A mutation that prevents paramutation in maize also reverses Mutator transposon methylation and silencing - PubMed (original) (raw)

A mutation that prevents paramutation in maize also reverses Mutator transposon methylation and silencing

Damon Lisch et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Both paramutation and Mutator (Mu) transposon inactivation involve heritable changes in gene expression without concomitant changes in DNA sequence. The mechanisms by which these shifts in gene activity are achieved are unknown. Here we present evidence that these two phenomena are linked mechanistically. We show that mutation of a gene, modifier of paramutation 1 (mop1), which prevents paramutation at three different loci in maize, can reverse methylation of Mutator elements reliably. In mop1 mutant backgrounds, methylation of nonautonomous Mu elements can be reversed even in the absence of the regulatory MuDR element. Previously silenced MuDR elements are reactivated sporadically after multiple generations of exposure to mop1 mutations. MuDR methylation is separable from MuDR silencing, because removal of methylation does not cause immediate reactivation. The mop1 mutation does not alter the methylation of certain other transposable elements including those just upstream of a paramutable b1 gene. Our results suggest that the mop1 gene acts on a subset of epigenetically regulated sequences in the maize genome and paramutation and Mu element methylation require a common factor, which we hypothesize influences chromatin structure.

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Figures

Figure 1

Diagram of the crosses used to generate the families segregating _mop1_-1 and silenced Mu elements that were analyzed in Table 1 and Fig. 2. Individual plants with the genotype indicated at the top of the diagram were self-fertilized. The resulting progeny from each family were segregating _mop1_-1 (darkly pigmented plants) and wild type (lightly pigmented plants) as indicated in A. The symbol Mop1/− indicates that the other allele could have been Mop1 or _mop1_-1. Dark and light individuals were crossed to generate the families shown in B. Several plants with the wild-type phenotype then were self-fertilized, and the resulting progeny were as indicated in C.

Figure 2

(A_–_C) Methylation status of Mu1 and MuDR elements in family 1162-3x, segregating for MuK and _mop1_-1. (A) A _Hin_fI digest of DNA from this family probed with a Mu1 internal fragment. The lanes marked “mop1” are samples from plants that were homozygous for _mop1_-1. The lanes marked “wild type” are samples from plants that were either _mop1_-1/Mop1 or Mop1/Mop1. The fragments expected for unmethylated Mu1 and slightly larger Mu1.7 elements are indicated. Individuals were scored as methylated (indicated by ●) if most of the Mu1 and Mu1.7 elements generated _Hin_fI fragments larger than the 1.4- and 1.7-kb fragment expected from complete digestion. The individual in lane 20 was retested, and the presence of methylated Mu1 elements was confirmed (data not shown). (B) To control for complete digestion by the restriction enzyme, the blot shown in A was stripped and rehybridized with a fragment of the a1 gene. The two fragments represent complete digestion by _Hin_fI. (C) A _Sac_I digest of the same DNA samples shown in A probed with an internal MuDR fragment. The 4.8-kb MuDR fragment diagnostic for the presence of unmethylated, intact MuDR elements is indicated. Smaller deleted versions of MuDR are indicated as dMuDR. The larger fragments observed in lanes 1–4 are sequences related to MuDR that are found in all maize lines (13), which serve as controls for loading differences and estimating the number of unmethylated, intact MuDR elements. (D_–_F) Reversal of Mu1 element methylation. (D) A blot of _Hin_fI-digested DNA samples from family 1511, segregating _mop1_-1 homozygotes (mop1) and _mop1_-1/Mop1_-1 heterozygotes (wild type) was probed with a Mu1 fragment. As controls, the last two lanes contain DNA of plants with Mu1 and carrying (lane 18) or lacking (lane 19) MuDR. (E) To control for complete digestion by the restriction enzyme, the blot shown in D was stripped and rehybridized with a fragment of the a1 gene. (F) The same blot was stripped and rehybridized with the SB probe from a region upstream of the b1 gene (see Fig. 5_C for location). The 1.8-kb fragment is the size expected for no methylation, and the larger fragment represents methylation of a particular site upstream of the b1 gene.

Figure 3

Reversal of Mu1 element methylation in the absence of MuDR. DNA was isolated from a family segregating for _mop1_-1/_mop1_-1 (mop1, lanes 1–3) and _mop1_-1/Mop1 or Mop1/Mop1 (wild type, lanes 4–6). In A and B, lane 7 contains DNA from a _MuDR_-active individual. The diagnostic fragments for a full-length unmethylated MuDR element and unmethylated Mu1 and Mu1.7 elements are indicated. (A) A _Hin_fI digest of DNA was blotted and hybridized with the Mu1 internal probe. The 1.7-kb fragment visible in lanes 1–6 represents an endogenous _Mu1.7_-homologous _Hin_fI fragment present in many maize lines (32). (B) DNA from the same family digested with _Sac_I, blotted, and probed with an internal fragment of MuDR. Digestions with additional methylation-insensitive enzymes confirmed that no intact MuDR elements were present (data not shown).

Figure 4

Reversal of Mu1 element methylation in _mop1_-2 plants. (A) DNA was isolated from a family segregating _mop1_-2 and wild type (_mop1_-2/Mop1 and Mop1, w.t.). (B) DNA was isolated from a family that was segregating _mop1_-2/_mop1_-1 and wild type (_mop1_-2/Mop1). A _Hin_fI digest of DNA was blotted and hybridized with the Mu1 interval probe. The diagnostic fragment for an unmethylated Mu1 element is indicated.

Figure 5

DNA blots and summary map of digests of DNA from mutant (mop1) and wild-type (w.t.) individuals using several methyl-sensitive enzymes. Hybridization was with the SB probe upstream of the b1 gene (shown in C). (A) _Apa_I/_Bam_HI digestions. Lanes 15 and 16 are _mop1_-1 homozygous and wild-type individuals, respectively, digested with only _Bam_HI. (B) _Sal_I/_Bam_HI and _Pvu_II/_Bam_HI digestions. The fragments marked with asterisks represent cutting at the _Bam_HI site in the b1 coding region and not cutting at one or more of the indicated upstream sites. (C) Restriction map of the region upstream of the start of transcription of _B_′. Sites marked with asterisks are methylated partially or completely in all genotypes tested (_B_′ Mop1 or Mop1/_mop1_-1 or _B_′ _mop1_-1). The other sites are unmethylated in all genotypes tested. A, _Apa_I; B, _Bam_HI; Pv, _Pvu_II; Hf, _Hin_fI; Sa, _Sal_I.

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A mutation that prevents paramutation in maize also reverses Mutator transposon methylation and silencing - PubMed (original) (raw)