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A SNF2‐like protein facilitates dynamic control of DNA methylation

A SNF2‐like protein facilitates dynamic control of DNA methylation DRD1 is a SNF2-like protein previously identified in a screen for mutants defective in RNA-directed DNA methylation of a seed promoter in . Although the initial study established a role for DRD1 in RNA-directed DNA methylation, it did not address whether DRD1 is needed for or maintenance methylation, or whether it is required for methylation of other target sequences. We show here that DRD1 is essential for RNA- directed methylation and acts on different target promoters. In addition, an unanticipated role for DRD1 in erasure of CG methylation was shown when investigating maintenance methylation after segregating away the silencing trigger. DRD1 is unique among known SNF2-like proteins in facilitating not only methylation of target sequences in response to RNA signals, but also loss of methylation when the silencing inducer is withdrawn. The opposing roles of DRD1 could contribute to the dynamic regulation of DNA methylation. RESULTS AND DISCUSSION INTRODUCTION Received 31 January 2005; revised 4 May 2005; accepted 5 May 2005; published online 10 June 2005 649 SNF2 - like protein and DNA methylation dynamic s T. Ka nno e t al scientifi c report a' promoter H complex (Fig 1 D, left). The level of methylation observed in NOS promoter - H FSc- PsPaEBa - P S B N wild-type F1 plants approximated that seen in plants in which the target complex and silencer complex had been together in the same genome for several generations (Fig 1 B, left). The similar methylation patterns indicate that the maximum attainable level of KIK ;-1-;DID RNA-directed methylation of the target a. ' promoter is essentially reached in the first generation containing both transgene complexes. By contrast, the target a. ' promoter did not acquire K IK;HI H;D/D detectable methylation after being combined with the silencer H complex in homozygous dr dl-6 plants (Fig 1 E, left ), which showed a hybridization pattern identical to that of the unmethy­ lated target a. ' promoter (Fig 1 A, left). The lack of methylation was NA KI K;HIH; dld not because of inadequate production of RNA signals, as indicated by the continued presence of a. ' promoter short RNAs in drdl-6 plants (Fig 3A). The target NOS promoter also becomes methylated de nova K l - ;H I - ;DID at CGs and non-CGs in wild-type plants after introducing the respective silencer H construct (Fig 1 D, right; Aufsatz et al, 2004), but it failed to acquire measurable methylation in the drdl-6 mutant (Fig 1 E, right). Bisulphite sequencing confirmed that no detectable methylation was induced at the target NOS promoter in dr dl-6 mutants, whereas methylation was observed at Cs in all sequence contexts within the region of RNA-DNA sequence Fig 1 I Analysis of de novo methylation. Methylation of the tar get ,:,, ' identity in wild-type plants (supplementary Fig 2 online). NOS pr omoter was analysed using r estr ic tion enzymes diagnostic fo r non- CG promoter short RNAs were detected in the F1 plants (Fig 3B), methylation (F, Sc , Ps, Pa, E, Ba) or CG methylation (H). Methylation of the nopaline synthase (NOS) pr omoter was analysed using r estr ic tion indicating that RNA signals were available but unable to induce methylation in the drdl-6 mutants. In contrast to the a. ' promoter, enzymes diagnostic fo r non- CG methylation (N), CG methylation (P, B) the level of NOS promoter methylation was less in F1 progeny or both (S). The r espec tive methylation- sensitive enzymes wer e added than in plants in which the target and silencer had been aft er a standar d digest with non- methylation- sensitive r estr iction together for several generations (compare Fig 1 D, right, with enzymes ('-' lanes). The position of the methylated fr agment is denoted Fig 1 B, right). Despite this diff erence, the results show that by the dots to the left of eac h blot. Shift s to the smaller fr agment( s) DRD1 is indispensable for RNA-directed de nova methylation indic ate no methylation at the site(s) tested. Maps of the pr omoter s of two distinct promoters. showing positions of r estr ic tion enzyme sites and the pr obes used fo r The eff iciency of maintenance methylation was examined in hyb r idization ar e depic ted in supplementar y Fig 1 online. Genotypes of wild-type and drdl-6 plants aft er crossing out the respective the plants analysed ar e shown to the r ight; dashes indic ate a hemizygous silencer H complexes to remove the source of the RNA signals. In tr ansgene loc us. Bold letter s to the left r epr esent boxed genotypes shown wild-type F2 progeny descended from DRD1 wild-type parents in the br eeding sc hemes in Fig 2. Abbr eviations: K, tar get c omplex; H, (Fig 2F), the target a. ' promoter lost both CG and non-CG silenc er c omplex; d, dr dl -6 mutant; D, wild type; NA, not applicable. methylation after segregating away the silencer complex (Fig 4F, Abbr eviations of enzymes and their r ec ognition sequenc es ( sensitivity to left ), resulting in a hybridization pattern that is indistinguishable C methylation indic ated by the super sc r ipt 'm'): r: pr omoter : B, BstUI m m m m from the unmethylated a. ' promoter (Fig 1 A, left). An identical ( c G CG); Ba, BamHI (GGAT c c ); E, EcoT22I (ATG CAT); m m m m pattern of methylation (Fig 4G, left) was observed in wild-type F2 F, Fnu4HI (G c NG c , if N is C); H, HpyCH4IV (A CGT); m m progeny descended from the drdl-6 mutant (Fig 2G). Bisulphite NOS pr omoter : N, Nh eI (GCTAG C); P, Psp 1406I (AA CGTT); m m m m m m sequencing confirmed the loss of non-CG methylation and Pa, Pagl (T CATGA); Ps, Pstl ( CTG CAG); S, SacII ( c c G CGG); m m showed only some residual methylation at two CG dinucleotides Sc , Scr Fl (C c NGG (if N is C). (supplementary Fig 3A online). Thus, in the a. ' promoter silencing system, almost all methylation is lost in wild-type progeny when as indicated by the persistent double band generated by the the source of the RNA signal is withdrawn. Unexpectedly, restriction enzyme abbreviated H (Fig 1 C, left). Similar findings for however, in drdl-6 progeny lacking the silencer H complex drdl-1 led to the suggestion that DRD1 is important primarily for (Fig 2H,I), substantial CG methylation was detected, even though non-CG methylation (Kanno et al, 2004). non-CG methylation was lost. Retention of CG methylation is To analyse whether DRD1 is needed for RNA-directed de nova again exemplified by the persistent double band generated by the methylation of target sequences, crosses were made to generate restriction enzyme abbreviated H. This double band was observed F1 plants in which a naive target K complex was combined with in dr dl-6 progeny that are homozygous (Fig 4H, left) and the silencer H complex in either wild-type (0/0; Fig 2D) or hemizygous (Fig 41, left) for the target K complex, indicating no homozygous dr dl-6 (di d) plants (Fig 2 E). Methylation of the target dependence on dosage of the target promoter. Bisulphite promoter was then examined in the resulting F1 progeny. sequencing confirmed the enhanced maintenance of CG methyla­ In wild-type F1 plants, the target a. ' promoter showed increased tion in the drdl-6 mutant (supplementary Fig 3B online) relative to methylation in CGs and in non-CGs aft er introducing the silencer wild-type plants (supplementary Fig 3A online). EMBO report s VOL 6 I NO 7 I 2005 ©2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION 651 Speculation 652 21: 99: 54: METHODS Plant material and genotyping. 13: 15: DNA methylation analysis. 24: 111: Short RNA analysis. 416: 22: Supplementary information 11: 653 117: 14: 431: 1677: 305: 303: 494: 241: 21: 5: http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png EMBO Reports Springer Journals

A SNF2‐like protein facilitates dynamic control of DNA methylation

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Publisher
Springer Journals
Copyright
Copyright © European Molecular Biology Organization 2005
ISSN
1469-221X
eISSN
1469-3178
DOI
10.1038/sj.embor.7400446
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Abstract

DRD1 is a SNF2-like protein previously identified in a screen for mutants defective in RNA-directed DNA methylation of a seed promoter in . Although the initial study established a role for DRD1 in RNA-directed DNA methylation, it did not address whether DRD1 is needed for or maintenance methylation, or whether it is required for methylation of other target sequences. We show here that DRD1 is essential for RNA- directed methylation and acts on different target promoters. In addition, an unanticipated role for DRD1 in erasure of CG methylation was shown when investigating maintenance methylation after segregating away the silencing trigger. DRD1 is unique among known SNF2-like proteins in facilitating not only methylation of target sequences in response to RNA signals, but also loss of methylation when the silencing inducer is withdrawn. The opposing roles of DRD1 could contribute to the dynamic regulation of DNA methylation. RESULTS AND DISCUSSION INTRODUCTION Received 31 January 2005; revised 4 May 2005; accepted 5 May 2005; published online 10 June 2005 649 SNF2 - like protein and DNA methylation dynamic s T. Ka nno e t al scientifi c report a' promoter H complex (Fig 1 D, left). The level of methylation observed in NOS promoter - H FSc- PsPaEBa - P S B N wild-type F1 plants approximated that seen in plants in which the target complex and silencer complex had been together in the same genome for several generations (Fig 1 B, left). The similar methylation patterns indicate that the maximum attainable level of KIK ;-1-;DID RNA-directed methylation of the target a. ' promoter is essentially reached in the first generation containing both transgene complexes. By contrast, the target a. ' promoter did not acquire K IK;HI H;D/D detectable methylation after being combined with the silencer H complex in homozygous dr dl-6 plants (Fig 1 E, left ), which showed a hybridization pattern identical to that of the unmethy­ lated target a. ' promoter (Fig 1 A, left). The lack of methylation was NA KI K;HIH; dld not because of inadequate production of RNA signals, as indicated by the continued presence of a. ' promoter short RNAs in drdl-6 plants (Fig 3A). The target NOS promoter also becomes methylated de nova K l - ;H I - ;DID at CGs and non-CGs in wild-type plants after introducing the respective silencer H construct (Fig 1 D, right; Aufsatz et al, 2004), but it failed to acquire measurable methylation in the drdl-6 mutant (Fig 1 E, right). Bisulphite sequencing confirmed that no detectable methylation was induced at the target NOS promoter in dr dl-6 mutants, whereas methylation was observed at Cs in all sequence contexts within the region of RNA-DNA sequence Fig 1 I Analysis of de novo methylation. Methylation of the tar get ,:,, ' identity in wild-type plants (supplementary Fig 2 online). NOS pr omoter was analysed using r estr ic tion enzymes diagnostic fo r non- CG promoter short RNAs were detected in the F1 plants (Fig 3B), methylation (F, Sc , Ps, Pa, E, Ba) or CG methylation (H). Methylation of the nopaline synthase (NOS) pr omoter was analysed using r estr ic tion indicating that RNA signals were available but unable to induce methylation in the drdl-6 mutants. In contrast to the a. ' promoter, enzymes diagnostic fo r non- CG methylation (N), CG methylation (P, B) the level of NOS promoter methylation was less in F1 progeny or both (S). The r espec tive methylation- sensitive enzymes wer e added than in plants in which the target and silencer had been aft er a standar d digest with non- methylation- sensitive r estr iction together for several generations (compare Fig 1 D, right, with enzymes ('-' lanes). The position of the methylated fr agment is denoted Fig 1 B, right). Despite this diff erence, the results show that by the dots to the left of eac h blot. Shift s to the smaller fr agment( s) DRD1 is indispensable for RNA-directed de nova methylation indic ate no methylation at the site(s) tested. Maps of the pr omoter s of two distinct promoters. showing positions of r estr ic tion enzyme sites and the pr obes used fo r The eff iciency of maintenance methylation was examined in hyb r idization ar e depic ted in supplementar y Fig 1 online. Genotypes of wild-type and drdl-6 plants aft er crossing out the respective the plants analysed ar e shown to the r ight; dashes indic ate a hemizygous silencer H complexes to remove the source of the RNA signals. In tr ansgene loc us. Bold letter s to the left r epr esent boxed genotypes shown wild-type F2 progeny descended from DRD1 wild-type parents in the br eeding sc hemes in Fig 2. Abbr eviations: K, tar get c omplex; H, (Fig 2F), the target a. ' promoter lost both CG and non-CG silenc er c omplex; d, dr dl -6 mutant; D, wild type; NA, not applicable. methylation after segregating away the silencer complex (Fig 4F, Abbr eviations of enzymes and their r ec ognition sequenc es ( sensitivity to left ), resulting in a hybridization pattern that is indistinguishable C methylation indic ated by the super sc r ipt 'm'): r: pr omoter : B, BstUI m m m m from the unmethylated a. ' promoter (Fig 1 A, left). An identical ( c G CG); Ba, BamHI (GGAT c c ); E, EcoT22I (ATG CAT); m m m m pattern of methylation (Fig 4G, left) was observed in wild-type F2 F, Fnu4HI (G c NG c , if N is C); H, HpyCH4IV (A CGT); m m progeny descended from the drdl-6 mutant (Fig 2G). Bisulphite NOS pr omoter : N, Nh eI (GCTAG C); P, Psp 1406I (AA CGTT); m m m m m m sequencing confirmed the loss of non-CG methylation and Pa, Pagl (T CATGA); Ps, Pstl ( CTG CAG); S, SacII ( c c G CGG); m m showed only some residual methylation at two CG dinucleotides Sc , Scr Fl (C c NGG (if N is C). (supplementary Fig 3A online). Thus, in the a. ' promoter silencing system, almost all methylation is lost in wild-type progeny when as indicated by the persistent double band generated by the the source of the RNA signal is withdrawn. Unexpectedly, restriction enzyme abbreviated H (Fig 1 C, left). Similar findings for however, in drdl-6 progeny lacking the silencer H complex drdl-1 led to the suggestion that DRD1 is important primarily for (Fig 2H,I), substantial CG methylation was detected, even though non-CG methylation (Kanno et al, 2004). non-CG methylation was lost. Retention of CG methylation is To analyse whether DRD1 is needed for RNA-directed de nova again exemplified by the persistent double band generated by the methylation of target sequences, crosses were made to generate restriction enzyme abbreviated H. This double band was observed F1 plants in which a naive target K complex was combined with in dr dl-6 progeny that are homozygous (Fig 4H, left) and the silencer H complex in either wild-type (0/0; Fig 2D) or hemizygous (Fig 41, left) for the target K complex, indicating no homozygous dr dl-6 (di d) plants (Fig 2 E). Methylation of the target dependence on dosage of the target promoter. Bisulphite promoter was then examined in the resulting F1 progeny. sequencing confirmed the enhanced maintenance of CG methyla­ In wild-type F1 plants, the target a. ' promoter showed increased tion in the drdl-6 mutant (supplementary Fig 3B online) relative to methylation in CGs and in non-CGs aft er introducing the silencer wild-type plants (supplementary Fig 3A online). EMBO report s VOL 6 I NO 7 I 2005 ©2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION 651 Speculation 652 21: 99: 54: METHODS Plant material and genotyping. 13: 15: DNA methylation analysis. 24: 111: Short RNA analysis. 416: 22: Supplementary information 11: 653 117: 14: 431: 1677: 305: 303: 494: 241: 21: 5:

Journal

EMBO Reports – Springer Journals

Published: Jul 1, 2005

Keywords: demethylation; de novo methylation; maintenance methylation; RNA‐directed DNA methylation; SNF2‐like protein

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