Pivotal role of AtSUVH2 in heterochromatic histone methylation and gene silencing in Arabidopsis - PubMed (original) (raw)

Comparative Study

. 2005 Apr 6;24(7):1418-29.

doi: 10.1038/sj.emboj.7600604. Epub 2005 Mar 17.

Affiliations

Comparative Study

Pivotal role of AtSUVH2 in heterochromatic histone methylation and gene silencing in Arabidopsis

Kathrin Naumann et al. EMBO J. 2005.

Abstract

SU(VAR)3-9 like histone methyltransferases control heterochromatic domains in eukaryotes. In Arabidopsis, 10 SUVH genes encode SU(VAR)3-9 homologues where SUVH1, SUVH2 and SUVH4 (KRYPTONITE) represent distinct subgroups of SUVH genes. Loss of SUVH1 and SUVH4 causes weak reduction of heterochromatic histone H3K9 dimethylation, whereas in SUVH2 null plants mono- and dimethyl H3K9, mono- and dimethyl H3K27, and monomethyl H4K20, the histone methylation marks of Arabidopsis heterochromatin are significantly reduced. Like animal SU(VAR)3-9 proteins SUVH2 displays strong dosage-dependent effects. Loss of function suppresses, whereas overexpression enhances, gene silencing, causes ectopic heterochromatization and significant growth defects. Furthermore, modification of transgene silencing by SUVH2 is partially transmitted to the offspring plants. This epigenetic stability correlates with heritable changes in DNA methylation. Mutational dissection of SUVH2 indicates an implication of its N-terminus and YDG domain in directing DNA methylation to target sequences, a prerequisite for consecutive histone methylation. Gene silencing by SUVH2 depends on MET1 and DDM1, but not CMT3. In Arabidopsis, SUVH2 with its histone H3K9 and H4K20 methylation activity has a central role in heterochromatic gene silencing.

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Figures

Figure 1

Figure 1

SU(VAR)3–9 homologous proteins in plants. (A) Four conserved groups of SUVH genes are found in angiosperms. Phylogenetic analysis of 21 SUVH protein sequences from Arabidopsis (AtSUVH), rice (OsSUVH), Pinus taeda (Pta), Physcomitrella patens (Pp) and Ceratopteris richardii (Cri). (B) Immunostaining of plants expressing myc fusion protein of SUVH1, SUVH2 and Drosophila SU(VAR)3–9 and HP1 in Arabidopsis interphase nuclei with α-myc. Heterochromatin association is found for the SUVH1 and SUVH2 proteins as well as for Drosophila SU(VAR)3–9 and HP1.

Figure 2

Figure 2

Differential effects of suvh1 and suvh2 mutations on histone and DNA methylation. (A, B) Immunohistochemical staining of wild-type, suvh1 and suvh2 interphase nuclei with antibodies recognizing specific histone and DNA methylation marks. In suvh2, but not in suvh1, mono- and dimethyl H3K9, mono- and dimethyl H3K27, monomethyl H4K20 and 5-methylcytosine (heterochromatic marks) are significantly reduced (A). No effects are found on the trimethyl H3K9, trimethyl H3K27, di- and trimethyl H3K36 and di- and trimethyl H4K20 euchromatic marks (B). (C) Western analysis of nuclear extracts of wild-type, suvh1 and suvh2 mutant plants. Only in suvh2 significant reduction of mono- and dimethyl H3K9 and monomethyl H3K27 is found. (D) In vitro recombinant SUVH2 shows H3 and H4 HMTase activity in assays with reconstituted nucleosomes.

Figure 3

Figure 3

Ectopic heterochromatization in SUVH2 overexpression lines. (A) Immunostaining of nuclei from 35S*∷mycSUVH1, 35S*∷mycSUVH2 with α-myc and GFP fluorescence analysis in dexamethasone-treated GVG_∷_SUVH1EGFP and GVG_∷_SUVH2EGFP plants. Only SUVH2 shows ectopic distribution. Electron microscopic analysis of nuclei from 35S*∷mycSUVH2 plants shows ectopic heterochromatin (arrowheads). (B) Immunocytological analysis of heterochromatic histone and 5-methylcytosine methylation in SUVH2 overexpression plants. Enhanced staining for all heterochromatic marks (mono- and dimethyl H3K9, mono- and dimethyl H3K27, monomethyl H4K20 and 5-methylcytosine) is found. (C) Western analysis of suvh2 mutant and 35S*∷mycSUVH2 overexpression plants. In suvh2, dimethyl H3K9 and monomethyl H4K20 are reduced. In 35S*∷mycSUVH2 overexpression plants, heterochromatic H3K9 dimethyl and H4K20 monomethyl are enriched, whereas euchromatic H3K9 trimethyl is reduced. (D) Immunostaining for euchromatic histone modification marks in SUVH2 overexpression plants. Staining for dimethyl H3K4, acetyl H3K9, trimethyl H3K27, dimethyl H3K36 and dimethyl H4K20 is significantly reduced.

Figure 4

Figure 4

Growth and developmental defects in SUVH2 overexpression plants. (A) SUVH2 overexpression causes mini-plant phenotype in 35S*∷mycSUVH2#5, #6, #22 and dexamethasone-treated GVG_∷_SUVH2EGFP lines. (B) SUVH2 overexpression seedlings show a curled cotyledon phenotype (upper panel). By introducing a suvh2 null allele, the mini-plant and curled cotyledon phenotypes are significantly rescued in suvh2/+; 35S*∷mycSUVH2#6/+ plants (lower panel). (C) Western analysis of extracts from 35S*∷mycSUVH2 lines with α-myc. The amount of mycSUVH2 protein correlates with the strength of growth defects. 35S*∷mycSUVH2#4 plants with a weak growth reduction expresses a lower amount of additional SUVH2 as compared to lines with a mini-plant phenotype.

Figure 5

Figure 5

Functional dissection of SUVH2 by transgene mutations. (A) Molecular nature of 35S*∷mycSUVH2#5 transgene mutations. Structure of SUVH2 with the conserved YDG, preSET, SET and postSET (p) domains. (B) Immunostaining with α-myc shows that only the N-terminus mutation 5-1 eliminates ectopic distribution of SUVH2. (C) All mutations eliminate ectopic H3K9 and H4K20 methylation. The N-terminus mutation 5-1, the YDG mutation 5-2 and mutation 5-3 eliminate ectopic DNA methylation, whereas in plants with the SET domain mutations 5-4, 5-5, 5-6 and 5-7 ectopic DNA methylation is observed. (D) Silencing of Athila transposons by SUVH2 overexpression is rescued by all transgene mutations independent of DNA hypermethylation (RT–PCR) (cf. Supplementary Figure 3 for bisulphite data).

Figure 6

Figure 6

Dosage-dependent modifier effects of SUVH2 on LUC2 transgene silencing. (A–C) Crosses of 35S*∷mycSUVH2#4/+ and 35S*∷SUVH2as#11/+ with LUC2 homozygous plants and backcrosses (BC) of F1 and F2 LUC2/+ plants to wild type (A). Structure and activity of the LUC2 repeated transgene in control plants (B). In 35S*∷mycSUVH2#4/+; LUC2/+ plants with SUVH2 overexpression, LUC2 silencing is enhanced, whereas in 35S*∷SUVH2as#11/+; LUC2/+ plants without SUVH2 LUC2 silencing is strongly released. The repressed or activated state of the LUC2 is maintained after a backcross of 35S*∷mycSUVH2#4/+;LUC2/+ and 35S*∷SUVH2as#11/+;LUC2/+ with wild-type plants, respectively. In LUC2/+ offspring from the second backcross generation, reversion of transgene silencing to the control level is found. Symmetric (red bars) and nonsymmetric (blue bars) DNA methylation at LUC2 transgenes was studied by bisulphite sequencing (A and B). Bisulphite sequence analysis of control LUC2/+, F1 35S*∷mycSUVH2#4/+; LUC2/+ and 35S*∷SUVH2as#11/+; LUC2/+ as well as LUC2/+ BC1 progeny plants. (C) Stars denote significantly changed symmetric CpG (red) and CpNpG (green) and nonsymmetric CpNpN (blue) cytosine residues (N, no G).

Figure 7

Figure 7

Genetic interaction of SUVH2 with MET1, CMT3 and DDM1. (A) The LUC7 transgene shows complete repression of LUC activity (no fluorescence). (B) Relaxation of gene silencing by met1-h1 dominates the enhancer effect of SUVH2 overexpression and significantly rescues the SUVH2 overexpression mini-plant phenotype. (C) The silencing enhancer effect of SUVH2 overexpression dominates the suppressor effect of cmt3-h1 on LUC7 silencing. (D) The met1-h1 and cmt3-h1 mutations confer reduced CpG and CpNpG methylation, respectively. _Hpa_II (H) and Msp_I (M) restriction map of LUC7 with LUC as a probe. (E) Suppression of SUVH2-dependent mini-plant phenotype in 35S*∷_SUVH2#6/+; ddm1–2 plants and rescue of ectopic dimethyl H3K9, monomethyl H4K20 and 5-methylcytosine methylation.

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