Robertson's Mutator transposons in A. thaliana are regulated by the chromatin-remodeling gene Decrease in DNA Methylation (DDM1)

Abstract

Robertson's Mutator transposable elements in maize undergo cycles of activity and then inactivity that correlate with changes in cytosine methylation. Mutator-like elements are present in the Arabidopsis genome but are heavily methylated and inactive. These elements become demethylated and active in the chromatin-remodeling mutant ddm1 (Decrease in DNA Methylation), which leads to loss of heterochromatic DNA methylation. Thus, DNA transposons in plants appear to be regulated by chromatin remodeling. In inbred ddm1 strains, transposed elements may account, in part, for mutant phenotypes unlinked to ddm1. Gene silencing and paramutation are also regulated by DDM1, providing support for the proposition that epigenetic silencing is related to transposon regulation.

Figure 1

Analysis of Mutator-like elements with long terminal inverted repeats (TIR-MULEs) in Arabidopsis. (A) Unrooted distance tree of the 22 TIR-MULEs identified in this study (Table 1A,B) based on terminal inverted repeat (TIR) sequences. Mutator-like elements with TIRs (TIR-MULEs) have been grouped by similiarty according to the cluster analysis. Elements that transpose in ddm1 strains are highlighted in red (AtMu1) and purple (AtMu6). Bootstrap values are indicated at nodes. Subgroups IA and IB (yellow) and group II (green) contain elements which are transcribed in ddm1 mutants (see Table 2). (B) Alignment of a 97-aa conserved region of the Arabidopsis MURA-like transposases with the maize MURA protein. Identical amino acids are boxed in dark grey, similar in light grey. Identical amino acids from the PROSITE signature pattern are shown below the corresponding sequence. (C) Alignment of TIR sequences of transcribed elements. Identical nucleotides are boxed in grey (TIRA, 5′ TIR; TIRB, 3′TIR).

Figure 1

Analysis of Mutator-like elements with long terminal inverted repeats (TIR-MULEs) in Arabidopsis. (A) Unrooted distance tree of the 22 TIR-MULEs identified in this study (Table 1A,B) based on terminal inverted repeat (TIR) sequences. Mutator-like elements with TIRs (TIR-MULEs) have been grouped by similiarty according to the cluster analysis. Elements that transpose in ddm1 strains are highlighted in red (AtMu1) and purple (AtMu6). Bootstrap values are indicated at nodes. Subgroups IA and IB (yellow) and group II (green) contain elements which are transcribed in ddm1 mutants (see Table 2). (B) Alignment of a 97-aa conserved region of the Arabidopsis MURA-like transposases with the maize MURA protein. Identical amino acids are boxed in dark grey, similar in light grey. Identical amino acids from the PROSITE signature pattern are shown below the corresponding sequence. (C) Alignment of TIR sequences of transcribed elements. Identical nucleotides are boxed in grey (TIRA, 5′ TIR; TIRB, 3′TIR).

Figure 1

Analysis of Mutator-like elements with long terminal inverted repeats (TIR-MULEs) in Arabidopsis. (A) Unrooted distance tree of the 22 TIR-MULEs identified in this study (Table 1A,B) based on terminal inverted repeat (TIR) sequences. Mutator-like elements with TIRs (TIR-MULEs) have been grouped by similiarty according to the cluster analysis. Elements that transpose in ddm1 strains are highlighted in red (AtMu1) and purple (AtMu6). Bootstrap values are indicated at nodes. Subgroups IA and IB (yellow) and group II (green) contain elements which are transcribed in ddm1 mutants (see Table 2). (B) Alignment of a 97-aa conserved region of the Arabidopsis MURA-like transposases with the maize MURA protein. Identical amino acids are boxed in dark grey, similar in light grey. Identical amino acids from the PROSITE signature pattern are shown below the corresponding sequence. (C) Alignment of TIR sequences of transcribed elements. Identical nucleotides are boxed in grey (TIRA, 5′ TIR; TIRB, 3′TIR).

Figure 2

Genomic locations of TIR-MULE transposons. Potentially intact elements with TIRs in the Columbia ecotype are shown to the left of each chromosome. Transposed AtMu1 elements in ddm1 strains are shown on the right (Table 3). Genetic markers and map positions, as well as pericentromeric (light grey) and nucleolar (dark grey) heterochromatin, are shown. TIR-MULE subfamilies are color-coded as in Fig. 1A.

Figure 3

Mutator transposons are methylated in Arabidopsis. DNA gel blot analysis of Arabidopsis DNA from (A) wild-type Landsberg erecta (La-er) and Columbia strains (Col-0) and (B) wild-type and ddm1 mutant plants (cv. Columbia) digested with EcoRII, BstNI, HpaII, and MspI. The blots were hybridized with probe 2 from AtMu1 (Fig. 4A).

Figure 4

Mutator transposons are activated in ddm1 mutants.(A) AtMu1 has 295 bp TIRs (grey boxes) and encodes three exons; probes 1 and 2 are indicated below. Restriction enzyme sites for HpaII (Hp), HindIII (H), EcoRI (R), and EcoRII (E) are shown. (B) DNA gel blot analysis of pooled wild-type and ddm1-mutant seedlings. DNA was digested with EcoRI and hybridized with probe 2. New bands (arrowheads) represent transposition of AtMu1. The preexisting elements T11I11.3, F21A20_a, and T3F12.12 are indicated by arrows on the left. The faint band in lanes 1 and 2 is partially methylated T3F12.12. Lane 1, Landsberg erecta (Ler); lane 2, Columbia wild-type (Col-0); lane 3, Columbia ddm1; lane 4, Landsberg erecta ddm1. (C) DNA gel blot analysis of individual Columbia ddm1 plants using HindIII and probe 1. New bands (arrowheads) are transposed AtMu1. (D) RT–PCR analysis (+) of transcripts using AtMu1 primers (top panel) and control RuBisCO primers (bottom panel). Alternate lanes (−) are mock reactions in the absence of reverse transcriptase. RuBisCO lanes 1 and 2 were amplified using Col-0 and Ler genomic DNA.