Two Arabidopsis methylation-deficiency mutations confer only partial effects on a methylated endogenous gene family

Abstract

In Arabidopsis a SWI2/SNF2 chromatin remodeling factor-related protein DDM1 and a cytosine methyltransferase MET1 are required for maintenance of genomic cytosine methylation. Mutations in either gene cause global demethylation. In this work we have assessed the effects of these mutations on the PAI tryptophan biosynthetic gene family, which consists of four densely methylated genes arranged as a tail-to-tail inverted repeat plus two unlinked singlet genes. The methylation mutations caused only partial demethylation of the PAI loci: ddm1 had a strong effect on the singlet genes but a weaker effect on the inverted repeat, whereas met1 had a stronger effect on the inverted repeat than on the singlet genes. The double ddm1 met1 mutant also displayed partial demethylation of the PAI genes, with a pattern similar to the ddm1 single mutant. To determine the relationship between partial methylation and expression for the singlet PAI2 gene we constructed a novel reporter strain of Arabidopsis in which PAI2 silencing could be monitored by a blue fluorescent plant phenotype diagnostic of tryptophan pathway defects. This reporter strain revealed that intermediate levels of methylation correlate with intermediate suppression of the fluorescent phenotype.

Figure 1

(A) PAI internal methylation patterns in methylation mutant strains. A genomic Southern blot of DNA prepared from whole 4-week-old plants of the indicated strains cleaved with either HpaII (H) or MspI (M) and probed with an internal PAI probe is shown. The WS ddm1 and WS met1 DNAs were extracted from a representative line of each strain inbred for six generations in the presence of the homozygous mutation. The WS ddm1 met1 DNA was extracted from a representative line that was inbred for one generation in the presence of both homozygous mutations. The C251Y (WS pai1C251Y) ddm1 and C251Y met1 DNAs were extracted from a representative line of each strain inbred for two generations in the presence of the homozygous mutation. The MspI restriction maps of the WS and Col PAI genes were previously described (13–15). The band sizes predicted for the PAI loci (P1–P4 is WS PAI1-PAI4, P1 is Col PAI1, P2 is WS or Col PAI2, P3 is WS or Col PAI3) are indicated in the left margin, with the fully cleaved species unmarked and the internally methylated species marked with an asterisk. (B) Centromere repeat methylation patterns. The same blot shown in (A) was stripped and reprobed with a fragment that detects the 180 bp centromere repeats. (C) Digestion pattern of the ASA1 gene. The same blot shown in (A) was stripped and reprobed with an ASA1 (anthranilate synthase α subunit 1) cDNA probe. Because the ASA1 region carries very little methylation, HpaII and MspI give similar restriction patterns. The probe detects a 4.9 kb band in WS and a 5.9 kb band in Col due to an MspI polymorphism. The ASA1 blot thus controls for equal loading and complete digestion of DNA samples

Figure 2

PAI promoter methylation patterns in methylation mutant strains. Bisulfite genomic sequencing of methylation patterns was performed for the top and bottom strands of the indicated PAI gene promoters in a representative line of four generation inbred WS ddm1 and a representative line of four generation inbred WS met1, with eight independent molecules sequenced for each strand. Vertical lines indicate the positions of cytosines, with the height of each line representing how many sequenced molecules had 5-methylcytosine (5-MeC) at that position. Black indicates cytosines in the context CG, blue indicates cytosines in the context CNG and red indicates cytosines in other contexts. Asterisks indicate sites where none of the sequenced molecules had a 5-MeC. The black horizontal line indicates the region of PAI identity and the gray horizontal line indicates flanking upstream heterologous sequence unique to each gene. For the purposes of comparison our previously published sequencing data for the wild-type WS PAI1 and PAI2 promoter regions are also shown; these data are reprinted from Luff et al. (15) with permission from Elsevier Science.

Figure 3

PAI steady-state transcript levels are not dramatically perturbed by methylation mutation-induced hypomethylation. Total RNA was prepared from 4-week-old plants of the same strains described in Figure 1. Duplicate northern blots prepared from these samples were probed with either the PAI cDNA 0.7 kb internal PstI fragment (3 day exposure) or with a β-tubulin probe (TUB) as a control for gel loading (16 h exposure). The ethidium bromide stained gel of the PAI northern blot (rRNA) is also shown as a control for gel loading.

Figure 4

The methylation mutants suppress PAI-deficient phenotypes of the WS pai1C251Y reporter strain. (A) Representative 2-week-old seedlings of the indicated genotypes photographed under visible (left) and UV (right) light are shown. (B) Representative 4-week-old plants of the indicated genotypes photographed under visible (left) and UV (right) light are shown. The C251Y ddm1 and C251Y met1 plants are from the F₅ generation, the same generation used to prepare DNA and RNA for molecular analysis (Figs 1 and 3).

Figure 5

Genetic pedigrees used to analyze the effects of ddm1 and met1 on the WS pai1C251Y reporter strain. The circled X symbol indicates self-pollination. In the F₄ generation phenotypes were assigned based on the average behavior of a population of 24 plants. sf indicates a strong fluorescent phenotype equivalent to that of the parental pai1C251Y strain. wf indicates a weaker fluorescent phenotype than displayed by the parental pai1C251Y strain.

Figure 6

A summary of methylation changes on the PAI genes in various mutant backgrounds. The organization of the four PAI genes in WS is shown. The arrows depict the direction of transcription for each gene, with the hatch marks between the PAI1-PAI4 locus and the PAI3 locus indicating that these loci are on the same chromosome but genetically unlinked. The Δ symbol represents deletion of the PAI1-PAI4 genes. The boxes around each gene locus indicate cytosine methylation. The approximate density of methylation at each locus is indicated as follows: a solid bold line indicates parental WS levels of methylation, a bold dashed line indicates a <50% reduction in methylation, a fine dashed line indicates a >50% reduction in methylation and no line indicates only trace levels or no detectable methylation. Density indications are based on combined results from HpaII/MspI Southern blot and promoter bisulfite sequencing analyses, except for the WS ddm1 met1 strain, which is based solely on Southern blot analysis.