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. 2005 Feb;17(2):404-17.
doi: 10.1105/tpc.104.028332. Epub 2005 Jan 19.

The Arabidopsis HOMOLOGY-DEPENDENT GENE SILENCING1 gene codes for an S-adenosyl-L-homocysteine hydrolase required for DNA methylation-dependent gene silencing

Pedro S C F Rocha  1 , Mazhar Sheikh, Rosalba Melchiorre, Mathilde Fagard, Stéphanie Boutet, Rebecca Loach, Barbara Moffatt, Conrad Wagner, Hervé Vaucheret, Ian Furner
Affiliations

The Arabidopsis HOMOLOGY-DEPENDENT GENE SILENCING1 gene codes for an S-adenosyl-L-homocysteine hydrolase required for DNA methylation-dependent gene silencing

Pedro S C F Rocha et al. Plant Cell. 2005 Feb.

Abstract

Genes introduced into higher plant genomes can become silent (gene silencing) and/or cause silencing of homologous genes at unlinked sites (homology-dependent gene silencing or HDG silencing). Mutations of the HOMOLOGY-DEPENDENT GENE SILENCING1 (HOG1) locus relieve transcriptional gene silencing and methylation-dependent HDG silencing and result in genome-wide demethylation. The hog1 mutant plants also grow slowly and have low fertility and reduced seed germination. Three independent mutants of HOG1 were each found to have point mutations at the 3' end of a gene coding for S-adenosyl-l-homocysteine (SAH) hydrolase, and hog1-1 plants show reduced SAH hydrolase activity. A transposon (hog1-4) and a T-DNA tag (hog1-5) in the HOG1 gene each behaved as zygotic embryo lethal mutants and could not be made homozygous. The results suggest that the homozygous hog1 point mutants are leaky and result in genome demethylation and poor growth and that homozygous insertion mutations result in zygotic lethality. Complementation of the hog1-1 point mutation with a T-DNA containing the gene coding for SAH hydrolase restored gene silencing, HDG silencing, DNA methylation, fast growth, and normal seed viability. The same T-DNA also complemented the zygotic embryo lethal phenotype of the hog1-4 tagged mutant. A model relating the HOG1 gene, DNA methylation, and methylation-dependent HDG silencing is presented.

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Figures

Figure 1.
Figure 1.
Location of Mutations of the HOG1 Gene (AT4G 13940) Coding for SAH Hydrolase. (A) Coordinates are given in amino acids from the start of the protein. The specific amino acid change is indicated for each of the three point mutants. The positions of the insertions are indicated for the two tagged mutants. The 5′ untranslated region, 3′ untranslated region, and the unique intron (according to The Arabidopsis Information Resource [TAIR] annotation) are indicated by white boxes and exons by black boxes. The arrow indicates the direction of transcription. (B) Partial alignment of SAH hydrolases of Arabidopsis with other eukaryotic hydrolases. The alignment is representative of hydrolases of the eukaryotic type as defined by Kurowski and Bujnicki (2003) and corresponds to the region to which the hog1 substitutions map. The substituted residues are indicated in the top row; numbering corresponds to SAH hydrolase 1. SAHH1, SAH hydrolase 1; SAHH2, SAH hydrolase 2; Dr, Danio rerio (GI 37,681,725); Rn, Rattus norvegicus (GI 38,197,378); Dm, Drosophila melanogaster (GI 7,293,098); Sc, Saccharomyces cerevisiae (GI 2,950,505); Pf, Plasmodium falciparum (GI 627050). An insertion in the D. melanogaster sequence is indicated. Invariant and highly conserved residues are highlighted.
Figure 2.
Figure 2.
Zygotic Lethality in the hog1-4 and met1-7 Mutants. (A) Wild-type (HOG1) silique. (B) Silique from a hemizygous hog1-4/HOG1 plant. Note the presence of aborted hog1-4 homozygote seeds. (C) Silique from a plant hemizygous for both hog1-4 and the complementing SAH hydrolase T-DNA. Note the decrease in zygotic lethality. (D) Silique from a hemizygous met1-7/MET1 plant. Note the presence of aborted met1-7 homozygote seeds.
Figure 3.
Figure 3.
Complementation of the hog1-1 Mutation. (A) RNA gel blots of total RNA prepared from the L5 (lane 1), L5-hog1-1 (lane 2), and L5-hog1-1-complementation (lane 3) lines. The probes are indicated at the bottom of the panel. (B) DNA gel blots of total genomic DNA prepared from L5 (lane 1), L5-hog1-1 (lane 2), and L5-hog1-1-complementation lines (lane 3) cut with methylation-sensitive restriction endonucleases as indicated. The DNA was also cut with HindIII. The probes are indicated at the bottom of the panel, and the numbers at left are the molecular weights in kilobases.
Figure 4.
Figure 4.
An Integrated Model to Explain the Mechanism of Action of the hog1 Mutants on C-Insert–Induced Silencing of the TT4 Locus. Also indicated in the model are proposed roles for genes corresponding to other mutants impaired in DNA methylation (ddm1, met1, and cmt3) that also relieve C-insert silencing.
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