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Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase
Author: James P. Jackson, Anders M. Lindroth,Xiaofeng Cao ,Steven E. Jacobsen
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"In vitro growth assay Cells were seeded in triplicate in 12-well plates at a concentration of 3 � 10 4 cells per well. Cells were collected at days 2, 4, 6 and 8, and viable cells, as assessed by trypan blue exclusion, were counted in a haemocytometer. Received 9 October 2001; accepted 14 February 2002. 1. Vogelstein, B. & Kinzler, K. W. The Genetic Basis of Human Cancer (McGraw-Hill Health Professions Division, New York, 1998). 2. Siegfried, Z. & Cedar, H. DNA methylation: a molecular lock. Curr. Biol. 7, R305?R307 (1997). 3. Bird, A. P. & Wolffe, A. P. Methylation-induced repression?belts, braces, and chromatin. Cell 99, 451?454 (1999). 4. Robertson, K. D. & Jones, P. A. DNA methylation: past, present and future directions. Carcinogenesis 21, 461?467 (2000). 5. Tycko, B. Epigenetic gene silencing in cancer. J. Clin. Invest. 105, 401?407 (2000). 6. Baylin, S. B. & Herman, J. G. DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet. 16, 168?174 (2000). 7. Ponder, B. A. Cancer genetics. Nature 411, 336?341 (2001). 8. Eads, C. A. et al. CpG island hypermethylation in human colorectal tumors is not associated with DNA methyltransferase overexpression. Cancer Res. 59, 2302?2306 (1999). 9. Schmutte, C., Yang, A. S., Nguyen, T. T., Beart, R. W. & Jones, P. A. Mechanisms for the involvement of DNA methylation in colon carcinogenesis. Cancer Res. 56, 2375?2381 (1996). 10. Li, E., Bestor, T. H. & Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915?926 (1992). 11. Okano, M., Bell, D. W., Haber, D. A. & Li, E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247?257 (1999). 12. Rhee, I. et al. CpG methylation is maintained in human cancer cells lacking DNMT1. Nature 404, 1003?1007 (2000). 13. Bestor, T. H. The DNA methyltransferases of mammals. Hum. Mol. Genet. 9, 2395?2402 (2000). 14. Kuo, K. C., McCune, R. A., Gehrke, C. W., Midgett, R. & Ehrlich, M. Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA. Nucleic Acids Res. 8, 4763?4776 (1980). 15. Feinberg, A. P., Gehrke, C. W., Kuo, K. C. & Ehrlich, M. Reduced genomic 5-methylcytosine content in human colonic neoplasia. Cancer Res. 48, 1159?1161 (1988). 16. Bachman, K. E. et al. Methylation-associated silencing of the tissue inhibitor of metalloproteinase-3 gene suggest a suppressor role in kidney, brain, and other human cancers. Cancer Res. 59, 798?802 (1999). 17. Herman, J. G., Graff, J. R., Myohanen, S., Nelkin, B. D. & Baylin, S. B. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl Acad. Sci. USA 93, 9821?9826 (1996). 18. Rainier, S. et al. Relaxation of imprinted genes in human cancer. Nature 362, 747?749 (1993). 19. Ogawa, O. et al. Constitutional relaxation of insulin-like growth factor II gene imprinting associated with Wilms? tumour and gigantism. Nature Genet. 5, 408?412 (1993). 20. Steenman, M. et al. Loss of imprinting of IGF2 is linked to reduced expression and abnormal methylation of H19 in Wilms? tumour. Nature Genet. 7, 433?439 (1994). 21. Cui, H., Horon, I. L., Ohlsson, R., Hamilton, S. R. & Feinberg, A. P. Loss of imprinting in normal tissue of colorectal cancer patients with microsatellite instability. Nature Med. 4, 1276?1280. (1998). 22. Uejima, H., Lee, M. P., Cui, H. & Feinberg, A. P. Hot-stop PCR: a simple and general assay for linear quantitation of allele ratios. Nature Genet. 25, 375?376 (2000). 23. Myohanen, S. K., Baylin, S. B. & Herman, J. G. Hypermethylation can selectively silence individual p16ink4A alleles in neoplasia. Cancer Res. 58, 591?593 (1998). 24. Okano, M., Xie, S. & Li, E. Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nature Genet. 19, 219?220 (1998). 25. Lyko, F. et al. Mammalian (cytosine-5) methyltransferases cause genomic DNA methylation and lethality in Drosophila. Nature Genet. 23, 363?366 (1999). 26. Santi, D. V., Garrett, C. E. & Barr, P. J. On the mechanism of inhibition of DNA-cytosine methyltransferases by cytosine analogs. Cell 33, 9?10 (1983). 27. Juttermann, R., Li, E. & Jaenisch, R. Toxicity of 5-aza-2 0 -deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation. Proc. Natl Acad. Sci. USA 91, 11797?11801 (1994). 28. Jackson-Grusby, L. et al. Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation. Nature Genet. 27, 31?39 (2001). 29. Chan, T. A., Hermeking, H., Lengauer, C., Kinzler, K. W. & Vogelstein, B. 14-3-3j is required to prevent mitotic catastrophe after DNA damage. Nature 401, 616?620 (1999). 30. Vertino, P. M., Yen, R. W., Gao, J. & Baylin, S. B. De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase. Mol. Cell Biol. 16, 4555?4565 (1996). Supplementary Information accompanies the paper on Nature?s website (http://www.nature.com). Acknowledgements We thank S. R. Lee and S. G. Rhee for assistance with the HPLC analysis. This work was supported by the Clayton Fund, the V Foundation, and by grants from the National Institutes of Health. Competing interests statement The authors declare competing financial interests: details accompany the paper on Natures?s website (http://www.nature.com).. Correspondence and requests for materials should be addressed to S.B.B. (e-mail: sbaylin@jhmi.edu) or B.V. (e-mail: vogelbe@welch.jhu.edu). .............................................................. Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase James P. Jackson, Anders M. Lindroth, Xiaofeng Cao & Steven E. Jacobsen Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, California 90095, USA ............................................................................................................................................................................. Gene silencing in eukaryotes is associated with the formation of heterochromatin, a complex of proteins and DNA that block transcription. Heterochromatin is characterized by the methyl- ation of cytosine nucleotides of the DNA, the methylation of histone H3 at lysine 9 (H3 Lys 9), and the specific binding of heterochromatin protein 1 (HP1) to methylated H3 Lys 9 (refs 1? 7). Although the relationship between these chromatin modifi- cations is generally unknown, in the fungus Neurospora crassa, DNA methylation acts genetically downstream of H3 Lys 9 methylation 8 . Here we report the isolation of KRYPTONITE,a methyltransferase gene specific to H3 Lys 9, identified in a mutant screen for suppressors of gene silencing at the Arabidopsis thaliana SUPERMAN (SUP) locus. Loss-of-function kryptonite alleles resemble mutants in the DNA methyltransferase gene CHROMOMETHYLASE3 (CMT3) 9 , showing loss of cytosine methylation at sites of CpNpG trinucleotides (where N is A, C, G or T) and reactivation of endogenous retrotransposon sequences. We show that CMT3 interacts with an Arabidopsis homologue of HP1, which in turn interacts with methylated histones. These data suggest that CpNpG DNA methylation is controlled by histone H3 Lys 9 methylation, through interaction of CMT3 with methylated chromatin. Heterochromatin contains a characteristic set of post-translat- ional histone modifications including H3 Lys 9 methylation, which is carried out by the SET domains of the Su(var)3-9 class proteins 1,2 . Genetic studies show that Su(var)3-9 proteins are essential for proper assembly of heterochromatin. For example, Drosophila su(var)3-9 mutants suppress a silencing phenomenon called pos- ition effect variegation (PEV) 10 , and clr4 mutants de-repress silent mating-type loci in Schizosaccharomyces pombe 11,12 . Mice lacking SUV39h genes show defects in pericentromeric heterochromatin, chromosome instabilities, and increased tumorigenesis 13 . The ?his- tone code? hypothesis 3,4 proposes that histone modifications direct the binding of specific proteins that mediate chromatin function. For instance, the chromo domain of HP1 has been shown to specifically bind methylated H3 Lys 9, and this binding is essential for heterochromatin formation in vivo 2,5 ?7 . Here we provide evidence that a histone code influences the enzymes that methylate DNA. We performed a screen for ethylmethane sulphonate (EMS)- induced suppressors of an epigenetic allele of the SUP locus (the clark kent-st allele, clk-st) 9,14 . clk-st plants show defects in the number of floral organs (Fig. 1a) owing to cytosine methylation and silencing of the SUP gene. In clk-st, SUP is methylated not only at CpG dinucleotides but also at CpNpG sites and asymmetric sites (cytosines not present in CpG or CpNpG contexts). We previously reported that nine clk-st suppressor mutants were loss-of-function alleles of the DNA methyltransferase CHROMOMETHYLASE3 (CMT3) 9 . cmt3 mutants reduce CpNpG DNA methylation and reactivate the expression of SUP, PAI2 and a subset of retrotrans- posons 9,15 . Further analysis of the clk-st suppressor mutants identi- fied three alleles of a new locus, which we have named KRYPTONITE (KYP) (Fig. 1a). These mutants, kyp-1 to kyp-3,are recessive and show similar phenotypes, suggesting that they are loss- of-function mutants. The kyp-2 mutant also suppresses a different letters to nature NATURE | VOL 416 | 4 APRIL 2002 | www.nature.com556 � 2002 Macmillan Magazines Ltd clark kent allele, clk-3 (ref. 14). Other than suppression of the clk phenotype, the kyp mutants did not exhibit morphological defects even after extensive inbreeding. The KYP gene was cloned and found to code for a protein of 624 amino acids containing a SET domain (Fig. 1b, c). A phylogenetic study of Arabidopsis SET proteins suggests that KYP is most similar to the Su(var)3-9 class of histone H3 Lys 9 methyltransferases 16 . This study described nine related Arabidopsis sequences listed as SU(VaR)3-9 homologues 1?9, with KYP listed as number 4. Align- ment of KYP with several H3 methyltransferases shows conserva- tion of sequences critical for in vitro methylase activity including the cysteine-rich pre-SET and post-SET motifs and specific residues within the SET domain (Fig. 1c) 1,2 . To test whether KYP methylates histones, we expressed a fusion protein of glutathione S-transferase (GST) and the KYP SET domain, and performed in vitro methylation assays. Similar to a GST?SUV39h1 control, GST?KYP methylated histone H3 (Fig. 2a). GST?KYP did not methylate histones H1, H2A, H2B or H4. KYP also methylated a GST fusion protein containing the 57 amino- terminal amino acids of histone H3, but not a mutant fusion protein in which Lys 9 was mutated to arginine (Fig. 2b) 17 . Thus, like other Su(var)3-9 class proteins, KYP is a H3 Lys 9 methyltransferase. All three kyp alleles are predicted to reduce or eliminate the function of the SET domain (Fig. 1c), suggesting that H3 Lys 9 methyltransfer- ase activity is critical for KYP function. We determined the effect of kyp on DNA methylation using both bisulphite genomic sequencing and Southern blot analysis using methylation-sensitive restriction enzymes. A bisulphite sequencing analysis of the SUP gene, comparing the methylation profiles of kyp- 1 with those of the cmt3-7 and met1 mutants (met1 is a recessive allele of the DNMT1-like MET1 CpG methyltransferase 18 ? 20 ), is shown in Fig. 3a. In the kyp-1 mutant, SUP showed a loss of DNA methylation in all sequence contexts, but the loss of CpNpG methylation and asymmetric methylation was stronger than that of CpG methylation. Thus the kyp methylation phenotype resembles that of cmt3 more than that of met1. The kyp mutants (like cmt3 mutants) did not develop the late flowering phenotype characteristic of reactivation of the normally methylated and silenced FWA locus 9,21 . In wild-type plants, FWA is methylated within two direct repeats of its promoter at CpG sites (88%) and to a lesser extent at CpNpG sites (20%) 21 . This contrasts with SUP, which is methylated predominantly at CpNpG sites 9 .To test whether kyp mutants affect CpG methylation at FWA,we performed a Southern blot analysis of two methylation-sensitive CfoI restriction sites that contain CpG within their recognition sequences (Fig. 3b). The pattern of enzyme digestion was similar in kyp-1, kyp-2 and clk-st, showing that neither kyp allele affects CpG methylation. We assayed CpNpG methylation at FWA with the Figure 1 KRYPTONITE mutants. a,Aclk-st flower containing nine stamens and a defective gynoecium and a kyp-1-suppressor mutant flower showing the normal six stamens and a normal gynoecium. b, KYP amino acids 1?332, showing a nuclear localization signal (underlined) and the YDG domain (blue) 16 . c, KYP amino acids 333? 624 constituting the pre-SET, SET and post-SET domains, aligned with four histone H3 methyltransferases, human SUV39h1 (NP_003164), Schizosaccharomyces pombe Clr4 (T43745), human G9a (NP_006700) and Neurospora DIM5 (AF419248). Asterisks in the pre-SET and post-SET domains mark conserved cysteines and those in the SET domain mark residues important for methylase activity 1 . Triangles mark splice junctions affected in kyp-1 and kyp-2. GST GST?KYP GST?SUV1GST?SUV1 (H320R) H1 H3 H2A H2B H4 a H3 Fluorogram Coomassie b H3N K9R Coomassie GST?KYP Fluorogram Figure 2 Methyltransferase activity of KRYPTONITE. a, Methyltransferase activity of GST, GST?KYP (amino acids 291?624), GST?SUV1 (SUV39h1, amino acids 82?412), and GST?SUV1(H320R) 1 fusion proteins with histone substrates and the methyl donor S-adenosyl-(methyl- 14 C)-L-methionine. Arrowhead marks Coomassie-stained purified proteins (top). Individual histones are indicated. Fluorography (bottom) indicates methyltransferase specificity for histone H3. b, GST?KYP methyltransferase assays using GST?H3 fusion protein substrates, H3N (57-amino-acid H3N terminus) and K9R (H3N terminus with a mutation to arginine at Lys 9). Arrowhead indicates GST?KYP fusion protein. Bullet indicates GST?H3 fusion proteins. Fluorogram shows that GST?KYP methylates wild-type but not Lys 9-mutant fusion proteins. letters to nature NATURE | VOL 416 | 4 APRIL 2002 | www.nature.com 557� 2002 Macmillan Magazines Ltd methylation-sensitive enzyme BglII, and found that kyp-1, kyp-2 and cmt3-7 all showed a significant increase in digestion relative to the control strain clk-st. Thus, like cmt3 mutants, the kyp mutants decreased CpNpG methylation but not CpG methylation of FWA. We confirmed these results by bisulphite sequencing the FWA locus from kyp-1 plants. We found levels of CpG methylation comparable to wild type, but lower CpNpG methylation. We next studied the effect of kyp on satellite methylation using a 180-base-pair centromere repeat probe 22 and the isoschizomers HpaII and MspI, which recognize 5 0 -CCGG-3 0 . HpaII is inhibited by methylation of either the inner (CG) or the outer (CCG) cytosine of the recognition site, whereas MspI is sensitive only to methylation of the outer cytosine, and thus detects CpNpG methylation. Similar to cmt3 mutants 9 , kyp-1 and kyp-2 mutants show a small but reproducible increase in MspI digestion but not HpaII digestion, indicating that kyp mutants reduce CpNpG but not CpG methyl- ation at centromeric repeat sequences (Fig. 3d). We also detected increased MspI but not HpaII cleavage in kyp-1, kyp-2 and cmt3-7 mutants at two additional sequences tested, a highly repetitive Athila retrotransposon long terminal repeat sequence (Fig. 3e) 9 and the single-copy Ta3 retrotransposon (Fig. 3f). Figure 3 Effect of kryptonite on DNA methylation and retrotransposon activation. a, Bisulphite sequence analysis showing the methylation of SUP in different mutant backgrounds. b, c, Southern blot of CfoI- (b) and BglII-digested (c) genomic DNAs probed with FWA 21 . The hypomethylated fwa-1 mutant 21 is included as an unmethylated control. kb, kilobases. d?f, Southern blot of HpaII- and MspI-digested genomic DNAs probed with a 180-base-pair centromeric repeat (d) 22 , an Athila long terminal repeat (e) 9 or Ta3 (f) 9 . g, RNA blot hybridized with a TSI probe. in pd ft in pd ftpd ft GST?CMT3 GST?CMT3GST 6His-LHP1 6His-GFP b a H3 H3m H3 H3m H3 H3m GST?HP1b chromo H3 H3m H3 H3m GST?CMT3 full GST?CMT3 chromo GST control H3 H3m GST?HP1b chromo GST?CMT3 chromo c d H3 H3m GST?LHP1 chromo H3 H3m GST?LHP1 chromo KYP CMT3 HP1 Histone methylation DNA methylation Me Figure 4 Interaction of CMT3 with histones and LHP1. a, Histone H3 peptide pull-down assays. Equivalent amounts of the indicated purified GST fusion proteins were mixed with matrices containing either unmodified histone H3 N-terminal peptides (H3) or Lys 9- methylated peptides (H3m) 5 , and bound proteins were detected with anti-GST antibodies. b, Biotinylated H3 N-terminal peptides (H3) or biotinylated Lys 9-methylated peptides (H3m) were mixed with glutathione-agarose-bound GST fusion proteins, and bound peptides were detected with streptavidin horseradish peroxidase (HRP). c, GST?CMT3 pull-down assays. Purified His-tagged LHP1 or His-tagged GFP fusion proteins were mixed with glutathione-agarose-bound GST fusion proteins, and bound proteins were detected with INDIA HisProbe-HRP. Approximately 5% of input proteins (in), 100% of bound proteins (pd), and 5% of flow-through (ft) fractions are shown. d, Model showing the proposed interaction of CMT3 with methylated histones by binding to LHP1. letters to nature NATURE | VOL 416 | 4 APRIL 2002 | www.nature.com558 � 2002 Macmillan Magazines Ltd Thus the methylation phenotype of kyp resembles that of cmt3:a loss of CpNpG methylation. However, the effect of kyp on CpNpG methylation was weaker than that of cmt3-7 at most loci tested (Fig. 3). Furthermore, kyp-1 showed a greater loss of CpG and asym- metric methylation at SUP (Fig. 3a), suggesting that, at some loci, its effects on DNA methylation are more general than those of cmt3-7. Similar to the cmt3 mutants, we found that kyp mutants induced a low level of expression of two retrotransposon-related sequences, an Athila sequence called TSI (ref. 23) (Fig. 3g) and the Ta3 sequence 9 (not shown). These results further demonstrate that KYP and CMT3 affect silencing at an overlapping set of loci. The close similarity in methylation and silencing phenotypes of kyp and cmt3 mutants prompted us to examine the relationship between CMT3 and methylated histones. CMT3, like HP1, contains a chromo domain 24 , suggesting the possibility that CMT3 binds directly to methylated H3 Lys 9, thereby directing the methylation of CpNpG sites. To examine this possibility, we first tested for the ability of an in vitro translated CMT3 chromo domain (amino acids 329?490) and full-length in vitro translated CMT3 to bind matrices containing either K9 di-methylated or unmethylated H3 N-terminal peptides. Under previously published conditions 5 , we did not detect specific binding to either matrix. Using a different approach, we expressed full-length CMT3, the CMT3 chromo domain (amino acids 366?448), and a mouse HP1b chromo domain (amino acids 10?80) as GST fusions in Escherichia coli.TheHP1b chromo domain fusion protein bound specifically to a matrix containing methylated histone H3 peptides, but the full-length CMT3 and the CMT3 chromo domain fusion proteins did not (Fig. 4a and not shown). Finally, we reversed the experiment and tested whether methylated or unmethylated biotinylated peptides could bind to the same GST fusion proteins affixed to glutathione-agarose beads. The H3 Lys 9-methylated peptide bound to the HP1b chromo domain fusion but not to the CMT3 fusion proteins (Fig. 4b). Thus, unlike HP1, the CMT3 chromo domain did not bind specifically to methylated histone H3 under the conditions tested. Several other chromo domains also do not bind methylated histones, including those in the proteins murine PC1 and Suv39h1 (ref. 5), M33 and Mi2 (ref. 6), and Esa1 (ref. 7). Furthermore, chromo domains are proposed to have other functions such as binding to RNA in the case of the histone acetyltransferase MOF 25 . Arabidopsis possesses a homologue of HP1 (LHP1) that, like HP1 from other organisms, localizes to discrete subnuclear foci and homodimerizes through its chromo shadow domain 26 . We found that the LHP1 chromo domain (a GST fusion protein containing LHP1 amino acids 54?183) specifically binds to methylated H3 Lys 9 (Fig. 4a, b). Thus a second possible explanation for the resem- blance of kyp and cmt3 is that CMT3 interacts with LHP1, thereby targeting CMT3 to methylated histones. To test this, we expressed His-tagged full-length LHP1 and performed pull-down assays with GST?CMT3. Figure 4c shows that 6His-LHP1 bound GST?CMT3 but not GSTalone. A control protein, 6His?GFP, did not bind to the GST?CMT3 matrix. We also found that in vitro translated full- length CMT3 bound to a GST?LHP1 fusion protein but not to GST alone, and that in vitro translated full-length LHP1 bound to GST? CMT3 but not GST alone (not shown). Therefore, CMT3 binds to LHP1 in vitro. This suggests a model (Fig. 4d) in which CMT3 is targeted to methylated chromatin through an interaction with LHP1. Human HP1 can distinguish between different types of H3 Lys 9-methylated heterochromatin, binding in vivo to constitutive heterochromatin such as centromeric regions, but not to facultative heterochromatin such as the inactive X chromosome 27 . Therefore, the interaction of CMT3 with LHP1 may serve to target CpNpG methylation to particular chromosomal domains. Our results show that the KYP histone H3 Lys 9 methyltransferase is required for maintenance of DNA methylation. Studies of the DIM5 H3 methyltransferase in Neurospora demonstrated an inti- mate relationship between histone methylation and DNA methyl- ation, because loss of DIM5, as well as mutation of Lys 9 of histone H3, resulted in a complete loss of DNA methylation in vivo 8 . Our results extend these findings to the plant kingdom, suggesting that a relationship between histone methylation and DNA methylation is conserved throughout higher eukaryotes. However, unlike the general loss of DNA methylation observed in dim5SQ14, kyp mutants show a specific loss of CpNpG methylation, a phenotype similar to that of loss-of-function alleles of the CMT3 DNA methyltransferase gene. One possible explanation for this finding is that, in plants, only CpNpG methylation is controlled by histone H3 Lys 9 methylation. Alternatively, the more subtle effects of kyp mutants on DNA methylation might be explained by the presence of eight additional KYP homologues in the Arabidopsis genome 16 , one or more of which might function in the control of DNA methylation in other contexts such as at CpG sites. Our data suggest that loss of CpNpG methylation in the kyp mutants results from lack of targeting of CMT3 to methylated histones, possibly through an interaction with an Arabidopsis HP1 homologue. The central role that HP1 plays in chromatin biology of animals and fungi suggests that recruitment of DNA methyltrans- ferases to chromatin by HP1 could be a general eukaryotic phenomenon. Methods Polymerase chain reaction (PCR) primers and PCR-based molecular markers are listed in Supplementary Information. Characterization of the kryptonite alleles We cloned the KYP gene by fine-scale mapping the kyp-2 allele and sequencing the candidate genes. kyp-2 was mapped by crossing to a Columbia sup-2 strain as described 9 . We analysed 1,436 chromosomes (718 kyp-2 homozygous F 2 plants) with a series of PCR- based molecular markers to narrow the KYP-containing region to between a marker on bacterial artificial chromosome (BAC) clone sequence MXE10 and one on BAC clone sequence MUA22. This defined an interval of 141 kilobases (kb) that contained only one additional BAC clone sequence, MAC12, which contains the KYP gene (MAC12.7; GenBank accession number AB005230). A description of the molecular nature of each kyp allele is provided in Supplementary Information. Histone methyltransferase assays A GST?KYP fusion construct was made by cloning KYP (amino acids 281?625) into the BamHI and EcoR1 sites of pGEX2TK by RT?PCR (PCR with reverse transcription of RNA) from DNase-treated total RNA of Landsberg erecta (Ler)-strain inflorescences, and confirmed by sequencing. The GST?SUV1 (82?412) and GST?SUV1(H320R) (a mutant version in which histidine 320 is mutated to arginine) constructs 1 were a gift of T. Jenuwein. Recombinant proteins were expressed and purified on glutathione-agarose beads (Pierce) as described 1 . In vitro histone methyltransferase reactions were performed as described 1 and proteins were separated by 18% SDS?PAGE and visualized by Coomassie staining and fluorography. Histone methyltransferase reactions on GST?H3 fusion proteins 17 (a gift of Y. Shinkai) were performed using similar reaction conditions. Analysis of DNA methylation Genomic DNA was isolated from leaves of 4-week-old flowering plants of all genotypes. For the results reported in Fig. 3a, bisulphite sequencing on the top strand of a 1,028- nucleotide region of SUP was performed as previously described 9 , with 15 independent cloned PCR products of each region analysed. kyp-1 and cmt3-7 were in a homozygous clk-st background and met1 was a previously described line in a clk background 9 . Detailed data supporting the graphical presentation in Fig. 3a can be found in Supplementary Information Table 1. Bisulphite sequencing of FWA was as previously described 21 , and the data reported in the text are from an analysis of eight cloned PCR products. RNA blot analysis For Fig. 3g, total RNAwas isolated from 4-week-old plants of each genotype, 40 mg of RNA was loaded per lane, and a blot was probed with a TSI probe 9 . As we observed previously with this probe 9 , there was a less-abundant TSI transcript of about 2.4 kb detected in the clk-st strain. Histone peptide binding assays GST fusion constructs were made by cloning fragments of CMT3, HP1b and LHP1 into the BamHI and EcoR1 sites of pGEX2TK, and clones were confirmed by DNA sequencing. Affinity matrices were generated by coupling unmodified H3 N-terminal (residues 1?20) or K9 dimethyl peptides (a gift of T. Jenuwein) to SulfoLink Coupling Gel (Pierce) as described by the manufacturer. Binding conditions, buffers, and wash conditions for letters to nature NATURE | VOL 416 | 4 APRIL 2002 | www.nature.com 559� 2002 Macmillan Magazines Ltd experiments shown in Fig. 4a, b were exactly as described 5 , except that the binding and washing buffer for the GST?LHP1 results shown in Fig. 4a consisted of 50 mM sodium phosphate and 25 mM NaCl at pH 6.0. For Fig. 4a, bound proteins were separated by 4? 20% gradient SDS?PAGE, blotted, and detected with an anti-GST monoclonal antibody (Pierce). For Fig. 4b, the bound biotinylated peptides (Upstate Biotechnology) were separated by 18% SDS?PAGE, blotted, and detected with streptavidin HRP conjugate (Upstate Biotechnology). Interaction of CMT3 with LHP1 A six-histidine fusion construct was made by cloning full-length LHP1 into the XhoI and PstI sites of pRSETB, and expressed in E. coli strain BL21. Proteins were purified on Ni- NTA-agarose (Qiagen) and eluted with 100 mM imidazole before mixing with glutathione-agarose-bound GST fusion proteins. Binding and wash buffers were the same as in the peptide binding assays 5 . Bound proteins were separated by 4?20% gradient SDS? PAGE, blotted, and detected with INDIA HisProbe-HRP (Pierce). Received 15 January; accepted 4 March 2002. Published online 17 March 2002, DOI 10.1038/nature731. 1. Rea, S. et al. Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406, 593?599 (2000). 2. Nakayama, J., Rice, J. C., Strahl, B. D., Allis, C. D. & Grewal, S. I. 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Mutations in LIKE HETEROCHROMATIN PROTEIN 1 affect flowering time and plant architecture in Arabidopsis. Development 128, 4847?4858 (2001). 27. Peters, A. H. F. M. et al. Histone H3 lysine 9 methylation is an epigenetic imprint of facultative heterochromatin. Nature Genet. 30, 77?80 (2002). Supplementary Information accompanies the paper on Nature?s website (http://www.nature.com). Acknowledgements We thank T. Jenuwein for the GST?Suv constructs and H3 N-terminal peptides, Y. Shinkai for the H3 N-terminal GST fusion constructs, A. Kouzarides for an HP1 construct, and S. Peyvandi for technical assistance. This work was supported by grants from the National Institutes of Health, the Beckman Young Investigator programme, and the Searle Scholars Foundation to S.E.J. J.P.J. was supported by an NIH training grant and A.M.L. by a post- doctoral fellowship from the Damon Runyon Walter Winchel Foundation. Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to S.E.J. (e-mail: jacobsen@ucla.edu). .............................................................. p63 and p73 are required for p53- dependent apoptosis in response to DNA damage Elsa R. Flores*, Kenneth Y. Tsai*?, Denise Crowley*�, Shomit Sengupta*�, Annie Yang?, Frank McKeon? & Tyler Jacks*� * Massachusetts Institute of Technology, Department of Biology and Center for Cancer Research, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA ? Harvard?Massachusetts Institute of Technology Division of Health Sciences and Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA ? Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA � Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, Maryland 20185, USA ............................................................................................................................................................................. The tumour-suppressor gene p53 is frequently mutated in human cancers and is important in the cellular response to DNA damage 1,2 . Although the p53 family members p63 and p73 are structurally related to p53, they have not been directly linked to tumour suppression, although they have been implicated in apoptosis 3?9 . Given the similarity between this family of genes and the ability of p63 and p73 to transactivate p53 target genes 10,11 , we explore here their role in DNA damage-induced apoptosis. Mouse embryo fibroblasts deficient for one or a combination of p53 family members were sensitized to undergo apoptosis through the expression of the adenovirus E1A onco- gene 12 ? 14 . While using the E1A system facilitated our ability to perform biochemical analyses, we also examined the functions of p63 and p73 using an in vivo system in which apoptosis has been shown to be dependent on p53. Using both systems, we show here that the combined loss of p63 and p73 results in the failure of cells containing functional p53 to undergo apoptosis in response to DNA damage. Previous work has shown that p53-deficient E1A mouse embryo fibroblasts (MEFs) treated with DNA-damaging agents are highly resistant to apoptosis 12 ? 14 . To determine whether p63 and/or p73 are involved in apoptosis induced by DNA damage, p63-deficient (p63 2/2 ) and p73-deficient (p73 2/2 ) E1A-expressing MEFs were generated and treated with doxorubicin for 0, 6, 12, 24 and 48 h (Fig. 1A; Supplementary Information, Fig. S1a), stained with annexin V coupled to fluorescein isothiocyanate, and analysed by flow cyto- metry. E1A MEFs lacking p63 or p73 exhibited a partial resistance to apoptosis in response to DNA damage; 70% of the p63 2/2 E1A MEFs and 80% of the p73 2/2 E1A MEFs were viable, compared with 42% of the wild-type E1A MEFs at 12 h. At 24 h, 50% of the p63 2/2 E1A MEFs and 65% of the p73 2/2 E1A MEFs were viable, compared with 5% of the wild-type E1A MEFs (Fig. 1A). Because p63-deficient and p73-deficient E1A MEFs exhibited a partial resistance to apoptosis, we hypothesized that they might cooperate with p53 or with each other in the DNA damage response. Therefore, double-homozygous E1A MEFs deficient for all pairs of combinations (p53 2/2 ; p63 2/2 , p53 2/2 ; p73 2/2 , p63 2/2 ; p73 2/2 ) were generated and treated with doxorubicin. As shown in Fig. 1A, all the double-knockout cells, including the p63 2/2 ; p73 2/2 E1A MEFs, were resistant to apoptosis in response to DNA damage. The p53 2/2 ; p63 2/2 and p53 2/2 ; p73 2/2 E1A MEFs were more resistant letters to nature NATURE | VOL 416 | 4 APRIL 2002 | www.nature.com560 � 2002 Macmillan Magazines Ltd "
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