Arabidopsis cmt3 chromomethylase mutations block non-CG methylation and silencing of an endogenous gene

Abstract

Plants maintain cytosine methylation at CG and non-CG residues to control gene expression and genome stability. In a screen for Arabidopsis mutants that alter methylation and silencing of a densely methylated endogenous reporter gene, we recovered 11 loss-of-function alleles in the CMT3 chromomethylase gene. The cmt3 mutants displayed enhanced expression and reduced methylation of the reporter, particularly at non-CG cytosines. CNG methylation was also reduced at repetitive centromeric sequences. Thus, CMT3 is a key determinant for non-CG methylation. The lack of CMT homologs in animal genomes could account for the observation that in contrast to plants, animals maintain primarily CG methylation.

Figure 1

Wassilewskija (WS) pai1C251Y cmt3 plant phenotypes. (A) Two-week-old seedlings of the indicated genotypes grown on agar medium are shown under visible (top) and UV (bottom) light. (B) Four-week-old adult plants of the indicated genotypes grown in soil are shown under visible (top) and UV (bottom) light. (C) Representative 2-week-old T2 generation transgenic seedlings of the indicated genotypes grown on agar medium are shown under visible (top) and UV (bottom) light. The phenotypes of the cmt3G456D allele shown are representative of the phenotypes observed with 10 other cmt3 alleles.

Figure 2

cmt3 mutations confer reduced PAI and CEN methylation. (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 (Bender and Fink 1995). (P1–P4) PAI1–PAI4; (P2) PAI2; (P3) PAI3; (P1) Columbia (Col) strain PAI1; asterisks indicate the positions of species methylated at internal HpaII/MspI sites (Bender and Fink 1995; Luff et al. 1999). The Col strain is included as a control for the positions of unmethylated PAI2 and PAI3 species. (B) The blot shown in A was reprobed with a 180-bp CEN repeat probe. The phenotypes of the cmt3G456D allele shown are representative of the phenotypes observed with 10 other cmt3 alleles.

Figure 3

Sequencing of PAI promoter methylation in the cmt3 mutant. Bisulfite genomic methylation sequencing was performed as described (Jeddeloh et al. 1998; Luff et al. 1999) for the top strands of the PAI1 and PAI2 promoters in Wassilewskija (WS) pai1C251Y cmt3G456D 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-methyl-cytosine. (Black) CG cytosines; (blue) CNG cytosines; (red) 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).

Figure 4

Positions of mutations in CMT3. The predicted amino acid sequences of Wassilewskiya (WS) CMT3, WS CMT2, and maize ZMET2 are shown aligned along their conserved C-terminal regions. The N termini, upstream of the backslash at the beginning of each sequence, are unrelated. CMT3 introns are indicated by inverted triangles above the sequence. CMT3 missense mutations are indicated above the affected residues. The stop mutation is indicated by an asterisk, and the splice donor and acceptor site mutations are indicated by an x to the left or right, respectively, above the affected introns. Residues identical between proteins are highlighted in boldface. Conserved sequence motifs are indicated under the alignment. GenBank accession nos. are: AF383170 for WS CMT3 and AF383171 for WS CMT2.