An Arabidopsis SET domain protein required for maintenance but not establishment of DNA methylation

Abstract

Cytosine methylation is critical for correct development and genome stability in mammals and plants. In order to elucidate the factors that control genomic DNA methylation patterning, a genetic screen for mutations that disrupt methylation-correlated silencing of the endogenous gene PAI2 was conducted in Arabidopsis: This screen yielded seven loss-of-function alleles in a SET domain protein with histone H3 Lys9 methyltransferase activity, SUVH4. The mutations conferred reduced cytosine methylation on PAI2, especially in non-CG sequence contexts, but did not affect methylation on another PAI locus carrying two genes arranged as an inverted repeat. Moreover, an unmethylated PAI2 gene could be methylated de novo in the suvh4 mutant background. These results suggest that SUVH4 is involved in maintenance but not establishment of methylation at particular genomic regions. In contrast, a heterochromatin protein 1 homolog, LHP1, had no effect on PAI methylation.

Fig. 1. PAI2 silencing is suppressed by suvh4 mutations. Representative 2.5-week-old plants of the indicated strains are shown under visible (upper panel) and UV (lower panel) light. The suvh4302* and cmt3i11a alleles were used. The suppressed phenotype of suvh4R302* is similar to those of the other six suvh4 alleles.

Fig. 2. suvh4 mutations confer reduced PAI2 methylation. (A) The HpaII (H) and MspI (M) restriction map of each WS PAI locus is shown, with the probed regions indicated by gray bars and the bisulfite sequenced regions indicated by hatched bars. (B) Genomic DNAs prepared from 4-week-old plants of the indicated genotypes were cleaved with either HpaII or MspI and used for Southern blot analysis with a PAI probe. P1-P4 is PAI1–PAI4, P2 is PAI2, and P3 is PAI3, with asterisks indicating the positions of species methylated at internal HpaII/MspI sites. Note that PAI3 is divergent from the probe sequence and thus gives a weaker signal than other PAI genes. (C) The blot shown in (B) was reprobed with a 180 bp CEN repeat probe. The phenotypes of the WS suvh4R302* allele shown are representative of the phenotypes observed with six other suhv4 alleles. The phenotypes of the WS pai1 suvh4R302* cmt3i11a double mutant line shown are representative of the phenotypes observed with three other independent double mutant lines.

Fig. 3. Bisulfite sequencing analysis of the PAI genes in a suvh4 mutant background. (A) Bisulfite genomic methylation sequencing was performed for the top strands of the PAI1 and PAI2 upstream regions in WS pai1 suvh4R302* or WS pai1 suvh4R302* cmt3i11a DNA. For each region, eight independent molecules were sequenced. Vertical lines indicate positions of cytosines, with the height of each line representing how many sequenced molecules had 5-methylcytosine (5-Me-C). Black indicates CG cytosines, blue indicates CNG cytosines, and red indicates other cytosines. Asterisks indicate sites with no methylation. The black horizontal line indicates the region of PAI identity, and the gray horizontal line indicates flanking upstream heterologous sequence unique to each gene. The exon and intron structures of PAI1 and PAI2 are shown as open boxes and dashed lines, respectively, under each sequence. These structures are based on full-length cDNA sequences for each gene (Melquist et al., 1999). (B) Within the region of PAI identity (black horizontal line in A), the percentages of available cytosines that are methylated in CG (black bars), CNG (white bars) or other (gray bars) sequence contexts are shown for either PAI1 (P1) or PAI2 (P2) in the indicated strains. Data for wild-type WS and WS pai1 cmt3 are from previous publications (Luff et al., 1999; Bartee et al., 2001). See also Supplementary figure 2.

Fig. 4. Positions of seven suvh4 alleles recovered as PAI2 silencing suppressors. The predicted amino acid sequence of WS SUVH4 is shown, with the positions of introns marked with black arrowheads. Missense mutations are indicated by the new amino acid above the affected codon. Nonsense mutations are indicated by an asterisk above the affected codon. Splice mutations are indicated by an x at the appropriate junction of the affected intron. The YDG, pre-SET, SET and post-SET domains as previously defined for SUVH4 relative to related proteins (Baumbusch et al., 2001; Jackson et al., 2002) are underlined. The WS genomic sequence of SUVH4 is available as DDBJ/EMBL/GenBank accession No. AF538715.

Fig. 5. A SUVH4 transgene can complement the suvh4 mutant defect. (A) Representative T₂ 2-week-old seedlings of the suvh4R302* mutant transformed with either a SUVH4 genomic clone or a CMT3 genomic clone (two independent lines each) are shown under visible (upper panel) or UV (lower panel) light. (B) Genomic DNAs prepared from single leaves of 4-week-old T₂ plants of the same lines shown in (A) were cleaved with either HpaII (H) or MspI (M) and used for Southern blot analysis with a PAI probe. P1-P4 is PAI1–PAI4, P2 is PAI2, and P3 is PAI3, with asterisks indicating the positions of species methylated at internal HpaII/MspI sites.

Fig. 6. The lhp1 mutation does not affect PAI or CEN methylation in WS. (A) Genomic DNAs prepared from 4-week-old plants of the indicated genotypes were cleaved with either HpaII (H) or MspI (M) and used for Southern blot analysis with a PAI probe. P1-P4 is PAI1–PAI4, P2 is PAI2, and P3 is PAI3, with asterisks indicating the positions of species methylated at internal HpaII/MspI sites. (B) The blot shown in (A) was reprobed with a 180 bp CEN repeat probe. The lhp1-1 allele, which displays characteristic developmental defects, was used for this analysis (Gaudin et al., 2001).

Fig. 7. The WS pai1–PAI4 inverted repeat locus triggers de novo methylation and silencing of an unmethylated Ler PAI2 gene in a SUVH4-deficient but not a CMT3-deficient background. (A) To the left, representative WxL wild-type (F₄), WxL suvh4 (F₅) or WxL cmt3 (F₅) 2.5-week-old seedlings are shown under visible (left panel) or UV (right panel) light. To the right, diagrams show the PAI genotypes and methylation phenotypes in these lines. PAI genes inherited from WS are indicated as black arrows. Note that none of the three WS PAI genes encodes functional PAI enzyme. The WS PAI1–PAI4 and PAI3 loci both lie on the upper arm of chromosome 1, indicated by a slash separating the two loci. The Ler PAI2 gene is indicated by a red arrow. Methylation is indicated by boxes around the affected genes, with a solid line representing dense methylation and a dashed line representing partial methylation. (B) Genomic DNAs prepared from 4-week-old plants of the indicated strains were cleaved with either HpaII (H) or MspI (M) and used for Southern blot analysis with a PAI probe. P1-P4 is WS PAI1–PAI4, P1 is Ler PAI1, P2 is PAI2, and P3 is PAI3, with asterisks indicating the positions of species methylated at internal HpaII/MspI sites. WxL wild-type, WxL suvh4 and WxL cmt3 DNAs were prepared from F₄, F₅ and F₅ generation plants, respectively.