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• W1968466047 abstract "Epigenetic gene silencing suppresses transposon activity and is critical for normal development [1Stancheva I. Caught in conspiracy: cooperation between DNA methylation and histone H3K9 methylation in the establishment and maintenance of heterochromatin.Biochem. Cell Biol. 2005; 83: 385-395Crossref PubMed Scopus (51) Google Scholar, 2Chan S.W. Henderson I.R. Jacobsen S.E. Gardening the genome: DNA methylation in Arabidopsis thaliana.Nat. Rev. Genet. 2005; 6: 351-360Crossref PubMed Scopus (680) Google Scholar]. Two common epigenetic gene-silencing marks are DNA methylation and histone H3 lysine 9 dimethylation (H3K9me2) [1Stancheva I. Caught in conspiracy: cooperation between DNA methylation and histone H3K9 methylation in the establishment and maintenance of heterochromatin.Biochem. Cell Biol. 2005; 83: 385-395Crossref PubMed Scopus (51) Google Scholar]. In Arabidopsis thaliana, H3K9me2, catalyzed by the methyltransferase KRYPTONITE (KYP/SUVH4), is required for maintenance of DNA methylation outside of the standard CG sequence context [3Jackson J.P. Lindroth A.M. Cao X. Jacobsen S.E. Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase.Nature. 2002; 416: 556-560Crossref PubMed Scopus (957) Google Scholar, 4Malagnac F. Bartee L. Bender J. An Arabidopsis SET domain protein required for maintenance but not establishment of DNA methylation.EMBO J. 2002; 21: 6842-6852Crossref PubMed Scopus (237) Google Scholar]. Additionally, loss of DNA methylation in the met1 mutant correlates with a loss of H3K9me2 [5Soppe W.J. Jasencakova Z. Houben A. Kakutani T. Meister A. Huang M.S. Jacobsen S.E. Schubert I. Fransz P.F. DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis.EMBO J. 2002; 21: 6549-6559Crossref PubMed Scopus (373) Google Scholar, 6Tariq M. Saze H. Probst A.V. Lichota J. Habu Y. Paszkowski J. Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin.Proc. Natl. Acad. Sci. USA. 2003; 100: 8823-8827Crossref PubMed Scopus (241) Google Scholar]. Here we show that KYP-dependent H3K9me2 is found at non-CG methylation sites in addition to those rich in CG methylation. Furthermore, we show that the SRA domain of KYP binds directly to methylated DNA, and SRA domains with missense mutations found in loss-of-function kyp mutants have reduced binding to methylated DNA in vitro. These data suggest that DNA methylation is required for the recruitment or activity of KYP and suggest a self-reinforcing loop between histone and DNA methylation. Lastly, we found that SRA domains from two Arabidopsis SRA-RING proteins also bind methylated DNA and that the SRA domains from KYP and SRA-RING proteins prefer methylcytosines in different sequence contexts. Hence, unlike the methyl-binding domain (MBD), which binds only methylated-CpG sequences, the SRA domain is a versatile new methyl-DNA-binding motif. Epigenetic gene silencing suppresses transposon activity and is critical for normal development [1Stancheva I. Caught in conspiracy: cooperation between DNA methylation and histone H3K9 methylation in the establishment and maintenance of heterochromatin.Biochem. Cell Biol. 2005; 83: 385-395Crossref PubMed Scopus (51) Google Scholar, 2Chan S.W. Henderson I.R. Jacobsen S.E. Gardening the genome: DNA methylation in Arabidopsis thaliana.Nat. Rev. Genet. 2005; 6: 351-360Crossref PubMed Scopus (680) Google Scholar]. Two common epigenetic gene-silencing marks are DNA methylation and histone H3 lysine 9 dimethylation (H3K9me2) [1Stancheva I. Caught in conspiracy: cooperation between DNA methylation and histone H3K9 methylation in the establishment and maintenance of heterochromatin.Biochem. Cell Biol. 2005; 83: 385-395Crossref PubMed Scopus (51) Google Scholar]. In Arabidopsis thaliana, H3K9me2, catalyzed by the methyltransferase KRYPTONITE (KYP/SUVH4), is required for maintenance of DNA methylation outside of the standard CG sequence context [3Jackson J.P. Lindroth A.M. Cao X. Jacobsen S.E. Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase.Nature. 2002; 416: 556-560Crossref PubMed Scopus (957) Google Scholar, 4Malagnac F. Bartee L. Bender J. An Arabidopsis SET domain protein required for maintenance but not establishment of DNA methylation.EMBO J. 2002; 21: 6842-6852Crossref PubMed Scopus (237) Google Scholar]. Additionally, loss of DNA methylation in the met1 mutant correlates with a loss of H3K9me2 [5Soppe W.J. Jasencakova Z. Houben A. Kakutani T. Meister A. Huang M.S. Jacobsen S.E. Schubert I. Fransz P.F. DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis.EMBO J. 2002; 21: 6549-6559Crossref PubMed Scopus (373) Google Scholar, 6Tariq M. Saze H. Probst A.V. Lichota J. Habu Y. Paszkowski J. Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin.Proc. Natl. Acad. Sci. USA. 2003; 100: 8823-8827Crossref PubMed Scopus (241) Google Scholar]. Here we show that KYP-dependent H3K9me2 is found at non-CG methylation sites in addition to those rich in CG methylation. Furthermore, we show that the SRA domain of KYP binds directly to methylated DNA, and SRA domains with missense mutations found in loss-of-function kyp mutants have reduced binding to methylated DNA in vitro. These data suggest that DNA methylation is required for the recruitment or activity of KYP and suggest a self-reinforcing loop between histone and DNA methylation. Lastly, we found that SRA domains from two Arabidopsis SRA-RING proteins also bind methylated DNA and that the SRA domains from KYP and SRA-RING proteins prefer methylcytosines in different sequence contexts. Hence, unlike the methyl-binding domain (MBD), which binds only methylated-CpG sequences, the SRA domain is a versatile new methyl-DNA-binding motif. We utilized chromatin immunoprecipitation (ChIP) and genomic bisulfite sequencing to further investigate the relationship between histone methylation and DNA methylation. In Arabidopsis, DNA methylation (5-methyl cytosine) occurs not only at CG residues, but also at CNG and asymmetric cytosines (CNN) [2Chan S.W. Henderson I.R. Jacobsen S.E. Gardening the genome: DNA methylation in Arabidopsis thaliana.Nat. Rev. Genet. 2005; 6: 351-360Crossref PubMed Scopus (680) Google Scholar]. METHYLTRANSFERASE 1 (MET1), a homolog of mammalian Dnmt1, maintains methylation at CG residues [7Finnegan E.J. Peacock W.J. Dennis E.S. Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development.Proc. Natl. Acad. Sci. USA. 1996; 93: 8449-8454Crossref PubMed Scopus (569) Google Scholar, 8Ronemus M.J. Galbiati M. Ticknor C. Chen J. Dellaporta S.L. Demethylation-induced developmental pleiotropy in Arabidopsis.Science. 1996; 273: 654-657Crossref PubMed Scopus (382) Google Scholar], whereas CHROMOMETHYLASE3 (CMT3) maintains the majority of CNG methylation and a small amount of the CNN methylation [9Lindroth A.M. Cao X. Jackson J.P. Zilberman D. McCallum C.M. Henikoff S. Jacobsen S.E. Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation.Science. 2001; 292: 2077-2080Crossref PubMed Scopus (630) Google Scholar, 10Bartee L. Malagnac F. Bender J. Arabidopsis cmt3 chromomethylase mutations block non-CG methylation and silencing of an endogenous gene.Genes Dev. 2001; 15: 1753-1758Crossref PubMed Scopus (309) Google Scholar]. A third enzyme, DOMAINS REARRANGED METHYLASE 2 (DRM2), which is mainly responsible for maintaining CNN methylation, plays a minor role in the maintenance of CNG methylation and is also required for de novo methylation of cytosines in all sequence contexts [2Chan S.W. Henderson I.R. Jacobsen S.E. Gardening the genome: DNA methylation in Arabidopsis thaliana.Nat. Rev. Genet. 2005; 6: 351-360Crossref PubMed Scopus (680) Google Scholar, 11Cao X. Jacobsen S.E. Locus-specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferase genes.Proc. Natl. Acad. Sci. USA. 2002; 99: 16491-16498Crossref PubMed Scopus (428) Google Scholar]. Genomic regions that are enriched in methylated DNA are also found associated with H3K9me2 [12Johnson L. Cao X. Jacobsen S. Interplay between two epigenetic marks. DNA methylation and histone H3 lysine 9 methylation.Curr. Biol. 2002; 12: 1360-1367Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 13Jackson J.P. Johnson L. Jasencakova Z. Zhang X. PerezBurgos L. Singh P.B. Cheng X. Schubert I. Jenuwein T. Jacobsen S.E. Dimethylation of histone H3 lysine 9 is a critical mark for DNA methylation and gene silencing in Arabidopsis thaliana.Chromosoma. 2004; 112: 308-315Crossref PubMed Scopus (228) Google Scholar, 14Gendrel A.V. Lippman Z. Yordan C. Colot V. Martienssen R.A. Dependence of heterochromatic histone H3 methylation patterns on the Arabidopsis gene DDM1.Science. 2002; 297: 1871-1873Crossref PubMed Scopus (344) Google Scholar]. It has previously been shown that mutations in KYP lead to reduction of non-CG DNA methylation, but not CG methylation, suggesting that non-CG methylation is dependent on histone H3K9me2 [3Jackson J.P. Lindroth A.M. Cao X. Jacobsen S.E. Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase.Nature. 2002; 416: 556-560Crossref PubMed Scopus (957) Google Scholar, 4Malagnac F. Bartee L. Bender J. An Arabidopsis SET domain protein required for maintenance but not establishment of DNA methylation.EMBO J. 2002; 21: 6842-6852Crossref PubMed Scopus (237) Google Scholar]. CMT3 is responsible for this non-CG methylation and has been shown in vitro to bind directly to H3 tails trimethylated at lysine 9 and lysine 27 [9Lindroth A.M. Cao X. Jackson J.P. Zilberman D. McCallum C.M. Henikoff S. Jacobsen S.E. Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation.Science. 2001; 292: 2077-2080Crossref PubMed Scopus (630) Google Scholar, 10Bartee L. Malagnac F. Bender J. Arabidopsis cmt3 chromomethylase mutations block non-CG methylation and silencing of an endogenous gene.Genes Dev. 2001; 15: 1753-1758Crossref PubMed Scopus (309) Google Scholar, 15Lindroth A.M. Shultis D. Jasencakova Z. Fuchs J. Johnson L. Schubert D. Patnaik D. Pradhan S. Goodrich J. Schubert I. et al.Dual histone H3 methylation marks at lysines 9 and 27 required for interaction with CHROMOMETHYLASE3.EMBO J. 2004; 23: 4286-4296Crossref PubMed Scopus (268) Google Scholar]. Conversely, the loss of DNA methylation in met1 mutants is also associated with decreased H3K9me2 at many loci [5Soppe W.J. Jasencakova Z. Houben A. Kakutani T. Meister A. Huang M.S. Jacobsen S.E. Schubert I. Fransz P.F. DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis.EMBO J. 2002; 21: 6549-6559Crossref PubMed Scopus (373) Google Scholar, 6Tariq M. Saze H. Probst A.V. Lichota J. Habu Y. Paszkowski J. Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin.Proc. Natl. Acad. Sci. USA. 2003; 100: 8823-8827Crossref PubMed Scopus (241) Google Scholar, 12Johnson L. Cao X. Jacobsen S. Interplay between two epigenetic marks. DNA methylation and histone H3 lysine 9 methylation.Curr. Biol. 2002; 12: 1360-1367Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 16Mathieu O. Probst A.V. Paszkowski J. Distinct regulation of histone H3 methylation at lysines 27 and 9 by CpG methylation in Arabidopsis.EMBO J. 2005; 24: 2783-2791Crossref PubMed Scopus (160) Google Scholar]. One possible explanation for this observation is that KYP is in a complex with MET1, such that loss of MET1 causes a loss of KYP activity. A second possibility is that DNA methylation itself directly affects KYP activity or targeting. To test these ideas, we examined H3K9me2 levels in different DNA methyltransferase mutants and at two loci with very different patterns of CG or non-CG methylation (see Figure S1 in the Supplemental Data available online). AtSN1 is a 170 bp SINE retrotransposon heavily methylated at non-CG and to a lesser extent at CG sites (∼13 versus 3 per element; Figure 1A, see also [17Xie Z. Johansen L.K. Gustafson A.M. Kasschau K.D. Lellis A.D. Zilberman D. Jacobsen S.E. Carrington J.C. Genetic and functional diversification of small RNA pathways in plants.PLoS Biol. 2004; 2: e104https://doi.org/10.1371/journal.pbio.0020104Crossref PubMed Scopus (1120) Google Scholar]). AtSN1 is also marked by H3K9me2, which is dependent on KYP, illustrated by its loss in a kyp null line, kyp-6 (Figure 1B). Elimination of most of the DNA methylation in the drm1 drm2 cmt3 triple mutant also results in significant loss of H3K9me2 methylation, showing a strong correlation between levels of DNA and histone methylation (Figure 1B). In contrast, the met1-3 mutation showed complete elimination of CG methylation from AtSN1, but only a minor effect on non-CG methylation (Figure 1A), and H3K9me2 was observed at wild-type levels (Figure 1B). This suggests that KYP activity is not affected by loss of MET1 per se and that histone H3 methylation can be maintained at genomic regions containing only non-CG methylation. Unlike AtSN1, AtCOPIA4 is a CG-rich retrotransposon [18Lippman Z. May B. Yordan C. Singer T. Martienssen R. Distinct mechanisms determine transposon inheritance and methylation via small interfering RNA and histone modification.PLoS Biol. 2003; 1: e67https://doi.org/10.1371/journal.pbio.0000067Crossref PubMed Scopus (322) Google Scholar]. Bisulfite sequencing of a 500 nucleotide segment revealed an average of 22 methylated CG residues, 6 methylated CNGs, and 3 methylated asymmetric cytosines in wild-type plants (Figure 1C, see also [19Henderson I.R. Zhang X. Lu C. Johnson L. Meyers B.C. Green P.J. Jacobsen S.E. Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning.Nat. Genet. 2006; 38: 721-725Crossref PubMed Scopus (415) Google Scholar]). This element is also associated with KYP-dependent H3K9me2 (Figure 1D; see also [18Lippman Z. May B. Yordan C. Singer T. Martienssen R. Distinct mechanisms determine transposon inheritance and methylation via small interfering RNA and histone modification.PLoS Biol. 2003; 1: e67https://doi.org/10.1371/journal.pbio.0000067Crossref PubMed Scopus (322) Google Scholar]). In drm1 drm2 cmt3, the CNG methylation was greatly reduced, while the CG and histone methylation levels were unchanged (Figures 1C and 1D). However, in met1-3, all DNA methylation was eliminated, as was most of the H3K9me2. Taking both the results at AtSN1 and AtCOPIA4 into account, we conclude that mutations—in particular DNA methyltransferases—may not directly affect histone methylation, but rather the level of either CG or non-CG DNA methylation is correlated with H3K9me2. One explanation for these results is that DNA methylation is important for recruitment or activity of KYP. The only recognizable domain in KYP besides the catalytic SET domain is the YDG (named for three conserved amino acids)/SRA (SET and RING associated) domain [20Baumbusch L.O. Thorstensen T. Krauss V. Fischer A. Naumann K. Assalkhou R. Schulz I. Reuter G. Aalen R.B. The Arabidopsis thaliana genome contains at least 29 active genes encoding SET domain proteins that can be assigned to four evolutionarily conserved classes.Nucleic Acids Res. 2001; 29: 4319-4333Crossref PubMed Scopus (242) Google Scholar]. We isolated a new allele of KYP (kyp-5) with a missense mutation in the SRA domain (E208K) as part of a previously described screen for suppressors of silencing of the SUPERMAN locus (Figure 2A) [9Lindroth A.M. Cao X. Jackson J.P. Zilberman D. McCallum C.M. Henikoff S. Jacobsen S.E. Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation.Science. 2001; 292: 2077-2080Crossref PubMed Scopus (630) Google Scholar]. Like null alleles [3Jackson J.P. Lindroth A.M. Cao X. Jacobsen S.E. Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase.Nature. 2002; 416: 556-560Crossref PubMed Scopus (957) Google Scholar, 5Soppe W.J. Jasencakova Z. Houben A. Kakutani T. Meister A. Huang M.S. Jacobsen S.E. Schubert I. Fransz P.F. DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis.EMBO J. 2002; 21: 6549-6559Crossref PubMed Scopus (373) Google Scholar, 13Jackson J.P. Johnson L. Jasencakova Z. Zhang X. PerezBurgos L. Singh P.B. Cheng X. Schubert I. Jenuwein T. Jacobsen S.E. Dimethylation of histone H3 lysine 9 is a critical mark for DNA methylation and gene silencing in Arabidopsis thaliana.Chromosoma. 2004; 112: 308-315Crossref PubMed Scopus (228) Google Scholar], this allele decreased H3K9me2 in chromocenters (Figure 2B). In addition, two previously reported mutant kyp alleles contained missense mutations (S200F and R260H) in the SRA domain, and transcriptional silencing, resulting from overexpression of the KYP-related gene SUVH2, was alleviated by mutation of its SRA domain [4Malagnac F. Bartee L. Bender J. An Arabidopsis SET domain protein required for maintenance but not establishment of DNA methylation.EMBO J. 2002; 21: 6842-6852Crossref PubMed Scopus (237) Google Scholar, 21Naumann K. Fischer A. Hofmann I. Krauss V. Phalke S. Irmler K. Hause G. Aurich A.C. Dorn R. Jenuwein T. et al.Pivotal role of AtSUVH2 in heterochromatic histone methylation and gene silencing in Arabidopsis.EMBO J. 2005; 24: 1418-1429Crossref PubMed Scopus (159) Google Scholar]. These results show that the SRA domain is critical for KYP function. The role of the SRA domain is unclear, but biochemical studies of a class of mammalian SRA- and RING-domain-containing proteins have been reported. In the murine protein Np95, the SRA domain was shown to bind histone H3 in vitro and to be required for chromatin association in vivo [22Citterio E. Papait R. Nicassio F. Vecchi M. Gomiero P. Mantovani R. Di Fiore P.P. Bonapace I.M. Np95 is a histone-binding protein endowed with ubiquitin ligase activity.Mol. Cell. Biol. 2004; 24: 2526-2535Crossref PubMed Scopus (152) Google Scholar]. However, a second study demonstrated that a human SRA-RING protein, ICBP90, bound methylated DNA in crude extracts, suggesting that the SRA either binds methylated DNA directly or binds indirectly through a methyl-DNA-binding domain protein present in the extracts [23Unoki M. Nishidate T. Nakamura Y. ICBP90, an E2F-1 target, recruits HDAC1 and binds to methyl-CpG through its SRA domain.Oncogene. 2004; 23: 7601-7610Crossref PubMed Scopus (212) Google Scholar]. We therefore tested whether the SRA domain from KYP was sufficient for binding to either histone H3 or methylated DNA. An in vitro translated SRA domain did not bind histone H3 (data not shown). However, analysis of DNA binding by mobility-shift assays with a purified GST (glutathione S-transferase)-tagged KYP-SRA fusion protein (amino acids 1–334, Figure 2C) revealed preferential binding to double-stranded oligonucleotides containing 5-methyl cytosine residues in all sequence contexts (all-mC) compared to unmethylated oligonucleotides with the same sequence (all-C) (Figure 3A). In contrast, a GST-tagged KYP-SET fusion protein (amino acids 335–601) did not bind to methylated or unmethylated DNA. The addition of an anti-GST antibody to the all-mC binding reaction blocked DNA binding (last two lanes in Figure 3A). These results indicate that the amino terminus of KYP containing the SRA domain binds methylated but not unmethylated DNA. Further analysis with various double-stranded oligonucleotides containing only CG, CNG, or CNN methylation revealed that the SRA domain in KYP binds to DNA with methylated cytosines in all contexts with a preference for CNG and CNN sequences (Figure 3B). This preference was supported by competition experiments (Figures 3C and 3H). To provide additional evidence that the SRA domain was indeed responsible for methyl-DNA binding, we engineered two different SRA mutations into the GST-KYP SRA construct (E208K and S200F). We observed weaker overall binding and a reduced ability of the mutant proteins to discriminate between methylated and unmethylated oligonucleotides (Figures 3D and 3E). These results strongly suggest that the SRA domain is responsible for methyl-DNA binding in the KYP amino terminus. In addition to KYP, SRA domains are found in the eight other histone methyltransferase genes (SUVH class) in Arabidopsis[20Baumbusch L.O. Thorstensen T. Krauss V. Fischer A. Naumann K. Assalkhou R. Schulz I. Reuter G. Aalen R.B. The Arabidopsis thaliana genome contains at least 29 active genes encoding SET domain proteins that can be assigned to four evolutionarily conserved classes.Nucleic Acids Res. 2001; 29: 4319-4333Crossref PubMed Scopus (242) Google Scholar]. We tested one of these, SUVH6, to determine whether it too could discriminate between methylated and unmethylated DNA. GST-SUVH6 full-length protein (amino acids 1–781) bound methylated, but not unmethylated, oligonucleotides (Figure S2A). Furthermore, competition experiments showed that GST-SUVH6 had a higher affinity for methylated CNG and CNN substrates than methylated CG, a preference very similar to that of KYP (Figure S2B). In order to engineer a fusion protein with just the SRA domain (Figure 2C), we first tried cloning the SRA domain predicted by SMART [24Schultz J. Milpetz F. Bork P. Ponting C.P. SMART, a simple modular architecture research tool: identification of signaling domains.Proc. Natl. Acad. Sci. USA. 1998; 95: 5857-5864Crossref PubMed Scopus (2885) Google Scholar] (delineated by arrows in Figure 2A) into vectors containing a His-tag and found that the tagged proteins were insoluble. GST or MBP (maltose binding protein) fusions were soluble, but inactive in binding DNA. We reanalyzed the SRA domain by aligning the nine different SUVH SRAs and two SRA-RINGs (see below) to determine whether any additional conserved regions outside the annotated SRA domain existed that could be important for its function. As shown in Figure 2A, a short stretch of amino acids upstream of the annotated SRA domain was found to be conserved. When this extended SRA domain from SUVH6 was fused to GST (SUVH6-SRA), it was completely soluble and highly active in binding methylated DNA. Importantly, like full-length SUVH6, SUVH6-SRA preferred methylated CNG and CNN over CG substrates (Figure 3F). Such a preference was confirmed by competition experiments with methylated CNG substrates and methylated CG or CNG oligonucleotides as competitors (Figure 3G). In addition to the histone methyltransferases, eight other Arabidopsis proteins contain the SRA domain. Two proteins consist of a single SRA domain, whereas a class of six proteins homologous to Np95 contains the SRA as well as PHD and RING domains. To determine whether the SRA domain found in the SRA-RING proteins also binds methylated DNA, we examined two Arabidopsis SRA-RING genes, which we named (ORTHRUS1) ORTH1 (At5g39550) and ORTH2 (At1g57820) (Figure 2A). Three GST fusion constructs were made from ORTH1: PNS contains the PHD domain, the N-terminal RING, and the SRA domain; PN contains the PHD and N-terminal RING domains; and SC contains the SRA and C-terminal RING domains (Figure 4A). In addition, a GST-tagged, full-length ORTH2 was made. Each of the proteins containing an SRA domain (PNS, SC, ORTH2) was able to bind methylated CG, CNG, or CNN substrates, but significantly less binding was observed to the unmethylated substrates (Figure 4B and Figures S3A and S3B). Interestingly, unlike the KYP and SUVH6 SRA domains that prefer CNG and CNN methylation, ORTH1 and ORTH2 show preferential binding to methylated CG substrates (Figures 4C and 4D, Figures S3C and S3D). Two different mutations introduced into the SRA domain in the PNS construct, S292F and R362H (corresponding to amino acids 200 and 260 based on the KYP sequence [Figure 2A]), resulted in loss of DNA binding (Figures S4A and S4B). These results implicate SRA-RING proteins in functions related to DNA methylation and further support the generality of the function of the SRA as a methyl-cytosine-binding domain. The SRA domain differs from the methyl-CpG-binding domain (MBD) found in several mammalian proteins such as MeCP2, because the MBD binds only methylated CG [25Meehan R.R. Lewis J.D. McKay S. Kleiner E.L. Bird A.P. Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs.Cell. 1989; 58: 499-507Abstract Full Text PDF PubMed Scopus (503) Google Scholar], but the SRA domain can recognize methylated DNA in any sequence context. Further, our data suggest that different SRA domains display preference for methylated cytosines in different sequence contexts. Our findings extend our understanding of DNA and histone methylation pathways in Arabidopsis thaliana. The observation that the SRA domain can bind directly to methylated DNA suggests that regions rich in either CG or non-CG DNA methylation may attract KYP, leading to H3K9 methylation. Alternatively, KYP could be targeted to heterochromatin by other mechanisms, and the binding of methylated DNA to the SRA domain may be somehow required for KYP activity. KYP-dependent histone methylation would then provide a binding site for CMT3 leading to CNG methylation, which would promote further action by KYP. This model suggests a self-reinforcing feedback loop for the maintenance of DNA and histone methylation and may help to explain the remarkable stability of epigenetic silent states." @default.
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• W1968466047 title "The SRA Methyl-Cytosine-Binding Domain Links DNA and Histone Methylation" @default.
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