeasy kit (Qiagen, Valencia, CA). DNA was completely performed, and linear regression equation and the calculation digested with HpaII or AciI. To analyze the methylation status for DNA amounts were established by using Prism 3.0 software of the genomic DNA, the following PCR primer pairs A6 (GraphPad, San Diego, CA) and Microsoft (Redmond, WA) (F1/R1), A6(F2/R2), A6(F3/R3), A6(F4/R4), A7(F5/R5), and Excel. Every ChIP experiment includes species-specific IgG A10 (F6/R6) were used, as listed in supporting information (SI) controls. The results have been calculated as percentage of Input Text. (which lay usually between 5% and 20%) and then expressed as PCRs were carried out as follows: 5 min at 94°C, 35 cycles of ratio of LshϪ/Ϫ over wild type. For better comparison, the 60 s at 94°C, 30 s at 60°C, and 60 s at 72°C, and finally 5 min at wild-type samples were set to one, and the values expressed as 72°C. The PCR products were electrophoresed on 1% agarose ratio of LshϪ/Ϫ samples over wild type. gels, stained with ethidium bromide, and photographed. Bisulphite Sequencing. Genomic DNA from MEF cells and brain RT-PCR. Total RNA was prepared from MEF cells, smashed tissue (day 18 of gestation) was subjected to bisulfite treatment embryos (day 18 of gestation), liver, and brain tissue (day 18 of by using CpGenome DNA modification kit (Chemicon Interna- gestation) using TRIzol reagent (Invitrogen), according to the tional, Temecula, CA) according to the manufacturer’s instruc- manufacturer’s instructions. Any genomic DNA present was tions. The PCR products were separated in agarose gels and eliminated with TURBO DNA-free Kit (Ambion, Austin, TX). purified by using the QIAEX II gel extraction kit (Qiagen). Approximately 1 ␮g of total RNA was reverse-transcribed by Amplified fragments were subcloned into the pCR2.1-TOPO using iScript reverse transcriptase (Bio-Rad, Hercules, CA). vector with the TOPO TA Cloning Kit (Invitrogen). Indepen- Omission of reverse transcriptase served as a negative control. dent clones for each fragment were sequenced by using the M13 cDNA was amplified by using Platinum PCR SuperMix (Invitro- gen). PCR followed by agarose gel electrophoresis using Hox F or M13 R and only sequences with individual fingerprint primers (17) was performed as follows: 5 min at 94°C, 35 cycles selected from analysis. The primers used are listed in SI Text. of 60 s at 94°C, 60 s at 56–59°C, and 60 s at 72°C, followed by one cycle of 5 min at 72°C. For real-time PCR, the following primers We thank Rodney Wiles and Terry Stull for excellent technical assis- tance. We thank Nancy Colburn and Peter Johnson for helpful discussion were used as listed in SI Text. of the manuscript. This project has been funded in whole or part with federal funds from the National Cancer Institute, National Institutes of ChIP. For ChIP, cells were cross-linked with 1% formaldehyde, Health, under Contract No. N01-C0-12400. This research was supported lysed, and sonicated on ice to generate DNA fragments with an by the Intramural Research Program of the National Institutes of Health, average length of 200–800 bp. After preclearing, 1% of each National Cancer Institute, Center for Cancer Research.

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