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. 2002 Dec 16;21(24):6832-41.
doi: 10.1093/emboj/cdf663.

HDA6, a putative histone deacetylase needed to enhance DNA methylation induced by double-stranded RNA

Affiliations

HDA6, a putative histone deacetylase needed to enhance DNA methylation induced by double-stranded RNA

Werner Aufsatz et al. EMBO J. .

Abstract

To analyze relationships between RNA signals, DNA methylation and chromatin modifications, we performed a genetic screen to recover Arabidopsis mutants defective in RNA-directed transcriptional silencing and methylation of a nopaline synthase promoter-neomycinphosphotransferase II (NOSpro- NPTII) target gene. Mutants were identified by screening for recovery of kanamycin resistance in the presence of an unlinked silencing complex encoding NOSpro double-stranded RNA. One mutant, rts1 (RNA-mediated transcriptional silencing), displayed moderate recovery of NPTII gene expression and partial loss of methylation in the target NOSpro, predominantly at symmetrical C(N)Gs. The RTS1 gene was isolated by positional cloning and found to encode a putative histone deacetylase, HDA6. The more substantial decrease in methylation of symmetrical compared with asymmetrical cytosines in rts1 mutants suggests that HDA6 is dispensable for RNA-directed de novo methylation, which results in intermediate methylation of cytosines in all sequence contexts, but is necessary for reinforcing primarily C(N)G methylation induced by RNA. Because CG methylation in centromeric and rDNA repeats was not reduced in rts1 mutants, HDA6 might be specialized for the RNA- directed pathway of genome modification.

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Figures

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Fig. 1. Transgene constructs. The target construct K (Kanamycin resistance) contains a nopaline synthase (NOS) promoter (white N)–neomycin phosphotransferase (NPTII) gene and a NOS gene. The silencing construct H (Hygromycin resistance) contains a hygromycin phosphotransferase (HPT) gene driven by the 19S promoter and a NOSpro IR, separated by 260 bp spacer containing the α′ promoter of the soybean storage protein β-conglycinin gene (Chen et al., 1986), under the control of the 35S promoter. The M (Mutator) construct contains a phosphinothricin acetyltransferase (PAT) gene, encoding resistance to Basta, under the control of the mannopine synthase promoter (white M). Four copies of the B1 enhancer of tobacco endogenous pararetrovirus (Mette et al., 2002a) on the right allow for possible activation tagging (Weigel et al., 2000) in addition to insertional mutagenesis. A bacterial origin of replication (pUC18) can be used to clone ‘M’ inserts by plasmid rescue. Abbreviations: LB, RB: left and right T-DNA borders, respectively; Kbac, a Kan-resistance marker used in bacterial cloning steps; T, NOS terminator; OT, octopine synthase terminator.
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Fig. 2. Growth of rts1 mutant seedlings on Kan. Seedlings that are triply homozygous for the target K locus, silencing H locus and the rts1 mutation (A) grow almost as well on medium containing 40 mg/l Kan as wild-type seedlings that are homozygous for the unsilenced target K locus (C). Wild-type seedlings that are doubly homozygous for the target K locus and the silencing H locus are sensitive to Kan (B).
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Fig. 3. RNase protection to analyze NPTII expression in rts1 mutants. Top: the map shows the location of the protected 190 bp NPTII fragment with respect to the NOSpro–NPTII gene. As input RNA, in vitro transcribed antisense RNA (black line) from a subcloned NPTII fragment upstream of the internal PstI (P) site was used. Bottom: NPTII expression was observed in wild-type T4 plants containing the unsilenced K locus (lane 2), but not detectable in T4 plants containing the silencing H locus (lane 3). Expression recovers 20–30% in rts1 plants from the M3, M4 and M5 generations (lanes 6–8), which is sufficient for resistance at the relatively low concentration of Kan used (40 mg/l). Silencing is rapidly reinitiated in first generation backcross (BC1) progeny, which are heterozygous for the rts1 mutation (lane 4, K and H loci are hemizygous; lane 5, K and H loci are homozygous). The percentage expression in rts1 mutants was determined by scanning blots with a Pharmacia Image Master using the residual input RNA after RNase digest (top band) as a reference standard. Lower band: positive control showing constitutive expression of the eIF-4A gene indicated by a 300 bp protected RNA fragment. The top band represents residual input RNA after RNase digest. R1 and r1: short versions of RTS1 and rts1, respectively.
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Fig. 4. NOSpro dsRNA and short RNAs in rts1 mutants. (A) The 0.3 kb NOSpro dsRNA, which is not made in plants lacking the silencing H locus (lane 1), is synthesized from the H locus in rts1 mutants (three shown, lanes 3–5) at levels comparable to those of wild-type plants (lane 2). (B) Sense and antisense NOSpro short RNAs 21–24 nt in length, which are not produced in plants lacking the silencing H locus (lanes 1 and 4) are somewhat enhanced in rts1 mutants (lanes 3 and 6) compared with wild-type plants (lanes 2 and 5). T4, M3 and M4 indicate the plant generation tested. Genotypes of plants are indicated above each lane. R1 and r1 are shortened versions of RTS1 and rts1, respectively. Arrows refer to the position of RNA size markers.
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Fig. 5. DNA methylation analysis. As shown in the map below panel C, methylation in the target NOSpro was examined by performing an EcoRI/PstI (E/P) double digest and adding as indicated a methylation-sensitive restriction enzyme: SacII (S) (mCmCGCGG), BstUI (B) (mCGmCG) or NheI (N) (top strand: GCTAGmCaa; bottom: GCT AGmCtg). (A superscript ‘m’ indicates a methylated C that can inhibit cleavage.) The probe consisted of NPTII-coding sequences. (A) Left: the target NOSpro at the K locus is normally unmethylated in the absence of the silencing H locus, as indicated by shifts to the smaller fragments when S, B or N are used. Right: when the silencing H locus is introduced, heavy methylation at the S and B sites and ∼50% methylation at the N site is observed. In individual rts1 plants of the M3 (B) and M4 (C) generations, methylation at the S and B sites is reduced on average to ∼50%. This is similar to the level of methylation at N sites, which remains unchanged in rts1 mutants compared with the wild-type. (D) CG methylation at centromeric and rDNA repeats was examined in rts1 and ddm1 mutants and wild-type (wt) plants using the isoschizomers HpaII (H) (mCmCGG) and MspI (M) (mCCGG). The patterns with H and M appear identical only in the ddm1 mutants, indicating loss of CG methylation. Genotypes of individual plants are indicated above each panel. R1 and r1 are shortened versions of RTS1 and rts1, respectively.
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Fig. 6. Summary of bisulfite sequencing of the target NOSpro in wild-type plants and rts1 mutants. Bottom: region of the NOSpro homologous to NOSpro dsRNA showing sites for methylation-sensitive restriction enzymes (Figure 5), the transcription start site (arrow) and start of the NPTII-coding region. Middle: cytosine methylation in the bottom DNA strand of the NOSpro (black: CG; blue: CNG; red: CNN). The height of the vertical lines shows the percentage of methylated Cs per total PCR clones sequenced. The dotted line indicates 50%. Top: bar graph showing reductions in methylation in the bottom strand for specific nucleotide groups in the rts1 mutant. In the top DNA strand, methylation was reduced from 64 to 55% (14%) in CGs; from 47 to 46% (2%) in CNGs; and was slightly increased (from 49 to 51%) in CNNs (not shown). Approximately 20 cloned PCR fragments were sequenced from each strand for both wild-type and rts1 mutant plants (see Supplementary data available at The EMBO Journal Online). DNA used for bisulfite treatment was isolated from pooled rts1 plants of the M4 generation. The data represent an average of this population.
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Fig. 7. Mapping of RTS1. The rts1 phenotype (Kan resistance in the presence of the H silencing locus) cosegregated with markers LFY3 and CER454886 at the bottom of chromosome 5. In a mapping population of 624 plants there were nine recombination events between LFY3 and RTS1 and six between RTS1 and CER454886. Five additional markers were created within the LFY3/CER454886 genetic interval for fine mapping of RTS1. There were two recombination events between CER456729 and RTS1 and one between RTS1 and CER455379, locating RTS1 on a 98 kb physical interval. This sequence is represented in overlapping BAC clones MBQ2, MJH22 and MDC12. The bottom part shows organization of the RTS1/HDA6 gene. The black boxes represent exons, the dashed lines denote introns. Coordinates of translational start and stop, exon–intron boundaries and the positions of the mutation in the rts1-1 and rts1-2 alleles are shown with respect to the numbering of BAC clone MDC12 (accession No. AB008265). The start and end of the mRNA are demarcated according to accession Nos AF195548 and AY072201. The direction of the black arrow indicates that RTS1/HDA6 is in complementary orientation to MDC12.
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Fig. 8. Complementation of the rts1 mutation with the wild-type HDA6 gene analyzed by RNase protection. Top: transformation of rts1-1 plants, which express the NOSpro–NPTII target gene, as evidenced by a 190 bp protected fragment (lane 1), with a 3.5 kb genomic HDA6 fragment results in re-silencing of the NPTII gene (lanes 2 and 3) comparable to wild-type plants (lane 4). Two representatives of ten complemented (+) T1 plants are shown. Wild-type expression of the NPTII gene in plants containing the unsilenced target K locus is shown in lane 5. Middle: positive control showing constitutive expression of the eIF-4A gene (300 bp protected fragment). Bottom: HDA6 genotypes. The rts1-1 allele contains a 37 bp deletion resulting in higher mobility of a PCR-amplified fragment on agarose gels (lane 1) compared with the PCR fragment from wild-type plants (lanes 4 and 5). Both bands can be detected in rts1 plants complemented with wild-type HDA6 (lanes 2 and 3). The K, H and RTS1 genotypes for individual plants are shown above each lane. R1 and r1 are shortened versions of RTS1 and rts1, respectively.
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Fig. 9. Hypothetical model for RTS1 activity. One or more de novo DMTases in conjunction with NOSpro RNAs (wavy lines) catalyze an intermediate level of uniform methylation (m) of Cs in all sequence contexts in the target NOSpro, resulting in partial silencing. Although a known de novo DMTase in Arabidopsis, DRM (Domain-Rearranged Methyltransferase; Cao and Jacobsen, 2002), remains a good candidate for this step, other candidate DMTases (MET1, CMT3, Dnmt2; Finnegan and Kovac, 2000) cannot yet be ruled out. HDA6 is envisioned to recognize the partially methylated DNA and to recruit maintenance (or reinforcement) DMTases such as MET1 and CMT3, which enhance CG (bold capital M) and to a lesser extent CNG (capital M) methylation. This step helps to lock in the silent state, probably in conjunction with additional histone modifications.

References

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